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Smart pH-Responsive Nanocubes Controlled Delivery of DNA Vaccine and Chemotherapeutic Drug for Chemoimmunotherapy Huu Thuy Trang Duong, Thavasyappan Thambi, Yue Yin, Jung Eun Lee, Young Kyu Seo, Ji Hoon Jeong, and Doo Sung Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21185 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Applied Materials & Interfaces

(Revised Manuscript for the ACS Applied Materials & Interfaces)

Smart pH-Responsive Nanocubes Controlled Delivery of DNA Vaccine and Chemotherapeutic Drug for Chemoimmunotherapy

Huu Thuy Trang Duonga,1, Thavasyappan Thambia,1, Yue Yinb, Jung Eun Leeb, Young Kyu Seoa, Ji Hoon Jeongb*, and Doo Sung Leea*

aSchool

of Chemical Engineering, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea bSchool of Pharmacy, Theranostic Macromolecules Research Center, Sungkyunkwan University, Suwon 16419, Republic of Korea

1These

authors contributed equally to this work.

*Corresponding authors: Ji Hoon Jeong, Ph.D. Tel.: +82-31-290-7783; Fax: +82-31-292-8800; e-mail: [email protected] Doo Sung Lee, Ph.D. Tel.: +82-31-299-6851; Fax: +82-31-299-6857; e-mail: [email protected]

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ABSTRACT The combination of chemotherapeutic agents with immune stimulating agents for treating degenerative diseases, called chemoimmunotherapy, has emerged as a promising cancer treatment modality. Despite the tremendous potential, chemoimmunotherapy by the combination of drugs and immune stimulators often suffers, due to the lack of controlled delivery nanostructures in the microenvironment. To this end, we show that by using pH-responsive smart nanocubes (NCs), cancer cells and tumor-associated immune cells can be precisely targeted with chemotherapeutic agent (Doxorubicin, DOX) and immune stimulating agent (plasmid OVA, pOVA) for enhanced chemoimmunotherapy. The pH-responsive smart NCs protect payloads from nuclease degradation and avoid renal clearance, and undergo super-sensitive structural change at the extracellular tumor regions that mediate efficient release. Concurrent release of pOVA vaccines encoding tumor-specific antigen laden with polyplexes were loaded on tumorassociated immune cells, and produce antigen-specific humoral immune response, whereas DOX enables the effective infiltration to the cancer cells, and is involved in the eradication of tumor tissues. The amount of Anti-OVA IgG1 antibody produced by the intravenous administration of NC formulation was similar to that of free OVA formulation. Importantly, the combined delivery of pDNA and DOX using NCs showed significantly enhanced antitumor efficacy in B16/OVA melanoma tumor xenograft, which remarkably outperforms the monotherapy counterparts. These results suggest that pH-responsive smart NCs laden with pDNA and DOX provide a promising nanostructure for chemoimmunotherapy that simultaneously involves cancer cell killing, and stimulates antigen-specific immune response to prevent cancer recurrence. Keywords: Chemoimminotherapy, pH-responsive copolymers, microenvironment, targeting and cancer therapy.


