Enzyme‐Responsive Branched Glycopolymer‐Based Nanoassembly for Co‐Delivery of Paclitaxel and Akt Inhibitor toward Synergistic Therapy of Gastric Cancer

Abstract Combined chemotherapy and targeted therapy holds immense potential in the management of advanced gastric cancer (GC). GC tissues exhibit an elevated expression level of protein kinase B (AKT), which contributes to disease progression and poor chemotherapeutic responsiveness. Inhibition of AKT expression through an AKT inhibitor, capivasertib (CAP), to enhance cytotoxicity of paclitaxel (PTX) toward GC cells is demonstrated in this study. A cathepsin B‐responsive polymeric nanoparticle prodrug system is employed for co‐delivery of PTX and CAP, resulting in a polymeric nano‐drug BPGP@CAP. The release of PTX and CAP is triggered in an environment with overexpressed cathepsin B upon lysosomal uptake of BPGP@CAP. A synergistic therapeutic effect of PTX and CAP on killing GC cells is confirmed by in vitro and in vivo experiments. Mechanistic investigations suggested that CAP may inhibit AKT expression, leading to suppression of the phosphoinositide 3‐kinase (PI3K)/AKT signaling pathway. Encouragingly, CAP can synergize with PTX to exert potent antitumor effects against GC after they are co‐delivered via a polymeric drug delivery system, and this delivery system helped reduce their toxic side effects, which provides an effective therapeutic strategy for treating GC.


Characterization methods
Characterizations of monomers and polymer conjugates were conducted by 1 H NMR (400 MHz Bruker Avance II NMR spectrometer, Germany) for their structure confirmation.The weight-average (Mw) and number-average (Mn) molecular weights of polymers were determined by Gel Permeation Chromatography (GPC) with a GPC column of Shodex Asahipak GF-510 HQ (7.5 mm ID × 300 mmL).The injection volume was 50 μL of 2 mg mL -1 sample solutions.The mobile phase was a 0.2 M lithium chloride sodium solution (H2O: DMF = 35:65, v/v) at a flow rate of 0.5 mL/min (45 °C).Mw, Mn and polydispersity index (PDI) were measured using Pullulan as a standard.High performance liquid chromatography (HPLC) analyses were performed on a Shimadzu prominence HPLC system.

Synthesis of MA-GFLG-MA
The Boc-GFLG-Boc compound was synthesized following a previously reported method. [4]bsequently, Boc-GFLG-Boc (690 mg, 1.09 mmol) was dissolved in 9 mL of dichloromethane (DCM) in an ice bath, followed by gradual addition of 9 mL of trifluoroacetic acid.The reaction mixture was mixed at room temperature for 5 h.Afterward, the solvent was removed by rotary evaporation, and the remaining residue was washed twice with diethyl ether.The solid residual was vacuum-dried, yielding a white solid product.

A B
The deprotection products of Boc-GFLG-Boc (400 mg, 0.63 mmol) was dissolved in a mixed solution of 6 mL acetonitrile (ACN) and water (1:4, v/v) under an ice bath.A trace amount of 4-methoxyphenol was added to the reaction mixture, followed by gradual addition of an ACN solution containing methylacrylyl chloride (MA-Cl) (0.183 mL, 1.89 mmol).
Concentrated sodium hydroxide (NaOH) was used during the reaction to maintain the pH of the reaction mixture at 8 to 9.After 1 h of reaction at 0 C, the reaction proceeded for an additional hour at room temperature.Upon completion, the organic solvent was removed by rotary evaporation.The remaining residue was diluted with a large amount of ethyl acetate.
Dilute hydrochloric acid was added to adjust the pH of the reaction mixture to be 2 to 3. The organic layer was collected, dried with anhydrous sodium sulfate, and concentrated to approximately 10 mL.The solution was left at 4 C for crystallization.Subsequently, the product was collected by filtration, yielding a white solid product of 0.34 g.

Synthesis of Maleimide-GFLG-PTX
Maleimide-GFLG-OH (500 mg, 0.92 mmol), PTX (943 mg, 1.1 mmol), and DMAP (7.1 mg, 0.06 mmol) were accurately weighed and placed into a round-bottom flask.Under a nitrogen atmosphere, 30 mL of DCM was added to ensure complete dissolution of the reactants.With continuous stirring over an ice bath, a solution of DIC (285 μL, 1.84 mmol) in DCM was slowly dripped into the reaction mixture.Subsequently, the reaction mixture was kept at 4 °C for 16 h.After removal of the solvent under a reduced pressure, the resulting residue was dissolved in 200 mL of DCM.The mixture was subjected to sequential washing with a saturated sodium bicarbonate solution, dilute hydrochloric acid, and a saturated sodium chloride solution.Each washing step was performed three times.Collect the organic layer, dry it with anhydrous sodium sulfate, and remove the solvent through vacuum distillation.The crude product was subjected to column purification, yielding a white solid product of 523 mg.

