Letter pubs.acs.org/NanoLett
Tumor Microenvironment Responsive Nanogel for the 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*,†,§,∥ †
Tongji School of Pharmacy, ‡Department of Pharmacy, Liyuan Hospital, §National Engineering Research Center for Nanomedicine, Hubei Engineering Research Center for Novel Drug Delivery System, Huazhong University of Science and Technology, Wuhan 430030, China ∥
S Supporting Information *
ABSTRACT: A biomimetic nanogel with tumor microenvironment responsive property is developed for the combinatorial antitumor effects of chemotherapy and immunotherapy. Nanogels are formulated with hydroxypropyl-β-cyclodextrin acrylate and two opposite charged chitosan derivatives for entrapping anticancer drug paclitaxel and precisely controlling the pH responsive capability, respectively. The nanogel supported erythrocyte membrane can achieve “nanosponge” property for delivering immunotherapeutic agent interleukin-2 without reducing the bioactivity. By responsively releasing drugs in tumor microenvironment, the nanogels significantly enhanced antitumor activity with improved drug penetration, induction of calreticulin exposure, and increased antitumor immunity. The tumor microenvironment is remodeled by the combination of these drugs in low dosage, as evidenced by the promoted infiltration of immune effector cells and reduction of immunosuppressive factors. KEYWORDS: Antitumor agents, chemo-immunotherapy, drug delivery, nanogel, tumor microenvironment
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Interleukin-2 (IL-2) is an important cytokine in regulating the survival, proliferation, and differentiation of activated T cells and natural killer (NK) cells, which are the main effector cells in tumor infiltrating lymphocytes (TILs).20,21 Moreover, IL-2 has been approved by FDA to treat metastatic melanoma and renal cell carcinoma for decades. However, effects of IL-2 treatment alone may be hindered owing to its side effects in high dosage and the immunosuppression in tumor microenvironment.22 Using low-dose IL-2 could be a proper way to avoid side effects, but the antitumor efficiency may be also reduced with insufficient induction of immune effector cells. Low-dose PTX with immune regulation effects, could activate DCs and reduce Tregs, which could further enhance the activation of immune effector cells induced by low-dose IL-2. Therefore, the combination of PTX and IL-2 in low dosage may be a promising strategy for improving the efficiency of antitumor immunity without producing obvious side effects. However, due to the different physicochemical properties and underlying mechanisms of PTX and IL-2, simultaneous administration of free drugs was faced with limitations (such as short half-lives, insufficient tumor accumulation, and unpredictable ratio of drugs in tumor) for realizing the real
hemo-immunotherapy is raising great attention for its enhanced antitumor effects by the synergism of chemotherapy and immunotherapy.1,2 There is mounting evidence to support that some chemotherapeutic drugs with certain dosage, such as paclitaxel (PTX), doxorubicin, and cisplatin, are capable of activating the immune system and regulating the immunosuppressive microenvironment in tumor.2−7 The immunogenic cell death of cancer cells induced by some chemotherapeutics could promote antigen releasing from tumors, taken up and presented by dendritic cells (DCs), and the subsequent activation of cytotoxic T lymphocytes (CTLs).8,9 It has been demonstrated that the pretreatment of chemotherapeutics could also make tumors more susceptible to lysis by activated CTLs.8,10,11 Moreover, a tumor immunosuppressive microenvironment can be remodeled via depleting suppressor cells like regulatory T lymphocyte (Treg) as well as inhibitory cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10).8,12,13 PTX is a wide spectrum antineoplastic agent against various cancers with water solubility of less than 0.3 μg/mL.14 Recent studies showed that low-dose PTX can improve the effect of cytokine immunotherapy by modulating the immunosuppressive tumor microenvironment, including cytokine network and inhibitory activity of Tregs.15−18 Moreover, low-dose PTX can also induce exposure of calreticulin (CRT) on tumor cells, stimulate DCs, and thus rebuild the immunosurveillance.13,15,19 © 2017 American Chemical Society
Received: July 26, 2017 Revised: August 26, 2017 Published: August 31, 2017 6366
DOI: 10.1021/acs.nanolett.7b03186 Nano Lett. 2017, 17, 6366−6375
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Nano Letters
Figure 1. Preparation of NRP+I and schematic illustration of chemo-immunotherapy.