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INTRODUCTION Cancer immunotherapy, which refers to the treatment of diseases through the modulation of immune cells that can recognize and kill cancer cells, has emerged as a promising strategy in cancer treatment.1-7 Nevertheless, accumulating evidence has suggested that the effectiveness of cancer immunotherapy is severely restricted by the microenvironment of developing tumors.8-10 Intelligent tumor cells selectively reprogram the metabolism to conquer their excessive energy requirement for survival and proliferation.11 Such metabolic reprogramming occurs within the tumor microenvironment, resulting in hypoxic and acidotic gradients across tumor tissues.12-15 The microenvironment architecture constructed by tumor cells and tumor-infiltrating immune cells is the hallmark of solid tumors, which is not only plays a critical part in tumor progression, survival, and metastasis, but is also effectively involved in the rejection of chemo and immune therapies.10, 16-19 The major reason for the treatment failure is the lack of effective immunization, resulting in insufficient memory response to fight against the recurring cancer cells. Recent studies have shown that solid tumors are effectively shielded by thick fibrous tissues, along with immune and interstitial cells.20-22 This fibrotic tissue plays a critical role in tumor development, and hinders the treatment efficacy.21 Therefore, the combination of chemotherapeutic agents with immune stimulating agents for treating degenerative diseases may be a promising strategy, and the immunological adaptive memory developed during the treatment may prevent cancer recurrence. Although effective immunization has been achieved by nanomaterial-mediated immunotherapy,23-24 the transportation of chemotherapeutic drugs and immune modulating agents to the tumor tissue is challenging, mainly due to the shielding of cancer-associated fibroblasts in the microenvironment.20 Owing to the aggressive nature of the tumor 3 ACS Paragon Plus Environment


microenvironment, tumor-specific cytotoxic CD8+ T cells (cytotoxic T cells) and CD4+ T cells activated during tumor progression have failed to reject the established tumors. Hence, the combination strategies effectively resolve the immunosuppressive nature of the tumor microenvironment.25 Furthermore, the adaptive immunity developed during the combination therapy prevents tumor recurrence. It is well known that certain chemotherapeutic drugs with appropriate dosage elicit immune response, and regulate the immunosuppressive tumor microenvironment.26-28 Therefore, on the one hand, the predominant population of tumorassociated immune cells and fibroblasts, a key barrier in cancer therapy, needs to be depleted first for effective therapy. On the other hand, surrounding tumor cells lose their physical barrier, and can enhance the passage of small molecule chemotherapeutic drugs, which may led to enhanced chemotherapy. Rational design of an intelligent nanocarrier that can carry both chemotherapeutics and immune-stimulating agents needs to be developed. In particular, the nanostructure that exhibits sharp structural change at the perivascular region of tumor cells, and delivers specifically immune-stimulating agents, with subsequent surface change that allows effective transfusion of chemotherapeutic agent-bearing nanomaterials into the tumor site, has rarely been studied. For instance, microneedle-assisted combined delivery of DNA vaccine and paclitaxel to dendritic cells substantially inhibited the tumor growth in B16 melanona murine model.29 Co-delivery of interleukin-15 and cisplatin using thermo-responsive injectable hydrogels exhibited synergistic anti-cancer effects in mice-bearing B16 melanoma tumors.30 Similarly, liposomal formulation co-loaded with docetaxel and PD-L1 antibody showed synergistic anti-tumor effect in B16 melanoma tumor-bearing mice with reduced systemic toxicity.31 As an emerging category of responsive nanomaterials, self-assembled polymeric 4 ACS Paragon Plus Environment

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nanostructures with different morphologies have gained great attention, due to their excellent solubility, ability to encapsulate multiple payloads, and ability to be excreted by glomerular filtration after performing their role in the body.21, 32-35 More importantly, the introduction of stimuli-responsive functional groups in the nanostructure may protect from therapeutic leakage at circulation, and deliver the payload to target tissues.14,

35-38

Herein, we propose a model

polymeric nanostructure strategy to differentially deliver immune stimulating agent (plasmid OVA, pOVA) and chemotherapeutic agent (Doxorubicin, DOX) to tumor-associated immune cells and tumor cells for chemoimmunotherapy. To harness the acidotic gradient feature of the tumor microenvironment, we formulated tumor microenvironment-responsive biodegradable pHresponsive smart nanocubes (NCs) to concurrently load pOVA and DOX. In aqueous solutions, the DOX was assembled in the nanocore through hydrophobic interaction, while the pOVA was ionically bound on the shells. Our previous studies on ultra-sharp pH-responsive nanocarriers demonstrated that they could exhibit hydrophobic-hydrophilic phase transition with change in surface properties to mild pH changes.39-41 We hypothesized that the ultra-sharp pH-sensitivity could be harnessed to deliver the pOVA at the perivascular regions; thereafter the change in surface characteristics would effectively be transfused into the deep tumor tissues, and release DOX to eradicate the cancer cells (Scheme 1). The pOVA would store the immunological memory, to evade the future occurrence of cancer cells.

