Hyaluronic acid-bilirubin nanomedicine-based combination chemoimmunotherapy

Despite significant advances in immune checkpoint blockade (ICB), immunosuppression mediated by tumor-associated myeloid cells (TAMCs) poses a major barrier to cancer immunotherapy. In addition, while immunogenic cell death (ICD) provides a viable approach to inducing anti-tumor immune response, it remains unknown how to effectively trigger ICD while addressing immunosuppressive TAMCs. Here, we show that SC144, a gp130 inhibitor that blocks the IL-6/gp130/STAT3 pathway, induces ICD of tumor cells and polarizes macrophages to M1-phenotype in vitro. However, as SC144 also induces killing of CD8+ T-cells, we sought to deliver SC144 selectively to tumor cells and TAMCs. Toward this goal, we have developed hyaluronic acid-bilirubin nanoparticles (HABN) that accumulate in CD44hi tumor cells and TAMCs. Systemic administration of SC144 loaded in HABN (SC144@HABN) induces apoptosis and ICD of tumor cells, increases the ratio of M1-like to M2-like macrophages, and decreases the frequency of myeloid-derived suppressor cells and CD4+ regulatory T-cells, while promoting anti-tumor CD8+ T-cells. Moreover, SC144@HABN combined with anti-PD-L1 ICB efficiently eliminates MC38 tumors and ICB-resistant 4T1 tumors. Overall, our work demonstrates a therapeutic strategy based on coordinated ICD induction and TAMC modulation and highlights the potential of combination chemoimmunotherapy.

