A dendritic cell-like biomimetic nanoparticle enhances T cell activation for breast cancer immunotherapy

Cancer immunotherapy has remarkably improved the therapeutic effect of melanoma and non-small cell lung cancer in the clinic. Nevertheless, it showed disappointing clinical outcomes for treating immunosuppressive tumors, wherein aggressive T cells are rather limited in tumor sites. Therefore, regulating the behavior of T cells in tumor sites to increase their attack ability for suppressing the immunosuppressive tumor is highly desirable. Inspiringly, we designed a dendritic cell-like biomimetic nanoparticle (DMSNs3@HA) to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors. In this work, anti-CD3 and anti-CD28 were responsible for mimicking dendritic cells to activate T cells, and anti-PD-1 for blocking the pathway of PD-1/PD-L1 to break the immune “brake”, which synergistically regulated the behavior of T cells to attack cancer cells. Experimental results indicated that DMSNs3@HA can effectively activate T cells and improve their immune response to significantly inhibit the growth of breast cancer. Moreover, it also proved that T cell activation combining immune checkpoint blocking induced the “1 + 1 >2” immunotherapy effect against immunosuppressive tumors. We expect that this strategy will provide new insights into tumor immunotherapy by modulating T cell behavior.

quickly and stirred for another 2 h. The obtained product was centrifuged and stirred in the saturated NaCl/MeOH for at least 48 h to remove the template. After that, the synthesized DMSNs were washed by ethanol and water for three times, and then dried for later use.
Synthesis of the DMSNs modified with amino and carboxyl groups. 40 mg of the prepared DMSNs, 4 mL of H 2 O and 10 mL of ethanol were homogeneously mixed.
Then 100 µL of ammonium hydroxide was added to the above solution to adjust the pH. After 30 min, 40 µL of APTMS was added and stirred overnight to modify the DMSNs with amino groups (DMSNs-NH 2 ). The DMSNs-NH 2 were centrifuged and washed by ethanol and water for three times. Ninhydrin assay was carried out to verify the modification of amino groups on DMSNs-NH 2 . 1 % of ninhydrin and DMSNs-NH 2 were mixed and subjected to boiling. That ninhydrin reacted with supernatant liquid of the centrifuged DMSNs-NH 2 solution served as the control group.
DMSNs-NH 2 were dispersed in anhydrous DMF, following with addition of excess succinic anhydride. The reaction continues overnight to obtain the DMSNs modified with carboxyl groups (DMSNs-COOH). The product were centrifuged and washed by ethanol and water for three times.
Synthesis of the DMSNs modified with antibodies. 1 mg of DMSNs-COOH were activated by 1.5 mg of EDC and 0.7 mg of NHS for 30 min. 12 µg of anti-PD-1 was added to the solution and stirred for 10 min. And then 24 µg of anti-CD3 and 24 µg of anti-CD28 were added to the above solution and stirred overnight at 4 ℃ to react with the activated DMSNs-COOH. DMSNs-COOH modified with only anti-PD-1 were named as DMSNs 1 . DMSNs-COOH modified with anti-CD3 and anti-CD28 were named as DMSNs 2 . DMSNs-COOH modified with anti-PD-1, anti-CD3 and anti-CD28 were named as DMSNs 3 .
Synthesis of the HA modified with amino groups. 1 g of HA monomer was dispersed in pH 6.0 MES buffer. And then 2.5 g EDC and 1.5 g NHS were added to the solution and stirred for 30 min to activate carboxyl groups. After that, 4.75 g of ethylenediamine was added and stirred overnight. The product (HA-NH 2 ) was dialysed by dialysis membrane (300 Da) and freeze-dried for further use. Ninhydrin assays were carried out to verify the modification of amino groups on HA-NH 2 .
Ethylenediamine, HA and HA-NH 2 were respectively mixed with 1 % of ninhydrin and subjected to boiling to observe the change of color.
Synthesis of the DMSNs 3 modified with HA. 1 mg of DMSNs 3 was dispersed in pH 6.0 MES buffer with addition of 2 mg of EDC and 1 mg of NHS for 30 min. And then 7 mg of HA-NH 2 was added and the reaction continued overnight to obtain DMSNs 3 @HA at 4 o C.

T cell activation. CD8 + T cells were harvested by MojoSort™ Mouse CD8 T Cell
Isolation Kit. Spleens were extracted from healthy mice, and single-cell suspension was prepared and dispersed in the cold buffer. After filtered by filter membrane (70 µm), the cell density was adjusted to 10 8 /mL. 10 µL of biotin-Antibody Cocktail was added into 100 µL of above cells. Mix and incubate on ice for 15 min. Then 10 µL of streptavidin was added and incubate on ice for another 15 min. After added with 2.5 mL of buffer, the mixed cell solution was placed in the magnet for 5 min. Collect the supernate and repeat the separation steps to obtain the CD8+ T cells with high purity for further use.
CD8 + T cells were divided into two groups and incubated with: 1) PBS; 2) 0.2 mg/mL DMSNs 3 for different time. Then the T cells were stained with anti-CD69-APC and anti-CD25-FITC and detected by flow cytometry to verify the activation of T cells.
CD4 + T cells were harvested by MojoSort™ Mouse CD4 T Cell Isolation Kit. The other steps were the same as above.
In vivo experiments. For the xenografts established from cultured cells00, 4T1 cells were suspended and harvested after trypsinization, and approximately 5 × 10 5 4T1 cells in 150 μL of serum-free RPMI 1640 medium were subcutaneously injected into the right flank of the mice. The tumor volume (V) was determined by measuring the length (L) and width (W) and was calculated as L × W 2 /2.
To study the antitumor efficacy, mice with tumors were randomly divided into six groups and subjected to different treatments: 1), PBS; 2), DMSNs@HA; 3), DMSNs 1 @HA; 4), DMSNs 2 @HA; 5), DMSNs 3 ; and 6), DMSNs 3 @HA. The intravenously injected dose was 50 mg/kg each time for 5 times in total. The tumor volumes and body weights of the mice were measured every other day for 14 days. Haematoxylin and eosin (H&E) staining of the five major organs (heart, liver, spleen, lung and kidney), routine blood tests and blood biochemical tests were carried out at 24 h post-treatment to prove that the body's immune response resulted in minimal side effects to major organs.
The carbazole assay. The samples were placed in the ice water bath. Gradually add in 5 mL of pre-cooling Na 2 B 4 O 7 /H 2 SO 4 (0.025 M) while stirring in the ice water bath.
Then the mixed solution was kept in boiling water for 20 min. Cool to room temperature and add in 0.2 mL of carbazole/ethanol (0.1%) to observe the color change after 2 h.
In vivo imaging. The fluorescence images were taken by an in vivo imaging system (IVIS). The Living Image software (Xenogen) was used to acquire the data at different times (0, 0.5, 2, 4, 12, 24, 36, 48 h) after treatments.