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Highly Integrated Nano-Platform for Breaking the Barrier between Chemotherapy and Immunotherapy Di-Wei Zheng, Jia-Li Chen, Jing-Yi Zhu, Lei Rong, Bin Li, Qi Lei, Jin-Xuan Fan, Mei-Zhen Zou, Cao Li, Si-Xue Cheng, Zushun Xu, and Xian-Zheng Zhang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 22, 2016

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Highly Integrated Nano-Platform for Breaking the Barrier between Chemotherapy and Immunotherapy Di-Wei Zheng†,‡,§, Jia-Li Chen†,‡,§, Jing-Yi Zhu†, Lei Rong†, Bin Li†, Qi Lei†, Jin-Xuan Fan†, Mei-Zhen Zou†, Cao Li‡, Si-Xue Cheng†, Zushun Xu‡ and Xian-Zheng Zhang†,*

†

Key Laboratory of Biomedical Polymers of Ministry of Education, Institute for Advanced Studies (IAS), Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

‡

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Key Laboratory for the Green Preparation and Application of Functional Materials of Ministry of Education, Hubei University, Wuhan, Hubei 430062, P. R. China

§ These authors contributed equally to this work.

KEYWORDS: Theranostic platform, Tumor therapy, Imaging, Target, Immune response

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Surgery operation, radiotherapy, chemotherapy and immunotherapy are effective methods for cancer therapy. However, for highly metastatic cancer types, such as triple negative breast cancer (TNBC), few patients are eligible for surgery or radiotherapy, and anthracycline-based chemotherapy is still a common treatment.1 Additionally, TNBC subtype elevates the expression of particular genes, which provides a list of therapeutic targets for immunotherapy. Most chemotherapies kill target cells by non-stimulatory apoptosis, and the induction of leukopenia inhibits the anti-cancer immune response (ACIR). Thus, immunotherapy and chemotherapy have always been regarded as unrelated or, even worse, antagonistic forms of therapy.2 Recently, some anticancer drugs (e.g. anthracyclines and oxaliplatin) have been reported to induce anti-cancer antigen release during apoptosis.3 However, chemotherapy induced ACIR is dosage dependent. A high dose of chemotherapeutic drug would cause leukopenia, and a low dosage of chemotherapeutic drug is inefficient to induce immune response.4,5 This contradiction is rather confused, and breaking this barrier would provide a new insight in achieving chemo-immuno combination therapy. Previously, in order to improve the chemotherapy efficiency, molecular targeted agents are carried out. However, some molecular targeted agents were found to inhibit the proper function of lymphocytes.7 As it is well known, nano drug delivery systems (DDS) could also improve the therapeutic efficiency with reduced side effect of chemotherapy.8,9 Most importantly, compared with small molecule drugs, this method offers considerable versatility, thus feasible of integrating a variety of functions, such as prolonging the circulation time, avoiding the early release during circulation, enhancing 2


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the tumor accumulation and improving cancer cell uptake.10 In nature, bio-machines based on peptides and proteins are highly potent and specific in a single function; learn from nature, supramolecular chemistry is a good strategy to design molecular machines, and most importantly, integrating different functions into a single platform for solving new problems.11,12 Among various supramolecular machines, rotaxane has attracted extensive interest for its distinct mechanical bond likes actin and myosin. Besides, the versatile molecular design makes rotaxane based DDS highly possible to integrate different functions and overcomes the limitation of chemotherapy. Among various type of DDS, mesoporous silica nanoparticles (MSN) has shown obvious advantages as a drug carrier, such as high drug loading capacity, good biocompatibility and feasibility of functionalization.13-15 Most importantly, MSN has no immunogenicity, which makes them a suitable drug carrier for inducing ACIR.16 In this study, doxorubicin loaded MSN (DOX@MSN) was prepared and its ability of inducing ACIR was carefully estimated. Because of its enhanced permeability and retention effect, DOX@MSN was found to amplify ACIR successfully. Inspired from this result, integrated MSN (IMSN) with a pH and GSH dual stimulated rotaxane as a molecular gate for avoiding the early release was synthesized. As illustrated in Figure 1a, β-cyclodextrin (β-CD) was threaded through a benzimidazole (Bz, pH-responsive)-polyethylene glycol (PEG)-ferricinium (Fc+, redox-responsive) rotaxane gate. β-CD in rotaxane was initially located at the Bz position, which blocked the drug release before cell uptake. However, in the intracellular environment, the low pH value led to the protonation of Bz, and the high GSH level induced the reduction of Fc+ to ferrocene (Fc). Thus, β-CD combined with 3


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Fc, resulted in drug release. Besides, on the basis of IMSN, the concept of “plug and play” was used to prepare highly integrated MSN (HIMSN) with active targeting and magnetic resonance/computed tomography (MRI/CT) imaging abilities.17 After comprehensive in vitro and in vivo studies, we found that the increase of MSN integration level gradually eliminated the leukopenia and significantly induced ACIR in high metastatic TNBC cell line (4T1).(18) The MRI/CT imaging ability of HIMSN also guided in vivo therapies.(19) In all, to realize that the satisfactory therapeutic effect of HIMSN were partly attributed to the induction of ACIR may allow us to design even more efficient nanomaterials for chemo-immuno combination therapy.

