Molecular Engineering of Electrosprayed Hydrogel Microspheres to Achieve Synergistic Anti‐Tumor Chemo‐Immunotherapy with ACEA Cargo

Abstract Molecular engineering of drug delivering platforms to provide collaborative biological effects with loaded drugs is of great medical significance. Herein, cannabinoid receptor 1 (CB1)‐ and reactive oxygen species (ROS)‐targeting electrosprayed microspheres (MSs) are fabricated by loading with the CB1 agonist arachidonoyl 2′‐chloroethylamide (ACEA) and producing ROS in a photoresponsive manner. The synergistic anti‐tumor effects of ACEA and ROS released from the MSs are assessed. ACEA inhibits epidermal growth factor receptor signaling and altered tumor microenvironment (TME) by activating CB1 to induce tumor cell death. The MSs are composed of glycidyl methacrylate‐conjugated xanthan gum (XGMA) and Fe3+, which form dual molecular networks based on a Fe3+‐(COO−)3 network and a C═C addition reaction network. Interestingly, the Fe3+‐(COO−)3 network can be disassembled instantly under the conditions of lactate sodium and ultraviolet exposure, and the disassembly is accompanied by massive ROS production, which directly injures tumor cells. Meanwhile, the transition of dual networks to a single network boosts the ACEA release. Together, the activities of the ACEA and MSs promote immunogenic tumor cell death and create a tumor‐suppressive TME by increasing M1‐like tumor‐associated macrophages and CD8+ T cells. In summation, this study demonstrates strong prospects of improving anti‐tumor effects of drug delivering platforms through molecular design.

(Changsha, China).All animal experiments were approved by the Institutional Animal Care and Use Committee of Xiangya Hospital (Approval No. 202009722) and complied with the ethical regulations.

Synthesis of XGMA
The conjugation of GMA to XG was based on a ring-opening polymerization reaction.Specifically, XG powder was dissolved in purified water and fully stirred to produce 0.6 wt% solution.Then, the solution's pH was adjusted to 4.2-4.8using hydrochloric acid, followed by dropwise addition of 1% (v/v) GMA to the XG solution.The resultant solution was stirred for 12 h at 80°C.Next, the mixture was collected and dialyzed for 3 days using dialysis membranes (molecular weight cut-off: 12-14 kDa) to remove unreacted residues.The dialysate was freeze-dried for 48 h to obtain XGMA.The DS of GMA to XG was calculated based on the ratio of area at peaks of δ = 5.65 and δ = 6.08 to that of δ =1.81 by 1 H NMR measurement.

Preparation of ACEA@CL(XGMA)-Fe(III) MS
The pre-gel solution was prepared by dissolving XGMA at different concentrations of 1, 1.25, and 1.5 wt% into a mixed aqueous solution containing 0.5 wt% I-2959 and 100 μM ACEA.Then, the pre-gel solution was stored in a 10 mL syringe with a 24G metal nozzle and electrosprayed with varying voltages at 7, 8, and 9 kV at the rate of 5 mm/min by an electrospinning machine (Yongkangleye Tech Co., Ltd., Beijing, China).The electrosprayed MSs were collected in 2.4 wt% FeCl3 solution, during which the first layer of Fe 3+ -(COO -)3 network was generated.Afterwards, the MSs were exposed to UV light to form the second layer of C=C addition reaction network.The resultant ACEA@CL(XGMA)-Fe(III) MSs were immersed in the sodium lactate buffer for 2 min and centrifuged to harvest the MSs.The encapsulation rate of ACEA was determined by the ratio of the total amount of incorporated ACEA in the MSs to the total quantity of ACEA added initially during the preparation.The loading capacity of ACEA was calculated by the ratio of the total amount of incorporated ACEA in the MSs to the total quantity of the MSs.

Characterization of ACEA@CL(XGMA)-Fe(III) MS
The morphology of ACEA@CL(XGMA)-Fe(III) MSs was observed in a microscope (CKX53, Olympus, Japan), and the size of MSs was analyzed with ImageJ (NIH, USA).The elements of freeze-dried MSs were measured with the energy dispersive spectroscopy (SU8220, HITACHI, Japan).The injectability of MSs in different sizes were evaluated by injection forces measured with a universal testing machine (MTS CMT2103, USA) where the MSs were placed into a 2-mL syringe with a 16G needle and injected at the rate of 10 mm/min.