Despite significant advances in immune checkpoint blockade (ICB), immunosuppression mediated by tumor-associated myeloid cells (TAMCs) poses a major barrier to cancer immunotherapy.In addition, while immunogenic cell death (ICD) provides a viable approach to inducing anti-tumor immune response, it remains unknown how to effectively trigger ICD while addressing immunosuppressive TAMCs.Here, we show that SC144, a gp130 inhibitor that blocks the IL-6/gp130/STAT3 pathway, induces ICD of tumor cells and polarizes macrophages to M1-phenotype in vitro.However, as SC144 also induces killing of CD8 + T-cells, we sought to deliver SC144 selectively to tumor cells and TAMCs.Toward this goal, we have developed hyaluronic acidbilirubin nanoparticles (HABN) that accumulate in CD44 hi tumor cells and TAMCs.Systemic administration of SC144 loaded in HABN (SC144@HABN) induces apoptosis and ICD of tumor cells, increases the ratio of M1-like to M2like macrophages, and decreases the frequency of myeloid-derived suppressor cells and CD4 + regulatory T-cells, while promoting anti-tumor CD8 + T-cells.Moreover, SC144@HABN combined with anti-PD-L1 ICB efficiently eliminates MC38 tumors and ICB-resistant 4T1 tumors.Overall, our work demonstrates a therapeutic strategy based on coordinated ICD induction and TAMC modulation and highlights the potential of combination chemoimmunotherapy.
Cancer immunotherapy aims to harnesses the immune system to eliminate cancer [1][2][3][4] .Immune checkpoint blockers (ICBs) targeting CTLA-4, PD-1, and PD-L1 have generated unprecedented anti-tumor responses in cancer patients [1][2][3] .However, only 10-30% of patients currently respond to ICBs 5,6 .The therapeutic efficacy of ICBs is generally thought to depend on pre-existing anti-tumor T-cell immunity.Thus, poor tumor-infiltration of T-cells and immunosuppressive tumor microenvironment (TME) observed in most advanced cancer patients is attributed to the limited patient response rate to ICB therapy 1,2 .Therefore, various strategies, including therapeutic vaccines, radiation therapy, and chemotherapy, are being developed to improve anti-tumor T-cell response and reverse immunosuppression in the TME so that combination immunotherapy with ICBs can promote stronger anti-tumor immunity [7][8][9] .
Notably, immunogenic cell death (ICD), a special form of tumorcell killing, induced by certain chemotherapeutic drugs, such as bleomycin, doxorubicin, and oxaliplatin, may contribute to anti-tumor immune response 10,11 .During ICD process, tumor cells expose "eat me", "danger", and "find me" signals.Once the "eat me" signals, such as calreticulin (CRT), are exposed on the surfaces of immunogenically dying tumor cells, they promote phagocytosis and presentation of tumor antigens by dendritic cells (DCs) in the context of major histocompatibility complex (MHC) class I or II, triggering antigenspecific T cell responses 12,13 .In addition, danger signals, such as highmobility group box 1 (HMGB1), released from immunogenically dying tumor cells can activate DCs via interactions with pattern-recognition receptors and facilitate antigen presentation by DCs, leading to enhanced antigen-specific T cell responses 11,14 .In parallel, "find me" signals, such as C-X-C motif chemokine ligand 10 (CXCL10), are released from immunogenically dying tumor cells, promoting intratumoral infiltration of anti-tumor T-cells 15,16 .Thus, ICD-inducing chemotherapeutic agents may provide an effective pathway to kill cancer cells while simultaneously eliciting anti-tumor T cell responses [16][17][18] .Indeed, clinical studies have shown anti-tumor immune responses induced by ICD-inducing drugs, such as doxorubicin, given either as a monotherapy or combined with immunotherapy 19 .However, there are still concerns of limited intratumoral accumulation of ICD agents as well as their off-target toxicities 7,8,20 .Moreover, ICD agents should be delivered to tumor cells while leaving other cells intact, since ICD agents taken up by immune cells in the TME can interfere with antitumor immune functions.
Another barrier in cancer immunotherapy is tumor-associated myeloid cells (TAMCs).TAMCs, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), are the major immune-suppressive component of TME, as they secrete various immune-regulatory factors, such as IL-6 and TGF-β, and inhibit activation, viability, and tumoral-infiltration of T-cells, thus leading to ICB resistance and poor prognosis [21][22][23] .Various studies have indicated that TAMC modulators, such as imiquimod, 852A, IMO-2055, and BLZ945, can improve ICB-induced anti-tumor immune response 21 .However, these TAMC modulators administered systemically lead to poor accumulation in TAMCs and cause significant off-target toxicities 7,24 .Hence, to overcome such resistance mechanisms and reduce the potential toxicity of TAMC modulators, a drug delivery approach for targeted delivery to TAMCs is needed.
To address these challenges, we sought to develop a general strategy for delivering an ICD agent and TAMC modulator in a manner compatible with ICB therapy.In particular, we previously reported the development of SC144, a small-molecule gp130 inhibitor that can block IL-6-induced nuclear translocation of STAT3, and shown their anti-tumor efficacy in murine xenograft tumor models [25][26][27][28] .STAT3 is an oncogenic transcription factor with potent immunosuppressive functions [29][30][31] , and activation of the IL-6/gp130/ STAT3 pathway is associated with tumor progression and M2polarized TAMs with pro-tumoral effects [32][33][34] .Moreover, it has been reported that STAT3 inhibitor can improve ICD of tumor cells [35][36][37] .Thus, here we examined the impact SC144 on ICD of tumor cells and polarization of macrophages.
Here, we report our discovery that SC144 induces ICD of tumor cells and polarizes immunosuppressive TAMCs into a less immunosuppressive phenotype.However, as we found that SC144 exhibited cytotoxicity among CD8 + T-cells, we sought to target the delivery of SC144 to tumor cells and TAMCs while sparing CD8 + T-cells.Toward this goal, we have employed hyaluronic acidbilirubin nanoparticles (HABN) that we have previously developed for CD44-targeted oral administration 38 .Here, we report that HABN administered intravenously (IV) accumulates in CD44-expressing tumor cells and TAMCs.HABN loaded with SC144 (SC144@HABN) allowed for efficient targeted delivery of SC144 to tumor cells and TAMCs.Importantly, SC144@HABN therapy induced ICD of cancer cells, converted TAMCs into less immunosuppressive phenotype, and increased anti-tumor T-cell response, leading to robust antitumor efficacy.Furthermore, SC144@HABN achieved robust synergy with anti-PD-L1 ICB therapy in both MC38 and 4T1 murine tumor models (Fig. 1a).Overall, our work demonstrates the potential of SC144@HABN-based induction of ICD and modulation of TAMCs for combination chemoimmunotherapy.
Next, we sought to understand what factors are important for the therapeutic efficacy of SC144@HABN.We first examined the effect of HA and BN on the anti-tumor efficacy of SC144@HABN.We compared the therapeutic efficacy of SC144@HABN with that of SC144 encapsulated into either PEGylated bilirubin nanoparticles (SC144@PEG-BN) or hyaluronic acid-cholesterol nanoparticles (SC144@HACN).The therapeutic efficacy of SC144@PEG-BN and SC144@HACN was significantly lower than that of SC144@HABN (Fig. 3k, l, Supplementary Fig. 19), indicating the crucial role of both BR and HA in the anti-tumor efficacy of SC144@HABN.Next, we examined the role of macrophages during SC144@HACN treatment by administering IgG antibody against colony-stimulating factor 1 receptor (anti-CSF1R) known to deplete macrophages 42 .Treatment with anti-CSF1R IgG significantly decreased the anti-tumor efficacy of SC144@HABN (Fig. 3m, n).Moreover, we observed SC144@HABN treatment lost its anti-tumor efficacy in mice bearing CD44-KO MC38 tumor cells pre-treated in vitro with CRISPR/Cas9 to knockdown the CD44 expression (Fig. 3m, n, Supplementary Fig. 20).Taken together, these results show the importance of HABNmediated delivery of SC144, modulation of macrophages, and CD44 expression among tumor cells for the observed anti-tumor efficacy of SC144@HABN.