synergistic antitumor efficacy.23 Therefore, a rational drug delivery platform was essential to be developed.24−26 Nanogel is an effective and safe drug delivery system for coencapsulation of hydrophilic and hydrophobic drugs by adjusting the chemical compositions.27,28 Herein, we designed erythrocyte membrane coated nanogels (NR) which can achieve the codelivery and controlled release of PTX and IL2 in response to tumor microenvironment. Chitosan is a natural polymer, widely applied in biomedical and biotechnological field with good biocompatibility, biodegradability, and low immunogenicity.29−32 By the modification of chitosan, two opposite charged chitosan derivatives, amphoteric methacrylamide N-carboxyethyl chitosan (CECm) and positive charged methacrylamide N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCCm), were obtained for constructing nanogels. The pH responsive capability to a weak acidic tumor microenvironment could be precisely controlled by adjusting the formulation of nanogel. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) is widely used as the solubilizing excipient for hydrophobic compounds in various pharmaceutical formulations.33,34 After modification, 2-hydroxypropyl-β-cyclodextrin acrylate (HP-β-CD-A) was introduced into nanogel via photocross-linking to improve the encapsulation efficiency (EE) of PTX as well as control the drug release behavior.35 To realize the encapsulation, protection, and delivery of IL-2, the red blood cell membrane (RBCm) was further coated on the nanogel (NG). The constructed NG-supported membrane could achieve “nanosponge” property and realize the enhanced adsorption, protection and delivery of IL-2 which is physical and chemical unstable.36−38 RBCm envelope could extend the in vivo circulation time.39,40 Moreover, the glycoprotein on RBCm which has a homologous structural property of IL-2 receptor could promote IL-2 binding.41 These were in favor of achieving well-controlled drug release and regulation of immune microenvironment.42 Upon accumulating in the tumor via the EPR effect, NR could respond to the acid
condition and swell quickly with exposing PTX loaded HP-βCD-A.43 PTX may be sustainedly released with the help of host−guest interaction for induction of CRT exposure on tumor cells and regulation of immunosuppressive microenvironment.32 After losing the support of inner core, the membrane could be disintegrated to constantly release IL-2 into tumor microenvironment for stimulating CTLs and NK cells. In this way, the responsively released agents could effectively play their roles and realize the promising synergistic antitumor effect (Figure 1). In this work, CECm, HTCCm, and HP-β-CD-A were synthesized and verified for the construction of NR. The structure, encapsulating capability, tumor microenvironment responsive drug release profiles, and pharmacokinetics of NR were investigated. Its capability for improving drug penetration, the induction of CRT exposure, and the synergistic antitumor effect against murine melanoma were then evaluated. The modulation of tumor microenvironment by NR mediated chemo-immunotherapy was further investigated by analyzing infiltration of immune effector cells and reduction of immunosuppressive factors. The synthetic routes are shown in Figure S1a−c (Supporting Information). The degree of substitution (DS) of chitosan derivatives was calculated by 1H NMR. From the spectra of Ncarboxyethyl chitosan (CEC) and CECm (Figure S1d), d = 1.8 ppm (H-f, CC−CH3), 1.9 ppm (H-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, 6), 4.4 ppm (H-1), and 5.4−5.6 ppm (H-e, CH2C−). From the spectra of N-(2-hydroxy)propyl-3trimethylammonium chitosan chloride (HTCC) and HTCCm (Figure S1e), d = 1.8 ppm (H-f, CC−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, 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 carboxyl group in CECm was 57.1%, and the quaternization in HTCCm was 62.5%. The peaks appeared at 6367
DOI: 10.1021/acs.nanolett.7b03186 Nano Lett. 2017, 17, 6366−6375
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Nano Letters 5.4−5.6 ppm (H-e, CH2C−) and the peak of 1.8 ppm (H-f, CC−CH3) originated from methacrylic anhydride further confirmed the coupling of methacryloyl group. The DS of methacrylation was found to be 11.5% and 11.2% for CECm and HTCCm, respectively. FT-IR spectra (Figures S2a and S2b) also confirmed the successful synthesis of CEC, HTCC, CECm and HTCCm. As shown in the FT-IR spectrum of CEC, peaks at 1568 and 1408 cm−1 corresponded to the asymmetric and symmetric stretching vibration of COO− contained in CEC, respectively. In the spectrum of HTCC, the peak corresponding to the methyl band of (2,3epoxypropyl)trimethylammonium chloride at 1480 cm−1 was presented. The peak at 1652 cm−1 in FT-IR spectra of CECm and HTCCm which was attributed to stretching vibration of CC also proved the successful coupling of the methacryloyl group. The DS of the vinyl group in HP-β-CD-A was measured by 1H NMR. As shown in Figure S 1f, d = 1.0 ppm (H-9), 3.2− 4.0 ppm (H-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-10, 11, 12) belonged to the vinyl group, and the DS was calculated as 35.3%. To prepare PTX loaded nanogel (NGP), PTX was first loaded in HP-β-CD-A, and the EE and drug-loading content (DLC) were then evaluated. As shown in Figure S3, the EE was 94.2 ± 2.1%, 93.2 ± 3.4%, 42.6 ± 1.0%, and 20.2 ± 0.5% with feeding 250, 500, 1000, and 2000 μg of PTX in 1 mg of HP-βCD-A, respectively, showing a decreasing tendency. However, owing to the limited cavity of HP-β-CD-A, DLC was up to 31.8 ± 0.8% at feeding 500 μg PTX in 1 mg HP-β-CD-A. To obtain the stable and pH responsive nanogel, the stoichiometric ratio of CECm, HTCCm, and HP-β-CD-A was investigated by turbidimetric titration. The formulation of nanogel depended on the electrical interaction between negative-charged CECm and cationic HTCCm at pH 7.4. As shown in Figure S4a, after adding around 5000 μL of CECm (1 mg/mL) into 1 mg of HTCCm (1 mg/mL), the transmittance (T) reached the minimum, indicating the strongest electrostatic interaction between CECm and HTCCm. Then, the proper proportion of HP-β-CD-A in nanogels was measured by turbidimetric titration as well (Figure S4b). When more than 1000 μL of HP-β-CD-A (1 mg/mL) was added into the above mixture of CECm and HTCCm, the transmittance was instantly increased, suggesting that excessive HP-β-CD-A (>1 mg) could affect the interaction between CECm and HTCCm. Therefore, the stoichiometric ratio of CECm, HTCCm, and HP-β-CD-A was selected as 5:1:1 for keeping effective PTX encapsulation without influencing the stability of nanogels. To further increase the stability and prevent the leakage of loaded drug, the formed nanogel was cross-linked by UV irradiation. As shown in Figure S5, about 36.4% of double bond (CH2C−) was reacted during UV cross-linking. Furthermore, in view of the low DS of methacrylation in chitosan, the cross-linking density may be too low with no significant influence on the pH sensitivity of NG. The pH-responsive capability of NG was further tested as exhibited in Figure S4c. Once the pH value was lower than 6.8, the transmittance was instantly increased (
