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Tumor Microenvironment Responsive Nanogel for Combinatorial Antitumor Effect of Chemotherapy and Immunotherapy Qingle Song, Yijia Yin, Lihuan Shang, Tingting Wu, Dan Zhang, Miao Kong, Yongdan Zhao, Yangzhou He, Songwei Tan, Yuanyuan Guo, and Zhiping Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03186 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on August 31, 2017
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Tumor Microenvironment Responsive Nanogel
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for Combinatorial Antitumor Effect of
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Chemotherapy and Immunotherapy
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Qingle Song1, Yijia Yin1, Lihuan Shang1, Tingting Wu1, Dan Zhang1, Miao Kong1, Yongdan
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Zhao1, Yangzhou He1, Songwei Tan1,*, Yuanyuan Guo1,2, and Zhiping Zhang1,3,4,*
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AUTHOR ADDRESS
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1
Tongji School of Pharmacy
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Department of Pharmacy, Liyuan Hospital
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3
National Engineering Research Center for Nanomedicine
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Hubei Engineering Research Center for Novel Drug Delivery System
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Huazhong University of Science and Technology, Wuhan 430030, China
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ABSTRACT: A biomimetic nanogel with tumor microenvironment responsive property is
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developed for the combinatorial antitumor effects of chemotherapy and immunotherapy.
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Nanogels are formulated with hydroxypropyl-β-cyclodextrin acrylate and two opposite charged
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chitosan derivatives for entrapping anticancer drug paclitaxel and precisely controlling the pH
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responsive capability, respectively. The nanogel supported erythrocyte membrane can achieve
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‘nanosponge’ property for delivering immunotherapeutic agent interleukin-2 without reducing
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the bioactivity. By responsively releasing drugs in tumor microenvironment, the nanogels
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significantly enhanced antitumor activity with improved drugs penetration, induction of
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calreticulin exposure and increased antitumor immunity. The tumor microenvironment is
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remodeled by the combination of these drugs in low dosage, as evidenced by the promoted
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infiltration of immune effector cells and reduction of immunosuppressive factors.
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KEYWORDS: Antitumor agents, Chemo-immunotherapy, Drug delivery, Nanogel, Tumor
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microenvironment
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Chemo-immunotherapy is raising great attention for its enhanced antitumor effects by the
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synergism of chemotherapy and immunotherapy.1, 2 There is mounting evidence to support that
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some chemotherapeutic drugs with certain dosage, such as paclitaxel (PTX), doxorubicin and
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cisplatin are capable of activating the immune system and regulating the immunosuppressive
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microenvironment in tumor.2-7 The immunogenic cell death of cancer cells induced by some
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chemotherapeutics could promote antigen releasing from tumors, taken up and presentation by
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dendritic cells (DCs) and the subsequent activation of cytotoxic T lymphocytes (CTLs).8, 9 It has
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been demonstrated that pre-treatment of chemotherapeutics could also make tumors more
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susceptible to lysis by activated CTLs.8,
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Moreover, tumor immunosuppressive
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microenvironment can be remodeled via depleting suppressor cells like regulatory T lymphocyte
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(Treg) as well as inhibitory cytokines such as transforming growth factor-β (TGF-β) and
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interleukin-10 (IL-10).8, 12, 13
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PTX is a wide spectrum antineoplastic agent against various cancers with water solubility of
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less than 0.3 µg/mL.14 Recent studies showed that low-dose PTX can improve the effect of
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cytokine immunotherapy by modulating the immunosuppressive tumor microenvironment,
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including cytokine network and inhibitory activity of Tregs.15-18 Moreover, low-dose PTX can
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also induce exposure of calreticulin (CRT) on tumor cells, stimulate DCs and thus rebuild the
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immunosurveillance.13,
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survival, proliferation and differentiation of activated T cells and natural killer (NK) cells, which
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are the main effector cells in tumor infiltrating lymphocytes (TILs).20, 21 Moreover, IL-2 has been
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approved by FDA to treat metastatic melanoma and renal cell carcinoma for decades. However,
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effects of IL-2 treatment alone may be hindered owing to its side effects in high dosage and the
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immunosuppression in tumor microenvironment.22 Using low-dose IL-2 could be a proper way
