Bortezomib‐Encapsulated Dual Responsive Copolymeric Nanoparticles for Gallbladder Cancer Targeted Therapy

Abstract Gallbladder cancer (GBC) is a rare but the most malignant type of biliary tract tumor. It is usually diagnosed at an advanced stage and conventional treatments are unsatisfactory. As a proteasome inhibitor, bortezomib (BTZ) exhibits excellent antitumor ability in GBC. However, the long‐term treatment efficacy is limited by its resistance, poor stability, and high toxicity. Herein, BTZ‐encapsulated pH‐responsive copolymeric nanoparticles with estrone (ES‐NP(BTZ; Ce6)) for GBC‐specific targeted therapy is reported. Due to the high estrogen receptor expression in GBC, ES‐NP(BTZ; Ce6) can rapidly enter the cells and accumulate near the nucleus via ES‐mediated endocytosis. Under acidic tumor microenvironment (TME) and 808 nm laser irradiation, BTZ is released and ROS is generated by Ce6 to destroy the “bounce‐back” response pathway proteins, such as DDI2 and p97, which can effectively inhibit proteasomes and increase apoptosis. Compared to the traditional treatment using BTZ monotherapy, ES‐NP(BTZ; Ce6) can significantly impede disease progression at lower BTZ concentrations and improve its resistance. Moreover, ES‐NP(BTZ; Ce6) demonstrates similar antitumor abilities in patient‐derived xenograft animal models and five other types of solid tumor cells, revealing its potential as a broad‐spectrum antitumor formulation.


Hydrophilic chain synthesis (ES-PEG2K-COOH):
To prepare the hydrophilic chain, polyethylene glycol 2000 (PEG2K) (5.0 g, 2.5 mmol) was dissolved in N,N-dimethylformamide (DMF) (30 mL). PEG-COOH was obtained by the reaction of PEG2K with succinic anhydride (0.3 g, 2.5 mmol), and estrone (0.7 g, 2.5 mmol) were then added to the mixture after dissolution as a receptor, respectively. The mixture was stirred and heated in an oil bath to 35 o C for 24 h. After 24 h, the reaction mixture was centrifuged to precipitate ES-PEG2K from DMF.
ES-PEG2K was purified by precipitation in ethanol (50.0 mL) 3 times. Finally, the precipitate was lyophilized to obtain the hydrophilic chain.
Hydrophobic chain synthesis (n-butylamine-P(Asp-DA)): The hydrophobic chain was synthesized using β-Benzyl-ʟ-aspartate N-carboxy anhydride (BLA-NCA) as a template. BLA-NCA was synthesized by the Fuchs-Farthing method with bis-(trichloromethyl)-carbonate (triphosgene). [1] Briefly, BLA (10.0g, 44.8 mmol) was suspended in tetrahydrofuran (THF) (100 mL) containing triphosgene (10.0g, 33.7 mmol) and stirred at 60 o C for 3 h under argon gas. The reaction mixture was cooled to room temperature and filtered. Next, the product was purified via precipitation with hexane (500 mL) three times, collected by filtration, and dried under vacuum. The resulting BLA-NCA is obtained as a white powder. To prepare the hydrophobic chain, monomer BLA-NCA (10.0 g, 40.0 mmol) was dissolved in DMF (30 mL) and was synthesized by ring-opening polymerization using n-butylamine (0.1 mL) as an initiator. The reaction mixture was stirred and heated in an oil bath to 40 o C for 48 h under argon gas. After 48 h, the reaction mixture was cooled to room temperature and the ammonolysis reaction of ammonia solution (1.0 mL) was added to the mixture and stirred at room temperature. The reaction mixture was centrifuged to remove the phenyl group from the solution, and the solution was collected. Catechol functional group was synthesized via dopamine, in which the precursor, dopamine hydrochloride (DA) (0.9 g, 5.0 mmol), was added to the solution and the reaction mixture was stirred and heated in an oil bath to 40 o C for 24 h under argon gas. After 24 h, the reaction mixture was cooled to room temperature and cool ethanol was added to precipitate n-butylamine-P(Asp-DA). n-butylamine-P(Asp-DA) was purified by precipitation in cool ethanol (50.0 mL) 3 times. Finally, the precipitate was lyophilized to obtain the hydrophobic chain.  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 o C in 5% CO2.
After incubating for 4 h, 12 h, and 24 h, cells were washed with PBS three times. The samples were fixed with 4% paraformaldehyde for 10 min and incubated with DAPI (0.01%) for 10 min. Cellular uptake was monitored by confocal microscopy (Zeiss LSM 880, Germany) at different time points.

Potential clinical applications and limitations
For deep-located abdominal tumors, our nanoparticles could also function similarly to those used in CDX or PDX models by delivering NIR to abdominal organs via interventional tools, such as optic fiber-assisted phototherapy. [2] Several studies have recently explored the application of interventional therapy-assisted phototherapy in deep in-situ tumors with encouraging results. [2,3] The same technique may also be applied to treat patients with GBC using currently available laparoscopic equipment. Additionally, our nanoparticles have practical applications based on the clinical characteristics of resected GBC. For instance, positive surgical margins for GBC are frequently associated with poor patient survival. [4] Hence, our nanoparticles combined with NIR irradiation of the margin can significantly help lower the positive rate of the margin during the surgical resection of GBC. In addition, GBC patients who underwent laparoscopic surgical treatment may develop a tumor on the surface of the body as a result of the incision implantation tumor. [5] Furthermore, skin permeability occurs between 620-850 nm, and higher wavelengths have stronger tissue penetration. [6] Thus, employing an 808 nm laser may be more advantageous in practice. Meanwhile, our results indicate that ROS levels generated by Ce6 under 808 nm laser irradiation maintained the effect of combination therapy. Naturally, it is undeniable that current researches on nanomaterials combined with NIR irradiation are usually verified on the surface of animals. Therefore, the treatment of deep tumors in the body with such materials requires further development and validation.

Statistical analysis:
The data in this study were expressed as mean ± SD of at least three independent experiments.
OriginPro 9.5.1 and GraphPad Prism 8 Software were used for the statistical analysis. Oneway analysis of variance (ANOVA) with Tukey's test was used to compare CCK-8 assay, ROS intensity, wound healing studies, colony-forming assays, tumor volume, body weight, etc., with different treatments. In all cases, the differences of statistics were considered at *P < 0.05, **P < 0.01. All P values were two-tailed.              Figure S15: Intracellular ROS observation of a) NOZ or b) GBC-SD cells exposed to normal media, BTZ, ES-NP(Ce6), or ES-NP(BTZ; Ce6) with/without 808 nm laser irradiation (2 W cm -2 , 5 min with every min interval). Scale bar = 100 µm.
Figure S17: 0-72 h viability of NOZ and GBC-SD cells exposed to different concentrations of BTZ. The data are represented as mean ± SD (n=3). Figure S18: 0-72 h growth curve of NOZ and GBC-SD cells exposed to normal media, BTZ, ES-NP(Ce6), and ES-NP(BTZ; Ce6) with and without 808 nm laser irradiation (2.0 W cm -2 , 5 min with every min interval). The data are represented as mean ± SD (n=3).