Detecting and explaining ROS-generating ability of the MS
To visualize ROS production in the ACEA@CL(XGMA)-Fe(III) MSs, the ROS probe was loaded in the pre-gel solution at the final concentration of 10 μM.The produced ACEA@CL(XGMA)-Fe(III) MSs loaded with the ROS probe were observed under UV exposure with a SpinSR confocal microscope (Olympus, Japan).The MSs without UV exposure were used as a control.
Moreover, to investigate the parameters affecting ROS production, we adjusted the sodium lactate buffer concentration to 0 mol/L, 0.5 mol/L (1×), and 0.75 mol/L (1.5×), and the UV light intensity to 0 W/cm 2 , 2 W/cm 2 , and 4 W/cm 2 .The MSs were dissolved in PBS containing the ROS probe at 10 μM.After different UV exposure times, ranging from 0 min to 4 min, 100 μL of PBS was collected for detection of the optical density (OD) at 490 nm using a multi-functional microplate reader (BioTek, USA).In addition, after a cycle of UV exposure, we divided the MSs into two equal parts and placed them into air atmosphere and nitrogen environment separately for 8 min.Then, the MSs were immersed in PBS containing the ROS probe and exposed to UV light again.Subsequently, the microplate reader was used to measure the OD of PBS at 490 nm to examine whether the MSs regained their ROS-generating ability when reoxidized by oxygen.To reveal the potential chemical mechanisms, the valence state change of Fe was evaluated based on XPS (Nexsa G2, Thermo Fisher Scientific, USA) according to the Gaussian-Lorentzian curve-fitting method.

Releasing profile of ACEA in vitro
After immersing 500 μL ACEA@CL(XGMA)-Fe(III) MSs in 1 mL PBS and exposing the mixture to UV light for 4 min followed by nitrogen protection, 50 μL of PBS was harvested at pre-determined time points of 0, 2, 4, 8, 16, 24, and 48 h.The MSs without UV exposure were used as a control.ACEA concentration in the PBS was measured using HPLC (LC20A, SHIMADZU, Japan).Water and acetonitrile were used as the mobile phase.

ROS generation and oxidative stress injury of SW480 cells
The ROS probe was also utilized to detect ROS levels of SW480 cells with the treatment of CL(XMGA)-Fe(III) MS + UV.Briefly, 4 × 10 5 cells were seeded into 12-well plates and incubated for 24 h.Then, around 100 μL of CL(XGMA)-Fe(III) MSs or PBS was added into six wells.On this basis, three of the six wells were placed under UV irradiation at 4 W/cm 2 for 1 min.After incubation for 6 h, the cells were incubated with the ROS probe.The PBS-treated cells without adding the ROS probe were used as a control.Moreover, SOD and MDA involved in the oxidative stress reaction were detected using a SOD kit (Jiancheng, Nanjing, China) and MDA kit (Solarbio, Beijing, China).The cells were processed as mentioned above.After lysis and centrifugation, the supernatant was collected for detection of SOD and MDA.

Real-time PCR
Cells or tissues were lysed by TRIzol (15596026, Thermo Fisher Scientific, USA), the total RNA was isolated, and cDNA synthesis was performed with a PrimeScript RT reagent kit (K1622, Thermo Fisher Scientific, USA) according to the manufacturer's instructions.Real-time PCR was performed using a BeyoFast™ SYBR Green qPCR Mix (1708882AP, Bio-Rad, USA).All primers are listed in Table 1.

Wound healing test
Cell migration was investigated by the wound healing test.First, 2 × 10 5 cells in 1 mL culture medium were seeded into each well of a 24-well plate and incubated for 24 h.Next, the monolayer cells were scratched vertically with a sterile pipette tip, and the cells were photographed with a microscope.Then, the cells were treated with 40 μL PBS, 40 μL ACEA at the concentration of 91.8 μM, 40 μL CL(XGMA)-Fe(III) MS + UV (365 nm, 4 W/cm 2 , 1 min), or 40 μL ACEA@ CL(XGMA)-Fe(III) MS + UV (365 nm, 4 W/cm 2 , 1 min).After being cultured for 24 h, the cells were photographed.The relative migration rate was determined by the ratio of cell movement distance to the initial gap length.