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to avoid side effects, but the antitumor efficiency may be also reduced with insufficient induction
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of immune effector cells. Low-dose PTX with immune regulation effects, could activate DCs
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and reduce Tregs, which could further enhance the activation of immune effector cells induced
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by low-dose IL-2. Therefore, combination of PTX and IL-2 in low dosage may be a promising
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strategy for improving the efficiency of antitumor immunity without producing obvious side
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effects. However, due to the different physicochemical properties and underlying mechanisms of
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PTX and IL-2, simultaneous administration of free drugs was faced with limitations (such as
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short half-lives, insufficient tumor accumulation and unpredictable ratio of drugs in tumor) for
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Interleukin-2 (IL-2) is an important cytokine in regulating the
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realizing the real synergistic antitumor efficacy.23 Therefore, a rational drug delivery platform
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was essential to be developed.24-26
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Nanogel is an effective and safe drug delivery system for co-encapsulation of hydrophilic and
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hydrophobic drugs by adjusting the chemical compositions.27, 28 Herein, we designed erythrocyte
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membrane coated nanogels (NR) which can achieve the co-delivery and controlled release of
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PTX and IL-2 in response to tumor microenvironment. Chitosan is a natural polymer, widely
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applied in biomedical and biotechnological field with good biocompatibility, biodegradability
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and low immunogenicity.29-32 By modification of chitosan, two opposite charged chitosan
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derivatives, amphoteric methacrylamide N-carboxyethyl chitosan (CECm) and positive charged
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methacrylamide N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCCm), were
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obtained for constructing nanogels. The pH responsive capability to weak acidic tumor
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microenvironment could be precisely controlled by adjusting the formulation of nanogel. 2-
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Hydroxypropyl-β-cyclodextrin (HP-β-CD) is widely used as solubilizing excipient for
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hydrophobic compounds in various pharmaceutical formulations.33,
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hydroxypropyl-β-cyclodextrin acrylate (HP-β-CD-A) was introduced into nanogel via photo-
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crosslinking to improve the encapsulation efficiency (EE) of PTX as well as control the drug
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release behavior.35 To realize the encapsulation, protection and delivery of IL-2, red blood cell
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membrane (RBCm) was further coated on the nanogel (NG). The constructed NG-supported
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membrane could achieve ‘nanosponge’ property and realize the enhanced adsorption, protection
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and delivery of IL-2 which is physical and chemical unstable.36-38 RBCm envelope could extend
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in vivo circulation time.39,
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structural property of IL-2 receptor could promote IL-2 binding.41 These were in favor of
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achieving well-controlled drug release and regulation of immune microenvironment.42 Upon
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After modification, 2-
Moreover, the glycoprotein on RBCm which has homologous
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accumulating in tumor via EPR effect, NR could respond to the acid condition and swell quickly
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with exposing PTX loaded HP-β-CD-A.43 PTX may be sustainedly released with the help of
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host-guest interaction for induction of CRT exposure on tumor cells and regulation of
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immunosuppressive microenvironment.32 After losing the support of inner core, the membrane
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could be disintegrated to constantly release IL-2 into tumor microenvironment for stimulating
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CTLs and NK cells. In this way, the responsively released agents could effectively play their
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roles and realize the promising synergistic antitumor effect (Figure 1).
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In this work, CECm, HTCCm and HP-β-CD-A were synthesized and verified for the
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construction of NR. The structure, encapsulating capability, tumor microenvironment responsive
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drug release profiles and pharmacokinetics of NR were investigated. Its capability for improving
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drug penetration, the induction of CRT exposure and the synergistic antitumor effect against
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murine melanoma were then evaluated. The modulation of tumor microenvironment by NR
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mediated chemo-immunotherapy was further investigated by analyzing infiltration of immune
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effector cells and reduction of immunosuppressive factors.