SC144@HABN and anti-PD-L1 combo elicits strong anti-tumor immunity
Based on SC144@HABN-mediated upregulation of PD-L1 on tumor cells (Fig. 3I, j), we sought to improve the anti-tumor efficacy of SC144@HABN by co-administration of anti-PD-L1 antibody.MC38 tumor-bearing mice were treated as in Fig. 3a with the addition of anti-PD-L1 antibody administered intraperitoneally after one day of each SC144-based therapy (Fig. 4a).

SC144@HABN and anti-PD-L1 combo exerts robust efficacy in 4T1 tumor model
To validate our approach, we examined the anti-tumor efficacy of SC144@HABN + anti-PD-L1 ICB therapy in BALB/c mice bearing 4T1 tumor, which is an aggressive breast cancer model that is resistant to anti-PD-L1 and other ICB therapies 22 .First, we evaluated the cytotoxicity of SC144@HABN in 4T1 cells.Both SC144 and SC144@HABN induced killing of 4T1 cells in vitro (Fig. 5a, b).Notably, SC144@HABN induced upregulation of CRT on 4T1 cells, indicating SC144@HABN-mediated ICD (Fig. 5c) Moreover, 4T1 cells expressed higher level of CD44, compared with MC38 cells (Supplementary Fig. 22), suggesting that CD44-expressing 4T1 cells may be a good target for HABNmediated delivery of SC144.
In addition, all survivors in the SC144@HABN + anti-PD-L1 combo group were resistant to re-challenge with 4T1 tumor cells performed on day 50, demonstrating establishment of long-term anti-tumor immunity (Fig. 5g).Lastly, we showed that SC144@HABN + anti-PD-L1 combination did not trigger any overt signs of systemic toxicity, autoimmunity, or pathologies in the major organs (Supplementary Fig. 23).
Taken together, these results demonstrate that SC144@HABN + anti-PD-L1 antibody exerts potent anti-tumor efficacy with long-lasting anti-tumor immunity with minimal toxicity.