Figure 1. (a) Structure of theranostic nano-platform and schematic diagram of in vivo diagnosis and treatment; (1) GSH and pH dual-stimulate “And” type logic gate of DOX@HIMSN; (2) Intravenous injection of DOX@HIMSN into mice via i.v. injection; (3) Endocytosis of DOX@HIMSN and intracellular drug release; (4) Anti-tumor immune response induced by DOX@MFMSN. (5) 4


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Tumor cell targeting, CT and MR imaging ability of DOX@HIMSN. (b) Cyclic voltammetry curve of Fc-PEG-Bz-MSN at various conditions. (c) Cell viability assay of 4T1 cells co-incubated with DOX@HIMSNs at various conditions. (d) CLSM study of DOX@HIMSN at various conditions. (e) In vitro phagocytosis of the PBS, DOX, or DOX@HIMSN treated 4T1 cells (red) by DC (green).

Here, we have successfully synthesized HIMSN for inducing anti-tumor immune responses. As observed by TEM (Figure S1), mono-dispersed spherical MSN with an average diameter of 125 nm was obtained and nanopores were distributed on the surface of MSN with a pore size of 2 nm. Bz-COOH, NH2-PEG-NH2 and Fc-COOH were grafted on the surface of MSN step by step to prepare IMSN (Figure S2). FT-IR spectra (Figure S3) and zeta potentials (Table S1) of nanoparticles confirmed the successful surface modification. The grafting ratio of electrochemical active ferrocene containing polymer determined by cyclic voltammetry was 1.14×10-6 mol/g (Figure S4). 1

H-NMR, ESI-MS and ICP-AES demonstrated the successful synthesis of the

functional β-CD derivatives. Finally, FA-β-CD, DOTA-β-CD and DA-β-CD were self-assembled with Fc-PEG-Bz-MSN to obtain HIMSN for imaging guided targeting therapy. In order to study the drug release property of HIMSN, in vitro drug release in the presence and in the absence of GSH at different pH values were compared (Figure S5). Approximately 45% of DOX was released within 5 h at pH 6.0 with 10 mM GSH. Whereas limited amount of drug (< 20%) was released in equal times at pH 7.4 and pH 6.0 in the absence of GSH. Because of the low lysosome pH and high GSH level within cells, DOX@HIMSN and 5


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DOX@IMSN were expected to have a favorable stimuli responsibility for intracellular drug release. Then, cyclic voltammetry test was carried out to study the rotaxane gate. As shown in Figure 1b, at pH 7.4/10 mM GSH, pH 6.0 and pH 7.4, a strong peak at 0.3 mV was observed, which indicated the existence of non-coupled Fc/Fc+. However, at pH 6.0 with 10 mM GSH, the peak was weakened, which was mainly due to the formation of host-derivate reduced the diffusing rate of Fc molecule. The decreased peak current can be evaluated numerically by the Randles-Savick equation: iP = 2.69×105n3/2D1/2v1/2Ac

(1)

where iP is the peak current, n is the electron transfer number, D is the diffusion coefficient, v is the polarizability, A is the electrode area and c is the concentration. This result was in agreement with the macroscopical drug release study. Because pH sensitive Bz was protonated at acidic environement and Fc+ was reduced to Fc under high GSH concentration.20,21 Thus, β-CD moved from Bz side to Fc side under intracellular environment and leaded to a drug release. To further study the tumor-triggered targeting ability, DOX@HIMSN was co-incubated with folate receptor positive 4T1 cells.22 As shown in Figure 1c, after being treated by DOX@HIMSN, cell viability assays for pH 7.4/10 mM GSH and pH 6.0 (without GSH) groups were significantly higher than the pH 6.0/10 mM GSH group. BSO induced GSH depletion and NH4Cl triggered restoration of intracellular pH, which also increased the 4T1 cell viability after the treatment of DOX@HIMSN (see Figure S6).23,24 Furthermore, confocal laser scanning microscope (CLSM) was used to study the intracellular drug release ability of DOX@HIMSN. After co-incubation with DOX@HIMSN for 8 h, 6


