Tumor Microenvironment Activated Membrane Fusogenic Liposome

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Tumor Microenvironment Activated Membrane Fusogenic Liposome with Speedy Antibody and Doxorubicin Delivery for Synergistic Treatment of Metastatic Tumor Hongzhang Deng, Kun Song, Xuefei Zhao, Yanan Li, Fei Wang, Jianhua Zhang, Anjie Dong, and Zhihai Qin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14683 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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ACS Applied Materials & Interfaces

Tumor

Microenvironment

Activated

Membrane

Fusogenic

Liposome with Speedy Antibody and Doxorubicin Delivery for Synergistic Treatment of Metastatic Tumor Hongzhang Deng, †, §, ξ, # , ‡ Kun Song, * Anjie Dong, §, ξ and Zhihai Qin†, #, *

#, ‡

Xuefei Zhao, §, ξ Yanan Li, # Fei Wang, † Jianhua Zhang, §, ξ,

†

The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou 450052, Henan Province, China. § Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ξ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China # Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China

KEYWORDS: tumor microenvironment, fusogenic liposome, antibody, DOX, metastasis

1

ACS Paragon Plus Environment


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ABSTRACT: Metastasis is the principal event leading to breast cancer death. Discovery of novel therapeutic approaches that are specific in targeting tumor metastasis factors at the same time for effective treatment of tumor is urgently required. S100A4 protein is a key player in promoting metastasis and sequestrating the effect of tumor-suppressor protein p53. Here, a tumor microenvironment activated membrane fusogenic liposome was prepared to deliver rapidly anti-S100A4 antibody and doxorubicin into the cytoplasm directly in a fusion-dependent manner in order to bypass the cellular endocytosis to avoid the inefficient escape and degradation in acidic endosome. After intracellular S100A4 blockage with anti-S100A4 antibody, the cytoskeleton of breast cancer 4T1 cells was rearranged and cell motility was suppressed. Meantime, the anti-tumor effect of doxorubicin enormously enhanced by reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. Importantly, both local growth and metastasis of 4T1 cells were inhibited in a xenograft mouse model. Together, speedy delivery antibody and DOX into cytoplasm based on a new membrane fusogenic liposome was an innovative approach for metastatic breast cancer treatment.

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INTRODUCTION Metastatic breast cancer is the leading cause for cancer-related death of the female population worldwide.1-6 Conventional chemotherapy, a major therapeutic approach for the treatment of tumor metastasis, mainly impaired initial primary tumor cells directly without targeting tumor metastasis factors.7-11 Furthermore, the neoplastic cells within metastases are intrinsically more resistance to anticancer treatments than the cells in primary tumors2,

12, 13

. Therefore, high dosage of

chemotherapeutic drug was required to keep their treatment concentration.14 Unfortunately, some researchers reported that large dosage of anti-tumor drugs destroyed the tumor microenvironment and induced tumor resistance and subsequent tumor metastasis.6, 15-19 Therefore, discovery of novel therapeutic approaches that are specific in targeting tumor metastasis factors rather than only debulking the main tumor mass is urgently required for metastasis cancer treatment.20 S100A4, a key player protein in promoting metastasis, is involved in the motility and metastatic capacity of breast cancer cells.21-26 Antibodies with specifically bind to a large variety of molecules are functionally used to analyze the expression and function of proteins.27-29 In order to block the S100A4 to suppress the tumor metastasis, large effort had been made to use antibody to neutralize the S100A4 directly. However, treatments rarely work. The target of antibody is limited to the cell exterior because antibody can’t cross the plasma membrane by passive transport. Meantime, systemically administration antibody could result immune-related adverse events which are serious autoimmune toxicities.30 Therefore, in order to block intracellular S100A4, we must find way to deliver antibody into tumor cells. To overcome the delivery limitation, several methods have been reported, containing the use of internalization delivery mediators.31-32 However, these methods have some drawbacks, such as electroporation or microinjection decreased cell viability and damaged the 3



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cell membrane. Some alternative approaches based on nanocarriers have been developed to improve the antibody delivery efficiency. For example, kataoka et al. reported the charge-conversional polyionic complex nanocarriers for antibody delivery into cytoplasm.29 However, the antibody delivery carriers must escape from endosomes before lysosomal degradation or other intracellular trafficking steps which were still the factors ultimately limiting the transduction of antibody.11, 33 Therefore, how to design the nanocarriers with the ability of fast delivery antibody into cytoplasm bypassing the cellular endocytosis was also a challenge. Although introducing the antibody into nanocarriers with specific in targeting tumor metastasis factors, how to enhance the anti-tumor metastasis efficiency of drug was also a challenge. The ultimate goal of drug delivery is to target sites. Nevertheless, we ignore a fundamental flaw: the fate of DOX after intracellular release. The p53 protein, a regulator of the cell cycle, is sensitive to any DNA damage caused during replication and induce apoptosis to prevent the production of defective. The increased DNA damage by drug will lead to p53 mediated cell death. However, the loss of p53 function was always appeared in metastasis tumor resulting the resistance of DNA damage.34 Although most investigations used the gene mediated method to increase the expression of p53 protein, the resistance of malignant cells to doxorubicin was not completely reversed. We suspected that the expression of p53 protein was inceased after gene expression, but the function of p53 was also limited. The interaction of S100A4 with p53 results in inhibition of p53 phosphorylation by PKC. Moreover, binding of S100A4 to p53 inhibited the complex formation between p53. The transcriptional activation capacity of p53 (induction and repression) could be altered by such mofification. The S100A4 protein was involved in the regulation of gain of function mutant p53 molecules.35 Therefore, synergistic interdiction the S100A4 by neutralization antibody with 4