Discussion
Here, we report our discovery that SC144 polarizes macrophages into M1-like phenotype (Supplementary Figs. 8, 10, 11) and induce ICD of cancer cells in vitro (Fig. 2f-i).Yet, SC144 also induced killing of CD8 + T-cells (Supplementary Fig. 16), which is a crucial cell type for anti-tumor immunity.Thus, in this work, we sought to deliver SC144 selectively to tumor cells and TAMCs, while sparing CD8 + Tcells from SC144-mediated cytotoxicity.Toward this goal, we have developed SC144@HABN.This was motivated by our observation that HABN administered IV accumulated in tumor cells and TAMCs in vivo (Fig. 1), potentially due to the interaction between HA coating of HABN and its ligand CD44 (Fig. 1i-l, Supplementary Figs.5-7) 41 .Indeed, SC144@HABN administered IV was taken up by TAMCs, especially among CD44 hi TAMCs, resulting in conversion of immunosuppressive TAMCs into a less immunosuppressive phenotype, as shown by an increased ratio of M1-like to M2-like macrophages and reduced frequency of MDSCs in the TME (Fig. 2a-e, Supplementary Figs. 5, 6).In addition, SC144@HABN triggered apoptosis and ICD of tumor cells, while encapsulation of SC144 within HABN shielded CD8 + T-cells from SC144-mediated cytotoxicity (Fig. 2f-l, Supplementary Figs.13-17), potentially due to the cytoprotective activity of HABN 38 .SC144@HABN treatment promoted strong tumor-infiltration of CD8 + T-cells and increased their proliferation and functionality in vivo, as shown by increased expression of Ki67 + and granzyme B + on CD8 + T-cells (Fig. 3d-f).
The TME-modulating effects of SC144@HABN led to significantly improved anti-cancer activity, as compared against SC144 or HABN monotherapy (Fig. 3a-c).Nevertheless, SC144@HABN treatment was associated with increased expression levels of PD-1 among CD8 + T-cells and PD-L1 on tumor cells (Fig. 3g, I, j), suggesting exhaustion of tumorinfiltrating CD8 + T-cells.To counter such responses, we combined SC144@HABN with anti-PD-L1 therapy, leading to amplification of antitumor CD8 + T-cell responses with robust anti-tumor efficacy in both MC38 and 4T1 tumor models (Figs. 4, 5).In addition, tumor-free survivors in both MC38 and 4T1 tumor models were resistant to tumor re-challenge, indicating long-lasting adaptive immunity (Figs.4l, m, 5g).Moreover, as 4T1 tumor model is highly resistant to ICB therapies 22 , our work highlights the potential of SC144@HABN to sensitize ICB-resistant tumors to ICB therapy.
In conclusion, our work shows that SC144@HABN converts TAMCs into a less immunosuppressive phenotype and triggers ICD of tumor cells, leading to robust anti-tumor effects and strong synergy with ICB therapy (Supplementary Fig. 24).While the exact molecular  mechanism of action for SC144-mediated polarization of macrophages and ICD induction is beyond the scope of this work, prior literature suggests that the IL-6/gp130/STAT3 axis plays a crucial role in tumor progression and immunosuppression [29][30][31][32][33][34] .Thus, our work based on a gp130 inhibitor, SC144, may be applicable to a wide range of cancers that are resistant to ICB therapy.In addition, our work represents a new, generalizable nanoparticle-based strategy that can be applied to other chemotherapeutic agents with ICD-inducing or TME-modulating properties.