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red fluorescence could be observed within 4T1 cells (Figure 1d), indicating DOX@HIMSN could be effectively endocytosed. However, NH4Cl or BSO treatment decreased the intracellular fluorescence intensity. For cells without NH4Cl or BSO treatment, DOX was accumulated preferentially in the nucleus domain of 4T1 cells, since free DOX intended to accumulate in the nuclei by the formation of DOX proteasome complex after passed through nuclear pores. Nevertheless, after the NH4Cl or BSO treatment, DOX was mainly distributed within the cytoplasm area, and almost no DOX could be observed in the cell nuclear. In the presence of free folic acid, the cellular uptake of DOX@HIMSN was significantly decreased. This result confirmed the active targeting ability of DOX@HIMSN for folate receptor positive tumor cells.25 Cell viability assay and CLSM of human tumor cells were shown in Figure S7 and S8, indicating that DOX@HIMSN is effective in treating various type of cancers. Because of

its

better anti-cancer

efficiency,

we estimated that

DOX@HIMSN should also have a better performance in inducing the release of chemotherapy associated anti-cancer antigen. After treatments of free DOX and DOX@HIMSN, an increased level of anti-tumor antigen high mobility group box-1 secretion was observed (Figure S9).26 Besides, red fluorescence labelled 4T1 cells was co-incubated with green fluorescence labelled dendritic cells (DCs), and a remarkable DCs uptake was observed in Figure 1e. Compared with PBS treated cells, increased mRNA level of interleukin-6 (IL-6) and interleukin-12P40 (IL-12P40) after DOX and DOX@HIMSN treatments was observed, which indicated the maturation of DCs (Figure S10).

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Figure 2. (a) Ex vivo fluorescence imaging for heart, lung, liver, spleen, kidney and tumor 24 h after i.v. injection of DOX@HIMSN. (b) CLSM images of tumors 24 h after i.v. Injection. (c) Quantitative analysis of in vivo distribution of free DOX after various treatments. (d) WBC count after various treatments. (e) Pharmacokinetics study of DOX@HIMSN. (f) In vitro MRI study of DOX@HIMSN. (g) In vivo MRI study of DOX@HIMSN. (h) In vivo CT imaging study of DOX@HIMSN. The in vivo tumor specific accumulation of DOX@HIMSN, DOX@IMSN and DOX@MSN was studied using 4T1 tumor bearing Balb/c mice as an animal model. As shown in Figure 2a, after the i.v. injection of DOX@HIMSN, most of the red fluorescence was accumulated in the tumor tissue, whereas limit amounts of fluorescence could be observed within primary metabolic organs. The very low DOX concentration in lymphoid organs suggested that the unfavorable effect of DOX@HIMSN on the immune system was limited.27 In addition, GSH depletion and restoration of intratumoral pH resulted in a decreased fluorescence accumulation in the tumor site. Compared with 8


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DOX@HIMSN, DOX@IMSN with no active targeting showed poor tumor targeting ability (Figure S11). Besides, DOX@MSN and free DOX was accumulated in the primary metabolic and lymphoid organs. The quantitative study of in vivo distribution and frozen section also agreed well with the above qualitative investigation (Figure 2b, 2c, and S12). Herein, levels of white blood cells were analyzed (Figure 2d). Free DOX treatment reduced 43% of white blood cells, whereas DOX@MSN, DOX@IMSN and DOX@HIMSN treatments reduced 2%, 14% and 27% of white blood cell, respectively. This result confirmed that the increased integration level alleviated leucopoenia. In vivo blood retention studies indicated that DOX@HIMSN significantly improved the bioavailability of free DOX. Since the plasma concentration-time curve (AUC24h) of DOX@HIMSN is approximately 10-fold greater than free DOX. Besides, t1/2 value of DOX@HIMSN was 20.9 times higher than DOX group. Then, the in vivo imaging ability of DOX@HIMSN was studied. As shown in Figure 2f and Figure S13, the r1 relaxivity was calculated to be 3.3 mM-1 s-1, which was superior to the commercialized Dotarems® (r1=3.1 mM-1 s-1).28 To evaluate the in vivo T1-weighted MRI imaging ability, Balb/c mice receiving i.v. injection of DOX@HIMSN were underwent MRI at different time points. As shown in Figure 2g and S14, before the injection of DOX@HIMSN, the tumor appeared as a hyper-intense area in the T1-weighted MRI images. After injection, a significantly enhanced signal was observed with time prolonging in T1 imaging.29 As shown in Figure 2h, CT scanning showed that after injection, the tumor displayed an enhanced positive-contrast, which was attributed to the strong X-ray absorption induced by DOX@HIMSN containing iodine.30 Our study demonstrated DOX@HIMSN as a dual model imaging agent, and 9


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DOX@HIMSN could further guide in vivo therapy.

