Microfluidics‐Enabled Nanovesicle Delivers CD47/PD‐L1 Antibodies to Enhance Antitumor Immunity and Reduce Immunotoxicity in Lung Adenocarcinoma

Abstract The CD47/PD‐L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune‐related adverse events (IRAEs) caused by the on‐target off‐tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics‐enabled nanovesicle using ultra‐pH‐sensitive polymer mannose‐poly(carboxybetaine methacrylate)‐poly(hydroxyethyl piperidine methacrylate) (Man‐PCB‐PHEP) is developed to deliver CD47/PD‐L1 antibodies (NCPA) for tumor‐acidity‐activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow‐derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD‐L1 antibodies accumulation, promoted tumor‐associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated.

WO2014199174A1 [1] . Briefly, D-mannose (4.5 g, 2.5 mmol) was dissolved by anhydrous tetrahydrofuran (20 mL) in a Schlenk flask equipped with a magnetic stirrer and the solution was bubbled by N2 for 10 min. After adding triethylamine into the flask, 2-bromoisobutyryl bromide (3.15 mL, 25 mmol) was transferred to the flask drop-wise to perform the reaction under N2 protection for 24 h. The mixture was dropped into an excess amount of cold petroleum ether to precipitate. After filtering and washing with petroleum ether, the solid was re-dissolved in chloroform and precipitated by cold petroleum ether twice. The solid was dried by vacuum to yield a white waxy product.

Synthesis of 2-hydroxyethyl piperidine methacrylate
A well-stirred, ice-cooled solution of 2-hydroxyethyl piperidine (1.32 mL, 10 mmol) in an aqueous sodium hydroxide (1.60 g, 20 mmol, 25 mL) was treated with dropwise addition of methacryloyl chloride (1.19 mL, 11 mmol) over a period of ∼15 min. After 30 min of stirring on ice, the reaction mixture was allowed to reach room temperature and stirred for a further 4 h. The mixture was adjusted to pH 7 using dilute HCl (0.1 M).
The resulting beige mixture was extracted with dichloromethane (DCM) and washed 6 times with NaCl saturated solution. The DCM solution was dried with anhydrous sodium sulfate over 0.5 h. After filtration, DCM was removed by evaporating to obtain a light yellow liquid product.

Synthesis of mannose-poly (carboxybetaine methacrylate) (Man-PCB)
The copolymer Man-PCB was synthesized by copper-free organic ATRP according to literature [2] . A 10 mL schlenk tube charged with a magnetic stirrer was added with initiator Man-Br (16.5 mg), catalyst dihydrophenazine PC™ B0301 (0.5 mg), monomer carboxybetaine methacrylate (200 mg), and solvent dimethylformamide (DMF) (10 mL). The tube was sealed and degassed by freeze-thawing three times. After 24 h reaction under white LED light, the mixture was dialysis against deionized water. The resulting solution was lyophilized to obtain a white product.

Synthesis of mannose-poly(carboxybetaine methacrylate)-poly(2-hydroxyethyl piperidine methacrylate) (Man-PCB-PHEP)
50 mg Man-PCB was dissolved by DMF in a 50 mL schlenk tube charged with a magnetic stirrer. Then, the catalyst dihydrophenazine PC™ B0301 (0.5 mg) and monomer HEPMA (2.50 g) were dissolved in DMF (10 mL) and transferred into the tube. The tube was sealed and degassed by freeze-thawing three times to process 24 h reaction under a white LED light. Afterward, the mixture was adjusted to pH 5 using dilute HCl (0.1 M) and dialysis against deionized water. The resulting beige solution was lyophilized to obtain a white yellow product.

Design and fabrication of microfluidic chip
The structure of the microfluidic chip for tumor-acidity responsive NCPA synthesis is shown in Figure S5, which has three inlets, two prismatic-ambulatory-plane channels, two straight mixing channels, one 5 loops double spiral mixing channel, and one outlet.
And the channel of this chip was 100 µm wide and 60 µm depth. The master mold and the chip were fabricated as previously described.

Characterization of synthetic polymers
1 H NMR spectra were obtained using an AVANCE III 400M spectrometer. D2O or CDCl3 was used as the solvent depending on their solubility. The molecular weight and molecular weight distribution of the polymers were estimated using a gel permeation chromatography (GPC) system in the water phase. The pKa of Man-PCB-PHEP was evaluated by titration according to the literature [3] .

Physicochemical characterization of NCPA
The hydrodynamic sizes and zeta potentials of NCPA were determined by dynamic light scattering (DLS). Measurements were performed at 25 °C using the Malvern Zetasizer Nano ZS instrument (Malvern Instruments Ltd, UK). The data of particle sizes and zeta potentials were collected on an auto-correlator with detection angles of scattered light at 175°, respectively. Transmission electron microscopy (TEM) was performed using an HT7700 operated at 120 kV. The samples were prepared by drying a drop (6 μL, 1 mg/mL) of the sample solution on a copper grid coated with amorphous carbon. For the negative staining of samples, a small drop of uranyl acetate solution (1 wt% in water) was added to the copper grid, which was then blotted with a filter paper after 1 min.
Finally, the grid was dried overnight in a desiccator before TEM observation.

Bone marrow isolation and Bone marrow-derived macrophages (BMDMs) culture
Progenitor cells were isolated from murine bone marrow and cultured in DMEM with M-GSF (20 ng/mL) according to methods described in the literature [4] . Briefly, the collected progenitor cells from the bone marrow of a mouse were seeded in a T75 cell culture flask. On day 3, media was aspirated and washed with PBS once and replaced with fresh DMEM with M-GSF (20 ng/mL). On day 7, the cells were dislodged by trypsin digestion in 10 minutes and counted with a hemocytometer for further experiments.

Isolation and culture of splenocytes
Splenocytes were isolated from the murine spleen and cultured in RPMI-1640 with IL-2 (20 ng/mL) according to methods described in the literature [5] . The collected splenocytes cells from a mouse spleen were seeded in a T75 cell culture flask. The media was replaced with fresh RPMI-1640 supplemented IL-2 (20 ng/mL) every two days. On day 7, the matured T cells were counted with a hemocytometer for further experiments.

Cell culture
Lewis lung carcinoma cells were cultured in Dulbecco's modification of Eagle medium (DMEM) supplemented with 10% (vol/vol) FBS, 1% L-glutamine, and 1% penicillin and streptomycin (P/S). And all cells were maintained at 37 °C and 5% CO2.

Tumor spheroid
The 3D tumor spheroid was established according to literature [6] . Briefly, target cells