EXPERIMENTAL PROCEDURES Materials Piperazine-1-ethanol (HP, 97%), 4-hydroxybutyl acrylate (HBA, 90%), methoxy poly(ethylene glycol) (mPEG, Mn=2,000 g/mol), dibutyltin dilaurate (DBTL, 95%), hexamethylene 5 ACS Paragon Plus Environment


diisocyanate (HDI, ≥99%), acroloyl chloride, L-glutamic acid 5-benzyl ester (BLG, 99%), triphosgene (98%), butylamine (99%), 2-hydroxypyridine (≥98%), diethylenetriamine (99%), doxorubicin·hydrochloride (DOX·HCl, 98%), methylthiazolyldiphenyl-tetrazolium bromide (MTT, ≥97.5%), dichloromethane (DCM, 99.5%), chloroform (CHCl3, 99.5%), and N,Ndimethylformamide (DMF, ≥99.9%) were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell culture media and reagents including Roswell Park Memorial Institute (RPMI) 1640 Medium, Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin-EDTA solution, penicillinstreptomycin solution (10,000 U/mL), and fetal bovine serum (FBS) were acquired from Thermo Fisher Scientific (Carlsbad, CA, USA). RAW 264.7 macrophage, B16/OVA melanoma, and A549 human lung carcinoma cells were acquired from The American Type Culture Collection (ATCC, Rockville, MD, USA). The nuclear staining dye 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) was purchased from Life Technologies (Carlsbad, CA, USA). BLG Ncarboxyanhydride (BLG-NCA) was prepared using our previously reported synthetic procedure.13 Synthesis of ultra-pH-sensitive copolymers Scheme 2 shows the synthetic route for the preparation of ultra-pH-sensitive copolymer. Synthesis of hydroxyl-terminated β-amino esters (HPB). HPB was synthesized according to our previously reported procedure.39 Preparation of mPEG-poly(β-aminoester urethane) (mPEG-PAEU). The diblock mPEGPAEU copolymer was synthesized through polyaddition polymerization reaction according to our previously reported procedure with slight modification.42 In short, mPEG (4 g, 2 mmol) was dried in a Schlenk flask under high vacuum at 110 °C for 3 h. The flask was then cooled to 60 °C,


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and then HPB (4.93 g, 18 mmol) was added, which was then dried under vacuum for another 1 h. Thereafter, the vacuum was replaced with nitrogen, followed by addition of anhydrous chloroform (90 mL). The polymerization was finally initiated by adding HDI (2.89 mL, 18 mmol) and DBTL catalyst (2.89 mL) and stirred at 60 °C for 3 h. Finally, the diblock mPEG-PAEU copolymer was precipitated in excess cold diethyl ether, followed by filtration and drying under vacuum for 2 days. Preparation of acrylate-terminated mPEG-PAEU (mPEG-PAEU-acrylate). mPEG-PAEU (2.34 g, 0.5 mmol) was solubilized in anhydrous chloroform (25 mL). Thereafter, acryloyl chloride (40 µL, 0.52 mmol) was slowly added to the solution at 0 °C, and then stirred for 12 h. The product was precipitated in excess diethyl ether at 0 °C, and dried under vacuum oven for 2 days. Preparation of butyl-poly(γ-benzyl-L-glutamate) (butyl-(PBLG)). The butyl-(PBLG) was synthesized using a ring-opening polymerization.43 In brief, BLG-NCA (5 g, 19 mmol) was dissolved in 50 mL of CHCl3. Butylamine (93 μL, 0.95 mmol) initiator was added to the solution and stirred for 3 days at room temperature. The product was then precipitated in cold ether, and dried for 3 days under vacuum. Preparation of butyl-poly(N-(N-(2-aminoethyl)-2-aminoethyl)-L-glutamate (butyl-(PN2LG)). Butyl-(PBLG) (3.8 g, 1 mmol) was solubilized in 38 mL of DMF at 55 °C under nitrogen. Next, 2-hydroxypyridine (9.52 g, 100 mmol) in DMF (100 mL) and diethylenetriamine (21.63 mL, 200 mmol) were added slowly, and continued the reaction for 3 days. Then, the butyl-(PN2LG) polymer was precipitated using diethyl ether. The crude product was dispersed in water and transferred to dialysis membrane tube (MWCO: 3,500 Da) and dialyzed against excess of 7 ACS Paragon Plus Environment