Figure 3. (a) Representative images of the tumors at the 25th day; (b) Relative tumor volume posttreatment; (c) In vivo tumor MRI images at the 20th day; (d,e) Representative images of the lungs and livers at the 25th day (f) In vivo metabonomics study of tumors 25 days posttreatment (1 PBS; 2 DOX; 3 DOX@MSN; 4 DOX@IMSN; 5 DOX@HIMSN). 10


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Inspired from above results, we propose to study the long-term image guided therapy of DOX@HIMSN in 4T1 tumor bearing Balb/c mice. Different agents after i.v. injections with a DOX dosage of 5 mg/kg were compared. As shown in Figure 3, DOX@HIMSN had a better therapeutic effect as compared with other agents. After 25-day treatment, the tumor volume of DOX@HIMSN group was 10 times smaller than that of the PBS group, 5 times smaller than the DOX group and 2 times smaller than the DOX@HIMSN group. Additionally, 1/3 of the DOX@HIMSN treated tumors were completely disappeared. Due to the low DOX dosage, the decrease of body weight was not observed of all groups (Figure S15). DOX@HIMSN and DOX@IMSN also increased the survival rate of tumor bearing mice. Additionally, the H&E staining was shown in Figure S16-S18. The in vivo therapy with s.c. injection for pre-experiment was shown in Figure S19-20, which was consistent with the study of i.v. injection. Besides, tumor progression was visualized by using 1H-MRI imaging in the 20th day. Intuitively, compared with other groups, the DOX@HIMSN group had a smaller tumor volume. Furthermore, in vivo 1H-MRS was used to make crosswise comparison among metabolites from lesions in real time.31-34 Changes in metabolic markers of all groups were presented in a heat map. Compared with other groups, an increase of α-glucose and β-glucose, together with the decrease of lactate, creatine, choline/lipid ratio, taurine, spermine and glutathione levels were observed in DOX@HIMSN group, indicating decrease of cell density and energy consumption. Since hepatic and pulmonary metastasis were common in TNBC, lungs and livers after different treatments were carefully examined.35 Images of lungs 11


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and livers were shown in Figure 3.No macroscopical pulmonary metastasis and hepatic metastasis could be observed after the DOX@HIMSN treatment. H&E staining indicated that there exist no obvious micrometastasis after DOX@HIMSN treatment, whereas, aggressive lung metastases were observed in other groups. Active targeting of FA enhanced the anti-metastasis ability of DOX@HIMSN. Then, the in vivo ACIR inducing ability of various materials was studied. Herein, immunofluorescence was used to further study the DOX@HIMSN induced immune response. As shown in Figure S22, a sizeable fraction of tumor cells underwent apoptosis, and caspases-3 immunofluorescence could be observed. To further studied the mechanism of chemotherapy induced immune response, CD86, CD8 and caspases-3 antibodies were used to visualize the intratumoral DCs, CD8+ T cells and dead cells respectively (Figure 4). As shown in Figure S23, quantitatively, DOX@HIMSN treated group had the strongest mean fluorescence intensity of both caspases-3 and CD86. Dead-cell proximity index (the ratio of the distance of CD86+ DCs from caspase-3+ tumor cells to the distance of CD86+ DCs from caspase-3– tumor cells) of DOX@HIMSN, DOX@IMSN and DOX@MSN treated groups were 70%, 75% and 82% of free DOX group, respectively.36 As shown in Figure 5a, Euclidean distance matrix analysis between CD86+ and caspase-3+ cells of the DOX@HIMSN, DOX@IMSN and DOX@MSN treated groups were 67%, 78% and 84% of free DOX treated groups, respectively. Dead-cell proximity index and Euclidean distance matrix analysis together demonstrated the initiation of cancer specific DCs. Additionally, an increased level of tumor-infiltrating CD8+ T cells was also observed after the DOX@HIMSN treatment. Compared with 12