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The synthetic routes were shown in Figure S1a-c (Supporting Information). The degree of
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substitution (DS) of chitosan derivatives was calculated by 1H NMR. From the spectrums of N-
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carboxyethyl chitosan (CEC) and CECm (Figure S1d), d= 1.8 ppm (H-f, C=C-CH3), 1.9 ppm (H-
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7), 2.3 ppm (H-a, -N-CH2-), 2.6 ppm (H-b, -CH2-CO2Na), 2.9 ppm (H-2), 3.2-4.0 ppm (H-3, 4, 5,
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6), 4.4 ppm (H-1) and 5.4-5.6 ppm (H-e, CH2=C-). From the spectrums of N-(2-hydroxy)propyl-
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3-trimethylammonium chitosan chloride (HTCC) and HTCCm (Figure S1e), d= 1.8 ppm (H-f,
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C=C-CH3), 1.9 ppm (H-7), 2.3-2.9 ppm (H-2, a, c), 3.1 ppm (H-d, -+N(CH3)3), 3.2-4.0 ppm (H-3,
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4, 5, 6), 4.2 ppm (H-b, CH-O-), 4.4 ppm (H-1) and 5.4-5.6 ppm (H-e, CH2=C-). The DS of
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carboxyl group in CECm was 57.1% and quaternization in HTCCm was 62.5%. The peaks
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appeared at 5.4-5.6 ppm (H-e, CH2=C-) and the peak of 1.8 ppm (H-f, C=C-CH3) originated
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from methacrylic anhydride further confirmed the coupling of methacryloyl group. The DS of
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methacrylation was found to be 11.5% and 11.2% for CECm and HTCCm, respectively. FT-IR
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spectrums (Figure S2a & S2b) also confirmed the successful synthesis of CEC, HTCC, CECm
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and HTCCm. As shown in the FT-IR spectrum of CEC, peaks at 1568 cm-1 and 1408 cm-1
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corresponded to the asymmetric and symmetric stretching vibration of COO- contained in CEC,
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respectively. In the spectrum of HTCC, the peak corresponding to the methyl band of (2,3-
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epoxypropyl)trimethylammonium chloride at 1480 cm-1 was presented. The peak at 1652 cm-1 in
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FT-IR spectrums of CECm and HTCCm which was attributed to stretching vibration of C=C
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also proved the successful coupling of the methacryloyl group. The DS of the vinyl group in HP-
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β-CD-A was measured by 1H NMR. As shown in Figure S1f, d= 1.0 ppm (H-9), 3.2-4.0 ppm (H-
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2, 3, 4, 5, 6, 7, 8), 4.8 ppm (H-1) and 5.9-6.4 ppm (H-10, 11, 12). The peaks of 5.9-6.4 ppm (H-
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10, 11, 12) belonged to the vinyl group and the DS was calculated as 35.3%.
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In order to prepare PTX loaded nanogel (NGP), PTX was first loaded in HP-β-CD-A, and the
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EE and drug-loading content (DLC) were then evaluated. As shown in Figure S3, the EE was
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94.2 ± 2.1%, 93.2 ± 3.4%, 42.6 ± 1.0% and 20.2 ± 0.5% with feeding 250, 500, 1000 and 2000
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µg PTX in 1 mg HP-β-CD-A, respectively, showing a decreasing tendency. However, owing to
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the limited cavity of HP-β-CD-A, DLC was up to 31.8 ± 0.8% at feeding 500 µg PTX in 1 mg
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HP-β-CD-A. To obtain the stable and pH responsive nanogel, the stoichiometric ratio of CECm,
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HTCCm and HP-β-CD-A was investigated by turbidimetric titration. The formulation of nanogel
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depended on the electrical interaction between negative-charged CECm and cationic HTCCm at
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pH 7.4. As shown in Figure S4a, after adding around 5000 µL of CECm (1 mg/mL) into 1 mg
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HTCCm (1 mg/mL), the transmittance (T) reached the minimum, indicating the strongest
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electrostatic interaction between CECm and HTCCm. Then, the proper proportion of HP-β-CD-
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A in nanogels was measured by turbidimetric titration as well (Figure S4b). When more than
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1000 µL of HP-β-CD-A (1 mg/mL) was added into the above mixture of CECm and HTCCm,
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the transmittance was instantly increased, suggesting that excessive HP-β-CD-A (> 1 mg) could
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affect the interaction between CECm and HTCCm. Therefore, the stoichiometric ratio of CECm,
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HTCCm and HP-β-CD-A was selected as 5:1:1 for keeping effective PTX encapsulation without
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influencing the stability of nanogels. To further increase the stability and prevent the leakage of
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loaded drug, the formed nanogel was crosslinked by UV irradiation. As shown in Figure S5,
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about 36.4% of double bond (CH2=C-) was reacted during UV crosslinking. Furthermore, in
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view of the low DS of methacrylation in chitosan, the crosslinking density may be too low with
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no significant influence on the pH sensitivity of NG. The pH responsive capability of NG was
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further tested as exhibited in Figure S4c. Once the pH value was lower than 6.8, transmittance
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was instantly increased (< 1 min), indicating that NG could precisely respond to weak acidic