deionized water for 3 days and lyophilized to obtain pure butyl-(PN2LG). Synthesis

of

ultra-pH-sensitive

mPEG-PAEU-poly(diethylenetriamine-L-glutamate)

(mPEG-PAEU-PN2LG) triblock copolymer. mPEG-PAEU-acrylate (1.37 g, 0.29 mmol) was solubilized in 15 mL of DMF at 50 °C under nitrogen. Next, butyl-(PN2LG) (1 g, 0.29 mmol) in DMF (50 mL) was added and continued the reaction for 24 h. The crude reaction mixture was cooled and transferred to dialysis membrane (MWCO: 3,500 Da), and purified by dialysis against distilled water for 2 days, followed by lyophilization to acquire final product.

Characterization 1H

NMR. The structural characteristics, including the molecular weight (Mn) and molecular

structure of synthesized monomers and polymers were performed using 1H NMR (Varian Unity Inova 500 instrument). Samples for NMR spectra measurement were prepared by dissolving the monomers and copolymers in CDCl3, DMSO-D6, or D2O at 10 mg/mL concentration. Zeta potential (ζ) and particle size measurements. ζ and particle size of copolymers at different pH’s were measured by dynamic light scattering (DLS, Zetasizer, model Nano-ZS90). The relative scattering intensity against the particle was set to 90°. The particle size and ζ measurements were performed at 25 °C. For size and ζ measurement, the copolymers were dispersed in de-ionized water at 1 mg/mL concentration, and the pH of the solution was altered to different values using HCl (0.1N) or NaOH (0.1N). The Smouluchowski equation was used to calculate the ζ values from the electrophoretic mobility values. In general, the ζ was calculated using the following Smouluchowski formula:

m= e.z/h 8 ACS Paragon Plus Environment

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Where, m, e, z and ζ is the mobility, dielectric constant, ζ, absolute viscosity, respectively, of solutions. Semi electron microscopy (SEM). SEM measurement was performed on a JSM-6390 (JSM6390, MA, USA) operating at 10 kV accelerating voltage. For measurement, copolymers (1 mg/mL) were mounted on a SEM sample stub, and each sample was sputtered with gold to make it conductive, and used for measurement.

DOX loading into the NCs Dialysis method was used to prepare DOX-loaded NCs (DOX-NCs). Firstly, DOX·HCl (10 mg), trimethylamine (3 eq), and pH-responsive copolymer (100 mg) were dissolved in 10 mL of DMF and stirred in the dark condition for overnight. The DMF and unloaded DOX were removed by dialysis with deionized water for 24 h. The DOX-loaded copolymer solution was filtered and lyophilized to obtain DOX-NCs. The content of DOX (DLC) and loading efficiency of DOX (DLE) in the DOX-NCs were calculated using the previously reported procedure.44 DOX-NCs were simply mixed with pOVA to prepare DOX-NCs/pOVA.

Electrophoretic mobility shift assay (EMSA) Complexation between copolymer/pOVA and DOX-loaded copolymer/pOVA was evaluated using EMSA. A series of formulations between pOVA and increasing amounts of either copolymers or DOX-loaded copolymers were prepared by simple mixing, and stirred at room temperature for 30 min. To confirm ionic complexes formation, they were run on 1% agarose gel in TAE buffer at 130 V at room temperature for 40 min. The EMSA images were visualized



under illumination of UV light.