Methods
Synthesis of hyaluronic acid-bilirubin conjugate (HA-BR) and hyaluronic acid-cholesterol conjugate (HA-Chol) Before synthesizing hyaluronic acid-bilirubin conjugate (HA-BR), an acid form of hyaluronic acid (HA) from hyaluronic acid sodium salt (Lifecore Biomedical) and an aminoethylene-bilirubin conjugate (AE-BR) were prepared.The acidic form of hyaluronic acid (HA) from hyaluronic acid sodium salt was prepared by dialysis against 0.01 M HCl for overnight, followed by lyophilization.To prepare AE-BR, 750 µmol of bilirubin (Lee Biosolutions) and 520 µmol of N-hydroxysuccinimide (NHS, Sigma-Aldrich) were added to 7.5 ml of dimethyl sulfoxide (DMSO) containing 0.225 µl of trimethylamine (TEA).Subsequently, 337.5 µmol of EDC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide] (Sigma-Aldrich) was added to the mixture.After stirring for 10 min at room temperature (RT), 562.5 µmol of ethylenediamine (EDA) was added to the mixture, and the reaction was allowed to proceed with stirring for 4 h at RT under nitrogen gas.50 ml of chloroform was added to the mixture and then washed twice with 50 ml of 0.1 M HCl, 0.1 M NaHCO 3 and then water.After evaporating the chloroform solution, 45 ml of methanol was added to the reaction mixture and then centrifuged at 3000 × g for 10 min.The supernatant was then evaporated to yield AE-BR.
To synthesize hyaluronic acid-bilirubin conjugate (HA-BR) or hyaluronic acid-cholesterol conjugate (HA-Chol), 80 µmol of an acidic form of HA, 40 µmol of NHS were added to 4.8 ml of DMSO and then, after adding of 140 µmol of EDC, the mixture was stirred for 10 min at RT. Subsequently, 20 µmol of AE-BR, or 5 µmol of cholesterol-PEG-NH 2 (Nanosoft Polymers) was added and stirred with the mixture for overnight at RT under nitrogen gas.The mixture was slowly poured into 30 ml of 0.01 M NaOH, and then dialysis was performed against 0.01 M NaOH for 5 h.Further dialysis was performed against 1:1 ratio of water/acetonitrile three times for 1 day, followed by distilled water three times for 2 days.The resulting solution was lyophilized, yielding HA-BR (native sodium salt form; 26.5 µg/ml of BR in 1 mg/ml of HABN) or HA-chol. 1 H-NMR spectra were obtained on a Varian 500 MHz system (Varian); chemical shifts represent ppm downfield from tetramethylsilane.Bilirubin portion of HA-BR or HA-Chol was calculated by measurement of UV/VIS spectra using Synergy TM NEO HTS multi-mode microplate reader (BioTek Instruments Inc).

Synthesis of PEGylated bilirubin (PEG-BR)
75 µmol of bilirubin and 33.75 µmol of EDC were added to 0.6 ml of DMSO containing 225 µl of TEA and 52 µmol of NHS for 10 min at RT.After then, 15 µmol of polyethylene glycol 20K-amine (PEG-NH 2, Nanocs) was added and mixed for 4 h under nitrogen gas.Subsequently, the mixture was added to 50 ml of chloroform, and the organic solvent was washed with 50 ml of 0.1 M HCl twice, followed by washing with 50 ml of 0.1 M NaHCO 3 twice and washing with 50 ml of distilled water twice.The organic layer was evaporated, and 50 ml of methanol was added to the residue.After centrifugation at 3000 × g for 10 min, the supernatant was collected and then evaporated.Dialysis was performed as described in the previous section.To yield PEGylated bilirubin (PEG-BR), lyophilization was performed.The final structure was confirmed by 1 H-NMR. 1 H-NMR spectra were recorded on a Varian 500 MHz system; chemical shifts represent ppm downfield from tetramethylsilane.