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tumor microenvironment. This may be attributed that the electrostatic interaction between CECm
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and HTCCm had been changed into electrostatic repulsion due to the protonation of amino on
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CECm. This ionic reaction could complete in quite short time. To precisely control the
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responsive property to weak acidic tumor microenvironment, we have also tested NG
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constructed by CECm with DS 50.2% or 65.5% (Table S1). The NG constructed by CECm with
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DS 50.2% was positive charged (3.36 ± 1.43 mV) which was not proper for RBCm coating.44
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The responsive pH value of NG by CECm with DS 65.5% was around 7.1, which was not
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appropriate for realizing the responsive release of agents in tumor microenvironment (pH 6.5-
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6.8) as the pH value of normal tissue is close to 7.2-7.4 (Figure S6).45
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For NRP preparation, NGP and RBCm were extruded through an 800 nm polycarbonate porous
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membrane for 7 times with an Avanti mini-extruder (Avanti Polar Lipids).46 To realize the
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encapsulation, protection and delivery of IL-2, NRP was then incubated with IL-2 to prepare
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PTX and IL-2 co-loaded NR (NRP+I). As comparison, NGP incubated with IL-2 was named as
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NGP+I. Free IL-2 was removed by ultrafiltration and measured by enzyme-linked immunosorbent
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assay (ELISA). As seen in Table S2, the EE of PTX in NGP+I and NRP+I was 86.8 ± 1.5% and
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73.5 ± 2.2%, respectively. In NRP+I, 89.3 ± 5.4% of IL-2 was adsorbed while only 61.2 ± 8.5%
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of IL-2 was loaded in NGP+I, showing an enhanced encapsulation efficiency of NR. As reported,
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nanoparticle supported RBCm exhibited ‘nanosponge’ feature which can be used for adsorbing
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pore-forming toxins.37 For NR, it is also capable of improving the EE and preventing the leakage
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of IL-2. As shown in Figure S7, compared to other formulations (PLGA nanoparticle, RBCm
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and NG), NR exhibited the highest EE (89.3 ± 5.4%) and the lowest leakage (9.0 ± 0.9%), which
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suggested the good loading and protecting capability for IL-2. It may be attributed to the
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glycoprotein on RBCm which is homologous with IL-2 receptor in amino acid sequence and in
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other structural properties.41 This protein may allow IL-2 to bind with RBCm. Therefore, the
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loading efficiency and protection of IL-2 on RBCm could be further enhanced and the leakage of
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IL-2 could also be reduced. In addition, IL-2 receptor on lymphocytes displayed higher affinity
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for IL-2 compared with the protein on RBCm. Thus, loading IL-2 on RBCm may not affect its
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subsequent binding to IL-2 receptor on lymphocytes and the bioactivity.
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In order to confirm the coating of RBCm, morphology of NRP+I was characterized by
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transmission electron microscopy (TEM) (Figure 2a). A core-shell structure of NRP+I was
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observed, indicating the successful coating of RBCm. To further confirm the coating of RBCm,
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NGP+I and NRP+I were incubated with membrane disruption agent (Triton X-100), and dynamic
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laser scattering (DLS) was used to monitor the change of diameter and zeta potential (Figure 2b).
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The diameter and zeta potential of NGP+I was 308.0 ± 8.4 nm and -16.0 ± 1.5 mV, respectively.
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After extrusion with RBCm, the diameter and zeta potential of NRP+I respectively increased to
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336.0 ± 6.2 nm and -7.1 ± 0.6 mV, which may be owing to the envelope of RBCm. After treated
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with 0.3% Triton X-100, the diameter and zeta potential of NRP+I decreased to 305.0 ± 6.4 nm
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and -13.4 ± 2.5 mV, respectively, which were similar to those of NGP+I. These results confirmed
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the envelope of RBCm in NR.
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The pH responsive capability is the decisive property of NR to realize the tumor
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microenvironment trigged drug release. As shown in Figure 2c, the diameter appeared a slight
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increase at pH 6.8 and was then hardly detectable at pH 6.5, suggesting a good responsive ability
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to weak acidic tumor microenvironment (pH 6.5-6.8).47, 48 TEM observation of NRP+I in pH 6.5
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buffer indicated that RBCm may be disrupted once the inner core disintegrated (Figure 2a). The
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pH responsive capability of NR may mainly depend on the protonation of chitosan polymers in
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the acidic condition, thus the response could be completed in very short time (