Release pattern of DOX and pOVA in vitro The release pattern of DOX and pOVA from NCs was investigated under different pH conditions (i.e., pH 7.4 and pH 5.5). For release test, DOX-NCs/pOVA (5 mg) was dispersed in 2.5 mL of PBS buffer (pH 7.4) or acetate buffer (pH 5.5), and transferred to a semi-permeable membrane dialysis tube (MWCO: 3,500 Da). The tube was then immersed in 47.5 mL of the same media, and placed in a water bath with 100 rpm at 37 °C. At 0, 1, 2, 4, 8, 12, 24 h time points, 2.5 mL of buffer solution was withdrawn and stored at 0 °C, whereas, 2.5 mL of fresh buffer was added to the sink and continued the release. The amounts of released DOX and pOVA were quantified by UV measurement at 480 nm and PicoGreen assay, respectively.

In vitro cytotoxicity test To examine the toxicity of prepared copolymers and their DOX-loaded counterpart, RAW 264.7 macrophages and A549 cancer cells were cultured in DMEM and RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin in a humidified atmosphere (5% CO2-95% air) at 37 °C in 96-well plates at a density of 1 × 104 cells/well. The cells were washed twice with PBS after 24 h, and incubated with copolymers, free DOX, DOX-NCs, and DOX-NCs/pOVA at different concentrations. After one day, MTT solution (20 μL, prepared at a concentration of 5 mg/mL in PBS) was added to each well, and the cells were further incubated at 37 °C for 2 h. The free MTT crystals and culture medium were removed, and DMSO (200 μL) was then added to each well, and further incubated at 37 °C for 30 min. The absorbance was measured using a microplate reader (Multiskan Go, USA) at 490 nm.


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Luciferase assay RAW 264.7 macrophages and A549 cancer cells were cultured in 12-well plates (15 × 104 cells/well). One day after the cell growth, they were washed twice with PBS, 450 μL serum-free culture media containing NCs/pOVA (50 μL, 1 μg pOVA) was added and incubated. After 6 h, the culture media were removed, and replenished with fresh media containing FBS and penicillin-streptomycin and incubated for 24 h. The commercial luciferase assay kit (Promega, Madison, WI) was used to observe the transfection efficiency in a GloMax 20/20 luminometer (Promega, Madison, WI). The luciferase activity was expressed as RLU/mg protein. The MicroBCA assay (Pierce, Rockford, IL) was used to determine protein concentration.

Cellular uptake RAW 264.7 macrophages and A549 cancer cells were used to confirm the cellular uptake ability of NCs. The cells were first seeded on confocal dishes at a density of 40 × 104 cells/dish and incubated for 24 h. After 1 day, the cells were incubated with 100 µL DOX-NCs/MFP488labeled pOVA in 900 µL of serum-free media for 4 h. The cells were stained with DAPI for 30 min, and washed with PBS to remove free dye, before adding 2 mL of 10% formaldehyde solution. Confocal laser scanning microscopy (Zeiss LSM 510) was used to observe the cellular uptake pattern of NCs.

In vivo biodistribution 7 week old BALB/c mice (Orientbio, Korea) were used to examine the in vivo distribution of NCs. The live animal experiments were performed in compliance with the relevant laws and



institutional committee guidelines of Sungkyunkwan University. The committees approved all of our animal experiments. To perceive the in vivo distribution, tumor-bearing mice were developed first. To develop tumor-bearing mice, B16/OVA cells (2 × 106 cells) were subcutaneously injected into the flank of mice. Ten days after the cancer cell inoculation, the mice were received DOX-NCs/pOVA through intravenous injection. After 24 h, the DOX fluorescence intensity was detected through an emission filter (580 nm). The biodistribution images were obtained using the optical imaging systm (eXplore Optix system).