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other groups, DOX@HIMSN had a better performance for DCs mutation and CD8+ T cells recruitment, and thus enhanced ACIR. The co-existence of caspase-3 and DOX was regarded as a characteristic of cytotoxicity in cell killing. Whereas, the co-existence of caspase-3 and granzyme-B was a feature of immunological clearance; the fluorescence overlap of caspase-3 and DOX indicated DOX@HIMSN treatment could efficiently induce apoptosis of tumor cells. Herein, the fluorescent overlap between granzyme-B and caspase-3 had a Mander overlap coefficient of 0.95, which were higher than that of caspase-3 and DOX fluorescence (with a Mander overlap coefficient of 0.90). Besides, compared with the DOX treatment (with a Mander overlap coefficient of 0.88), DOX@HIMSN increased the efficiency of immunological cells killing with a better caspase-3/granzyme-b overlap and a higher mean fluorescence intensity (Figure 5b and S22). These dates suggested that in the DOX@HIMSN treatment group, ACIR played an important role in killing cancer cells as much as the cytotoxicity of drugs itself. With the increase of integration level, both cytotoxic and immunogenic cell killing effect was increased, which was in consistent with above studies. An increase of spleen, tumor and serum interleukin-1β (IL-1β), IL-12P40, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), CD4, CD8 and CD86 levels was also observed after DOX@HIMSN treatment, when compared with other groups (Figure 5c).37 After DOX@HIMSN accumulated in tumor tissue and was endocytosed by cancer cells, DOX release was triggered by the intracellular environment and the released DOX led to apoptosis. Then, antigen such as high mobility group box-1 was released from the dying cells and the released antigen further 13


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activated professional antigen-presenting DC cells. A better cell killing efficiency of materials with a high integration level provided a better DC activation. Additionally, antigens were cross-presented to cytotoxic lymphocyte and the active cytotoxic lymphocyte further eliminated cancer cells. The reduced immunosuppression of DOX@HIMSN also ensured a better ACIR effect.

Figure 4. Dual-labeling immunofluorescence images of caspase-3/CD-86, caspase-3/CD-8, caspase-3/DOX and caspase-3/Granzyme-B after PBS, DOX, DOX@MSN, DOX@IMSN and DOX@HIMSN treatments. As far as we know, the efficiency of DDSs always lacks a quantitative assessment standard. To establish a reliable standard for evaluating the therapeutic effect of DDSs, a comprehensive assessment was established to quantitatively evaluate the therapeutic efficiency of DDSs. As shown in Figure 5d, compared with free DOX, DOX@HIMSN achieved enhanced anti-primary 14


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tumor ability, anti-lung metastasis ability, anti-liver metastasis ability, immune response, immune suppression and tumor targeting.

Figure 5. (a) Dead-cell proximity index and Euclidean distance matrix analysis index of CD-86/caspase-3 after various treatments. (b) Fluorescence colocalization

of

immunogenic

and

cytotoxic

cell

killing

effect.

(c)

Semiquantitative analyze of TNF-α, IFN-γ, IL-1β, IL-12, CD-4, CD-8 and CD-86 content within serum, spleen and tumor, respectly. (d) A total of 6 indices, were used to make a comprehensive percentile evaluation between DOX (blue) and DOX@HIMSN (red). In this study, we demonstrate an integrated theranostic nano-platform for chemo-immuno combination therapy. In our design, HIMSN promotes the intratumoral drugs accumulation of DOX, and reduces the leukopenia. Then, the death of cancer cells initiates ACIR to achieve a better therapeutic effect. 1

H-MRS, CT and 1H-MRI are used to monitor the tumor progression and

provides valuable information to guide the treatment. The theranostic nano-platform developed in our study provides a promising strategy for the efficient treatment of highly metastatic tumors such as TNBC. Most importantly, 15


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our research indicates that using well-design nano-DDSs should be considered as a new method to achieve chemo-immuno combination therapy, which shows great clinical potentials.

ASSOCIATED CONTENT Supporting Information. Synthesis, characterization of DOX@HIMSN and other experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51233003, 51533006 and 21474077).

ABBREVIATIONS β-CD, β-cyclodextrin; Fc, Ferrocene; Fc+, Ferricinium; Bz, Benzimidazole; PEG, Polyethylene glycol; FA, Folic acid; DA, Diatrizoic acid; DOTA, 16


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1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium complex; DDS, Drug delivery system; MSN, Mesoporous silica nanoparticles; IMSN, Integrated mesoporous silica nanoparticles; HIMSN, Highly integrated mesoporous silica nanoparticles; GSH, Glutathione; BSO, L-buthionine sulfoxide; High mobility group box-1; IL-1β, Interleukin-1β; IL-6, Interleukin-6; IL-12P40, Interleukin-12P40; IFN-γ, Interferon-γ; TNF-α, Tumor necrosis factor-α; DC, Dendritic cell; DOX, Doxorubicin; MRS, Magnetic resonance spectrum; TNBC, Triple negative breast cancer; ACIR, Anti-cancer immune response.

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(7) Y. Lu, Q.; Hu, Y.; Lin, D.; Pacardo, B.; Wang, C.;

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