Synthesis of LAEMA-based branched polymeric prodrug
A cathepsin B-sensitive branched polymer prodrug (branched poly(PLAEMA)-GFLG-PTX, BPGP) was synthesized through a combination of RAFT polymerization and thiol-ene reaction.First, a high-molecular-weight chain transfer agent based on LAEMA was prepared.
Subsequently, this agent was copolymerized with MA-GFLG-MA, a GFLG-functionalized crosslinker, and MA-PySS, resulting in the formation of a branched polymer scaffold.After deprotection and exposure of thiol groups on the branched polymer scaffold, the maleimidemodified GFLG-functionalized PTX prodrug was covalently coupled to the polymer via thiolene reaction, yielding the target product.The synthetic route is illustrated in the Scheme S2.

IHC and score
Paraffin-embedded clinical tissue sections were subjected to antigen retrieval; endogenous peroxidase was blocked using 3% hydrogen peroxide; and BSA was evenly applied to cover the tissue sections and incubated at room temperature.A primary antibody was applied on the tissue sections for overnight incubation at 4°C in a humidified chamber.After washing with PBS, the tissues were incubated with an HRP-conjugated secondary antibody at room temperature.After washing with PBS, DAB was used for color development, and the reaction was terminated by rinsing with tap water.Counterstaining was performed with hematoxylin, followed by dehydration and slide sealing.Image acquisition and scoring analysis were conducted.
The immunohistochemical scoring was determined by multiplying the percentage of positive cells and the staining intensity.The scoring system for the percentage of positive cells was set as follows: 0 score for less than 5% of positive cells; 1 score for greater than 5% but less than 25% of positive cells; 2 score for greater than 25% but less than 50% of positive cells; 3 score for greater than 50% but less than 75% of positive cells; and 4 score for greater than 75% of positive cells.The staining intensity was scored as 0 for negative (no color), 1 for weak positive (light yellow), 2 for moderate positive (light brown), and 3 for strong positive (brown). [5]Table S3 lists the scoring criteria for IHC.
After incorporating the immunohistochemical scoring and survival rates into the X-tile software, [6] we calculated the score that maximizes the survival difference and designated it as the cut-off value.Basing on the score, we classified the patients into an AKT low-expression group and an AKT high-expression group.

Tumor regression grading (TRG)
The TRG was evaluated by two experienced pathologists independently according to the CAP TRG classification. [7]TRG was classified as follows: Grade 0 (complete response) for the absence of viable residual cancer cells; Grade 1 (moderate response) for a small number of residual cancer cells; Grade 2 (minimal response) for residual tumors outgrown by fibrosis; and Grade 3 (poor response) for abundant residual cancer.

Figure S14 .
Figure S14.Semi-quantitative analysis of mean fluorescence intensity (MFI) of Cy5 in MFC cells after exposure to Cy5-labelled BPGP@CAP for 1 h, 2 h and 4 h via Image J (n = 3).Data were expressed as mean ± SD.The statistical significance was displayed by two-sided unpaired Student's t-test, ***p < 0.001.

Figure S15 .
Figure S15.Semi-quantitative analysis of MFI in MFC cells after exposure to Cy5-labelled BPGP@CAP for 1 h, 2 h and 4 h (n = 3).Data were presented as mean ± SD.Statistical significance was determined using a two-sided unpaired Student's t-test, ***p < 0.001.

Figure S16 .
Figure S16.Flow cytometry diagram and MFI of Cy5-labelled BPGP@CAP uptaken by MFC cells after exposure to different inhibitors or at a low temperature (n=3).Data were shown as mean ± SD.The statistical significance was displayed by two-sided unpaired Student's t-test, ***p < 0.001.

Figure S17 .
Figure S17.Microfilament changes in MFC cells induced by various treatments for 24 h.Green for Actin-Tracker Green and blue for cell nuclei stained by DAPI.Scale bar = 20 μm.

Figure S18 .
Figure S18.Heatmap of differentially expressed genes (DEGs) in MFC cells after different treatments measured by RNA-seq.Red for upregulation and green for downregulation.The P adjusted value was < 0.05.

Figure S19 .
Figure S19.Total radiant efficiencies of tumors in the MFC tumor-bearing mice after treatment with free Cy5 and Cy5-labelled BPGP@CAP at different time points.

Figure S20 .
Figure S20.Photographs of tumors harvested from the mice at the end of the treatment schedule (A) and tumor growth inhibition (TGI) values in different treatment groups (B) (n = 5).Data were shown as mean ± SD.Statistical significance was determined using a two-sided unpaired Student's t-test, ***p < 0.001.

Table S1 .
The relationship between AKT expression and neoadjuvant chemotherapy response in gastric

Table S2 .
Pharmacokinetic parameters for mice treated with BPGP@CAP and free Cy5 by fitting the data to a non-compartment model by PKSolver 2.0 software.