OVA antigen expression in vivo To quantify the antigen expression, BALB/c mice (7 weeks old, Orientbio, Korea) were intravenously injected with OVA, free pOVA, NCs/pOVA, and DOX-NCs/pOVA. Three days after intravenous injection, blood was collected into the heparin containing tube through cardiac puncture. The plasma was separated by centrifuge at 4 °C with 13,500 rpm for 30 min. OVA ELISA kit (MyBioSource, San Diego, CA) was used to determine the amount of OVA in the supernatant. Furthermore, the total amount of protein in the supernatant was also calculated using BCA assay.

Antigen-specific OVA antibody detection Anti-OVA IgG antibody was detected by ELISA method. BALB/c mouse was intravenously administrated with OVA, free pOVA, NCs/pOVA, and DOX-NCs/pOVA, and blood sampling was done 2 weeks after administration. To quantify the anti-OVA IgG antibody, 20 µg/mL of OVA antigen were coated on 96-well plate. 12 h after incubation, the plate was washed with PBST buffer, and incubated with blocking buffer at 37 °C. After for 2 h, the buffer was removed,


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and incubated with collected plasma samples for 1 h at 37 °C. The plate was then rinsed with PBST buffer, and secondary antibody was added and incubated for 30 min at 37 °C. After washing with PBST, 3,3’,5,5’-tetramethylbenzidine was added to the plate for staining, and the absorbance at 450 nm was detected on a microplate reader (Multiskan Go, USA).

Antigen challenge in vivo Four weeks after immunization, the mice were challenged with the intravenous administration of OVA antigen. At day 5, the blood samples were collected, and the amount of OVA antibodies was measured by ELISA. Prior to the OVA challenge the blood samples were collected, and used as control.

Tumor inhibition For tumor challenge, C57BL/6 female mice (7 weeks old Orientbio, Korea) were indiscriminately divided into 7 groups with 5 animals in a group7 groups with 5 mice per group as follows: (i) Negative control, (ii) Free OVA (50 mg/mL), (iii) Free pOVA (50 mg/mL), (iv) Free DOX (50 mg/mL), (v) DOX-NCs (50 mg/mL of DOX), (vi) NCs/pOVA (50 mg/mL of pOVA), and (vii) (DOX-NCs)/pOVA (40 µg of DOX and 10 µg pOVA in 1 mL). The tumor was prepared by subcutaneous injection as mentioned in the previous in vivo distribution procedure. 200 μL of formulation solution was intravenously injected when the tumor volumes reach (50100) mm3. The injection was done every 2 days for 14 days. The tumor-bearing mice were intravenously injected with through the tail vein. The body weight and tumor volume were recorded every other day for 20 days. The tumor volume was calculated by measuring length and width of tumors (the largest and smallest diameters, respectively) using digital caliper. Tumor



volume = (width2 × length)/2. The tumor inhibition ratio of the antitumor efficacy among test groups was calculated using the following formula:

Vc and Vt represent the mean tumor volume of negative control group and treatment group, respectively. The time period of treatment was 20 days.

Statistical analysis Statistical analysis was determined by SigmaPlot. Statistical significances were evaluated with the paired Student t test. The values were presented as the mean ± SD. The P values of 0.05 or less were considered significant.

RESULTS AND DISCUSSION Synthesis and characterization of mPEG-PAEU-PN2LG triblock copolymer As shown in Scheme 2, the pH-sensitive mPEG-PAEU-PN2LG copolymer was synthesized through a seven-step process. At first, Michael-addition reaction was used to prepare HPB monomer. Figure S1 of the Supplementary Information (SI) shows the 1H NMR spectrum of HPB. Appearance of new characteristic peaks at 1.7, 2.5 and 3.7 ppm illustrated new methylene protons, which indicated successful conjugation of HBA to HP. Then, mPEG-PAEU was synthesized by the simple polyaddition polymerization using HDI, HPB, and mPEG using DBTL 14 ACS Paragon Plus Environment