Synthesis of HA-BR-Cy5.5 conjugate
Prior to conjugating HA-BR with Cy5.5 amine (AAT Bioquest), a native sodium salt form of HA-BR was converted into an acidic form of HA-BR, HA-Chol or free HA by dialysis processes for efficient dissolution in DMSO. 10 µmol of each power form was dissolved into 5 ml of distilled water.Dialysis was performed against 0.1 M HCl for overnight, and then the solution was dialyzed against distilled water three times for 1 day.The resulting powder was obtained through a lyophilization step.10.5 µmol of the acidic form of HA-BR, HA-Chol, or free HA was dissolved in 0.8 ml of DMSO for overnight.2 µmol of NHS and 2 µmol of EDC were added to the mixture.After mixing for 10 min at RT, 0.1 µmol of Cy5.5-NH 2 was further added to the reaction mixture.After stirring for overnight at RT, dialysis was performed once more against 0.01 M NaOH three times for 1 day, followed by distilled water twice for 2 days.After lyophilization, the native sodium salt form of HA-BR-Cy5.5 conjugate was obtained.
Preparation of SC144-loaded HABN (SC144@HABN) SC144-loaded HABN (SC144@HABN) was prepared using an o/w emulsion method.Briefly, 15 mg of HABN was dissolved in 1.5 mL of sterilized distilled water and stirred for 5 min at room temperature.After adding 60 μl of SC144 (5 mg/100 μl DMSO) dropwise to the solution, ultra-sonication (140 W, 26 Hz, 2 s on and 3 s off of short interval) was performed for 5 min at 4 °C.The solution was dialyzed against an excess amount of distilled water and acetonitrile with a dialysis bag overnight, followed by centrifugation at 14,000 rpm for 10 min to concentrate.Before further analysis, the solution was filtered through a 0.45 μm pore-sized microporous membrane.The size and zeta potential of the nanoparticles were obtained using a Nanosizer ZS90 (Malvern Instruments Ltd).Morphology was examined by Transmission Electron Microscopy (TEM) using JEOL 1400-plus TEM (JEOL USA), and TEM images were acquired with AMT602 software (JEOL USA).The resulting nanoparticles were diluted in PBS or culture medium for in vitro and in vivo experiments.SC144-loaded HACN and SC144-loaded HACN were also prepared using the same o.w emulsion method as described above.The amount of SC144 in each nanoparticle formulation was determined by using HPLC and calculated as shown below.
Drug loading percentage (%) = [weight of drug in particle/(weight of drug in particle + weight of HABN added initially)] × 100.

Animals
Animals were cared for following federal, state, and local guidelines.All work performed on animals was in accordance with and approved by the Institutional Animal Care & Use Committee (IACUC) at University of Michigan, Ann Arbor and Ewha Womans University (EWHA IACUC 21-068-4).All animals were obtained from the Jackson Laboratory (Bar Harbor, ME) and the Laonbio (South Korea) as mixed littermates.All animals were housed under pathogen-free conditions with controlled temperature (20-26 °C), humidity (40-60%), and lighting (12-h lightdark cycle) in the animal facility at the North Campus Research Complex of University of Michigan and the College of Pharmacy, Ewha Womans University, respectively.Mouse tumor size and survival were monitored every 2-4 days.Tumor size was calculated based on the equation: volume = length × width 2 × 0.5.Animals were euthanized when the tumor reached 2.0 cm in diameter or when they became moribund with more than 20% weight loss or unhealing ulceration.During all the animal studies, the maximum tumor size permitted by the IACUC at University of Michigan, Ann Arbor and Ewha Womans University was not exceeded.

In vivo IVIS imaging
To check ability of HABN to target the tumor tissue, C57BL/6 mice were inoculated subcutaneously with 3 × 10 5 MC38 cells on day 0. After treating animals of with 10 mg/kg of HABN-Cy5.5 (having the equivalent mass of free Cy5.5) or 0.25 mg/kg of free Cy5.5 on day 25, whole body fluorescence intensities were monitored at predetermined times using a Xenogen IVIS Lumina in vivo imaging system (PerkinElmer) with a Cy 5.5 filter channel and an exposure time of 5 s.In vivo images were acquired and analyzed using IVIS Lumina Living Image Software (v.4.5.5, PerkinElmer).After 24 h of treating animals of each group, mice were euthanized, and organs including heart, lung, kidney, spleen, liver, and tumor were excised.Fluorescence intensity in organs from each group was analyzed using a Xenogen IVIS Lumina in vivo imaging system (PerkinElmer) with a Cy 5.5 filter channel and an exposure time of 5 s.For the HABN-Cy5.5uptake studies in vivo, animals were euthanized on day 12, and tumor tissues harvested, followed by flow cytometric analysis.