In vivo tumor models and treatment
To evaluate the therapeutic effects of ACEA@ CL(XGMA)-Fe(III) MS + UV, 1 × 10 6 SW480 cells were injected subcutaneously into the left flank of the BALB/c nude mice.Nine days later, when the tumor volume reached ~100 mm 3 , mice were randomly divided into five groups (n = 5) as follows: PBS (as control); ACEA (1.5 mg/kg/d); ACEA@CL(XGMA)-Fe(III) MS; CL(XGMA)-Fe(III) MS + UV; and ACEA@CL(XGMA)-Fe(III) MS + UV.The ACEA in the MSs was used in the same dose as that injected directly.For the two groups treated with UV, the mice were treated with 5 min of UV irradiation (365 nm, 4 W/cm 2 , interval time: 6 h) 3 times per day for 3 d at the tumor sites.Tumor volume and body weight of mice were measured at 0, 3, 6, 9, 12, 15, and 18 d.Finally, the mice were sacrificed, and the tumor and major organ tissue were harvested for subsequent analysis.
To compare the profile of controlled ACEA release from MSs in the ACEA@CL(XGMA)-Fe(III) MS group and ACEA@CL(XGMA)-Fe(III) MS + UV group, from the day 9 to day 19 after the injection of MSs, the skin covered on tumors was cut.The residual MSs were taken out carefully, and then weighed followed by the placement in tubes.The amount of drug in the residual hydrogel was determined by HPLC.Water and acetonitrile were used as the mobile phase.The degradation of MSs and the accumulated release of ACEA in the two groups were calculated by the following equations: In vivo MS degradation = (Mms@0 -Mms@n) / Mms@0 × 100%; Accumulated ACEA release = (Mdrug@0 -Mdrug@n) / Mdrug@0 × 100%; Mms@0 is the initial mass of MSs injected to tumor sites, and Mms@n is the residual mass of MSs on the day n of injection; Mdrug@0 is the initial mass of ACEA in the MSs injected to tumor sites, and Mdrug@n is the residual mass of ACEA in the MSs on the day n of injection.
The ACEA in the MSs was used in the same dose as that injected directly.For the two groups treated with UV, the mice were treated with 5 min of UV irradiation (365 nm, 4 W/cm 2 , interval time: 6 h) 3 times per day for 3 d at the tumor sites.Two days later, the tumors were resected for subsequent analysis.

Hematoxylin & eosin (HE) staining
The tumors and organ tissues, including heart, liver, lung, kidney, and spleen, were collected, and fixed with formalin, dehydrated, and then embedded in paraffin.Serial sections were prepared, subjected to HE staining, and photographed with a microscope.

TUNEL staining
The tumors were fixed in formalin, embedded in paraffin, and sectioned.A TUNEL kit (TUN11684817, Roche, Swiss) was used to detect the apoptosis of the tumor tissue according to the manufacturer's instructions.

Flow cytometry in vivo
The tumors harvested from mice were divided into pieces, and cell

Statistical analysis
Statistical analysis was performed using Origin Pro and GraphPad Software 9.0.All data were presented as the mean value ± standard error of the mean (SEM), and analyzed with one-way ANOVA (when there were more Figure S16.

Figure S5 ,
Figure S5, related to Figure 2f.Detection of ACEA concentration using HPLC.(a) Determination of ACEA retention time as 5.547 min.(b, c) Preparation of ACEA concentration gradients (b) to establish of a standard concentration-area curve (c).HPLC: high performance liquid chromatography.

Figure S6 ,
Figure S6, related Figure 3j.Quantitative analysis of early and late apoptotic cell proportions after different treatments based on the outcome of flow cytometry.n = 3. ****, P < 0.0001.

Figure S9 ,
Figure S9, related to Figure 4b.Photography of tumors in different groups before (upper panel) and after (lower panel) surgery.G: group.

Figure S10 .
Figure S10.HE staining of heart, liver, lung, kidney, and spleen in different groups reveals non-toxicity to these important organs by different treatments.G: group.

Figure S16 .
Figure S16.Gating strategy of flow cytometry to detect T cells (CD3 + ) and the subtypes of CD4 + T cells and CD8 + T cells.G: group.