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catalyst. Figure S2 of the SI shows 1H NMR of mPEG-PAEU copolymer. The methylene peaks of PEG appeared at 3.64 ppm, whereas the methylene group protons of β-aminoester were appeared at 4.2 and 2.6 ppm. Furthermore, the protons from 1.30 to 1.60 ppm and 3.1 ppm correspond to the characteristics peaks of HDI. For the copolymer preparation, the feed ratio of mPEG/HPB/HDI was 1/9/9 with the conversion of 67%, and the Mn of the prepared copolymers calculated from 1H NMR spectrum was found to be 4,684 g/mol. This result illustrated that mPEG-PAEU copolymer had been successfully synthesized. Thereafter, acrylation reaction was carried at the chain end of hydroxyl groups for further conjugation with biodegradable poly(amino acid), butyl-(PN2LG). The peak of acryloyl groups was clearly shown at 5.83-6.45 ppm, indicating the successful acrylation (Figure S3 of the SI). For butyl-(PN2LG) polypeptide preparation, BLG-NCA was first prepared by the cyclization reaction of BLG using triphosgene. The well cyclized BLG-NCA was obtained as a white crystal and the 1H NMR shows the successful synthesis (Figure S4 of the SI). Then, the polypeptide polymer was synthesized by the BLG-NCA ring-opening polymerization using butylamine. Figure S5 of the SI shows the 1H NMR spectrum of butyl-PBLG copolymer. The polymerization degree of PBLG in the polymer was determined by comparing the integral ratio of butylamine methyl proton at 0.86 ppm and benzylic methylene proton of glutamate derivative at 5.09 ppm. The feed ratio of butylamine/BLG-NCA is 1/20 with the conversion of 85%. Then, butyl-(PN2LG) polymer was synthesized by the aminolysis of butyl-PBLG using diethylenetriamine. The 1H NMR spectrum shows the disappearance of the benzyl methylene group and aromatic group at 5.1 ppm and 7.3 ppm, respectively, indicating the effective aminolysis (Figure S6 of the SI). Finally, the butyl-(PN2LG) was conjugated to the acrylterminated mPEG-PAEU through the Michael-addition reaction. The 1H NMR spectrum of



mPEG-PAEU-PN2LG shows the clear disappearance of acryl protons at 5.83-6.45 ppm, which indicates the effective conjugation of butyl-(PN2LG) to mPEG-PAEU (Figure S7 of the SI).

Characterization of NCs In our study, we synthesized a biodegradable tumor acidic pH-sensitive copolymer based on mPEG-PAEU-PN2LG triblock copolymer to construct an optimal delivery platform with high translational promise in clinics. At the physiological condition (pH 7.4), this amphiphilic copolymer assembles into NCs, and the mean size of the NCs was found to be ~155 nm (Figure 1A). Such small clustered nanostructures can effectively extravasate into the tumor matrix. At the tumor site, the characteristic tumor acidic pH ionizes the poly(β-amino esters) and PN2LG, which leads to increase in ζ, which in turn facilitates the uptake of the ionized NCs (Figure 1B). The pH-sensitive characteristics of the NCs were studied under different pH conditions. The light scattering result indicates no obvious size changes under basic pH conditions, suggesting that NCs would be stable when administered intravenously. Interestingly, incubation of the NCs at acidic pH conditions showed significant increase in size and ζ. The increase in size started at the neutral pH, and gradually increased at pH 6.5. This strongly explains the tertiary amine characteristics of poly(β-amino esters), and the pKa value is about ~6.5. At an acidic pH condition (~pH 5.5), the pH-responsive smart copolymers have been partially ionized due to the presence of multiple amine groups. The electrostatic repulsion between the ionized amine groups and aggregation between the hydrophobic components enlarged the particles size. On the other hand, lowering the pH to

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