In vivo ELISA analysis
For determining the concentrations of cytokines in the serum, blood was collected at indicated time points and allowed to clot for 30 min, and the serum was collected by centrifugation for 10 min at 3000 × g in the serum separator microtainer (BD Science).CXCL-10 concentration in the serum was measured by enzyme-linked immunosorbent assay (ELISA) at the Cancer Center Immunology Core of the University of Michigan.HMGB1 was measured using a mouse HMGB1 ELISA kit (LifeSpan BioSciences Inc.).

In vitro ELISA analysis
For determining the concentrations of cytokines (IL-1β, IL-6, TNF-α, IL-10, TGF-β), BMDM cells were treated with SC144 (10 μM), HABN (40 μg/ml), or SC144@HABN (10 μM of SC144; 40 μg/ml of HABN), or fresh medium for 24 h in the absence or presence of 20 ng/ml of IL-4.As M0 and M1 control groups, the cells were treated with 100 ng/ml of LPS and 10 ng/ml of IFN-γ for M1 induction, or control medium for M0 control.The levels of cytokines in the resulting supernatants or medium were measured by ELISA at the Cancer Center Immunology Core of the University of Michigan.FACS analysis was also performed to check macrophage phenotypes.To measure the release of CXCL10 and HMGB1 from dying tumor cells, MC38 cells seeded in 96-well plates (5 × 10 3 /well).After 1 day incubation, cells were incubated with SC144 (10 μM), HABN (40 μg/ml) or SC144@HABN (10 μM of SC144; 40 μg/ml of HABN) for 24 h.After incubation, each supernatant was collected and centrifuged at 1000 × g for 20 min.HMGB1 was measured using a mouse HMGB1 ELISA kit (LifeSpan BioSciences Inc.).

CCK-8 assay
BMDM, CT26, MC38, or 4T1 cells in culture medium were grown in 96well plates (0.7 × 10 4 /well) for 24 h at 37 °C.After medium was removed, SC144 (10 μM), HABN (40 μg/ml), SC144@HABN (10 μM of SC144; 40 μg/ml of HABN), or fresh medium was added to each well and plates were incubated for 24 h.After incubation, cells were washed with fresh culture medium and 100 µl of fresh culture medium was added to each well, followed by the addition of 10 µl of CCK-8 (Dojindo Molecular Technologies.Inc.) and after incubation for 1 h at 37 °C, the absorbance was measured at 450 nm using a 96-well plate microreader.

In vivo toxicity test
Six-weeks old female C57-BL/6 mice were housed in groups of five mice per cage and acclimatized for 1 wk before inclusion in the study.30 mg/kg of HABN (10 kDa, 100 kDa, or 700 kDa) or PBS was administered via an oral route on day 0, 2, 4, and 6.Mice were observed over a 1 wk period for changes in behavior or weight.Blood was collected from jugular vein and immediately sent to the ULAM Pathology Core for Animal Research for blind blood assessment.Mice were sacrificed, and their major organs (heart, liver, lung, kidney, spleen, and colon) were collected for histopathological analysis.Each organ was fixed with 4% (v/v) buffered formalin and 70% (v/v) alcohol, and embedded in paraffin.Tissues were sectioned, stained with H&E, and examined by microscopy.Histology images were acquired using Matra Quantitative Pathology Workstation (v.1.0.3) and analyzed by inForm image analysis software (v.2.3.0).All histological assessments were performed in a blinded manner to prevent observer bias.

Statistical analysis
The results are expressed as means ± s.e.m.A one-way or two-way ANOVA, followed by Tukey's HSD multiple comparison post hoc test was used for testing differences among groups.Data were approximately normally distributed and variance was similar between the groups.No samples were excluded from analysis.Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA) was used for statistical analyses.All experiments were repeated at least twice with similar results.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.