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chemotherapeutics was a novel method with specific in targeting tumor metastasis factors and enhanced chemotherapy effect for metastasis cancer treatment. Here,

a

new

type

of

1,2-distearoyl-sn-glycero-

membrane

fusogenic

liposome

(MFLp)

3-phosphoethanolamine-N-Arg-Arg-Arg-Arg

was

prepared

from

(DSPE-4A)

and

3-phosphoethanolamine-N- benzaldhyde-[methoxy(polyethyleneglycol)-2000] (DSPE-Hy-PEG2k) with pH sensitively deshelling PEG. In tumor acidic microenvironment, the PEG chains detached from the surfaces of the MFLp and four arginines with superlative cell penetrating efficiency exposed, which facilitated the MFLp fusing with tumor cells membrane bypassing the cellular endocytosis and quickly released the package into cytoplasm. Therefore, MFLp loaded anti-S100A4 antibody and DOX were synergistic treatment of metastasis tumor with specific in targeting tumor metastasis factors and enhanced chemotherapy effect for metastasis cancer treatment as shown in Figure 1. In vitro real-time visualization of internalization indicated that only 30 s the membrane fusogenic liposome could delivery package into cytoplasm. The cell invasion and wound healing assays indicated that targeted neutralization S100A4 by antibody significantly suppressed the tumor metastasis. Furthermore, in vitro and in vivo antitumor studies demonstrated the synergistic effect of inhibition tumor growth by reversing the effect of S100A4 on the sequestration of tumorous suppressor protein p53. Together, speedy delivery antibody and DOX into cytoplasm based on a new membrane fusogenic liposome was an innovative approach for metastatic breast cancer treatment. RESULTS AND DISCUSSION Preparation of MFLp and drug loaded MFLp. In this work, DSPE-4A was synthesized according our previously reported literature.14 DSPE-Hy-PEG2k with pH sensitive linker was prepared in our lab.15 The detail structure characterization of DSPE-Hy-PEG2K was shown in supporting information 5



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(Figure S1). Furthermore the pH sensitive property have been studied in our previously report.37 MFLp loaded anti-S100A4 antibody (S-mAb) and/or DOX were prepared by a double emulsion-solvent evaporation technique, as previously described.36, 38, 39 As shown in Figure 2a and 2b, the morphology of the drug-free MFLp and DOX and/or S-mAb loaded MFLp was examined using TEM and the laser particles size analyzer measurements. To test the loading capacity of anti-S100A4 antibody (S-mAb), S-mAb loaded MFLp was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions (native PAGE) to allow the electrophoretic migration of unbound proteins from the formulations. As shown in Figure 2c, we observed that the S-mAb had been encapsulated into the MFLp. The ability of BSA adsorption on MFLp was investigated as shown in Figure 2d indicating that there is no obvious adsorption of BSA. Therefore, this MFLp had good stability in blood circulation due to the protection of PEG. Furthermore, FRET pair (DiO, donor and DiI, acceptor) was loaded in MFLp to study the stability of MFLp.40-42 After incubation with 10% fetal bovine serum (FBS) in phosphate buffer saline (PBS) for 48 h, the fluorescence intensity remained constant (Figure 2e), demonstrating the integrity of MFLp structure. After MFLp liposome decomposition by methanol, FRET disappeared because DiI and DiO could not been closely retained anymore. The S-mAb encapsulation efficiency was further studied using a fluorescence quenching menthod. Gold nanoparticles (AuNPs) are known to quench the FITC fluorescence without diffusing through a MFLp layer due to large size. The fluorescence of free FITC was gradually quenched with addition of AuNPs (Figure S2). However, adding AuNPs to the FITC-S-mAb loaded MFLp solution, the fluorescence only slightly reduced as shown in Figure 2f. These results indicated that the FITC-S-mAb was encapsulated into MFLp layer with the nanoassembly structure. In the meantime, as shown in Figure 2g, DOX was also encapsulated into 6


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MFLp. The characteristic of the DOX-loaded liposome were summarized in Table S1. The in vitro release of DOX and S-mAb from MFLp was investigated at 37 oC under the condition of pH 7.4, and 6.0, respectively (Figure S3). Furthermore, the expression levels of the S100A4 in different cells was evaluated by western blot analysis as shown in Figure S4. The membrane fusogenic property of MFLp. The membrane fusogenic property of liposome containing DSPE-4A had been evaluated in our previous study.36 However, the speed of package release into the cytoplasm was no extraordinary enhanced. It may be due to the restriction of PEG. In order to obtain speedy membrane fusogenic property and quick release, the combination shelling PEG and exposure four arginines was used to facilitate the liposome. A membrane fusogenic liposome was prepared from 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-Arg-Arg-Arg-Arg (DSPE-4A)

and

3-phosphoethanolamine-N-benzaldehyde-[methoxy(polyethyleneglycol)-2000]

(DSPE-Hy-PEG2k). The detail process contained three steps: tumor acidic microenvironment actiavated PEG chains were detached from the surfaces of the liposome, four arginines were exposed with superlative cell penetrating efficiency after exposure and then the liposome fused with tumor cells membrane bypassing the cellular endocytosis and quickly released the antibody and DOX into cytoplasm. FITC labeled MFLp was used to study the interaction between MFLp and 4T1 cell. As shown in Figure 3A, the observation of cells incubated with FITC labeled MFLp over 1 h revealed that fluorescence on the cell membrane was much bright and no fluorescence was observed in the lysosome and cytoplasm. FITC labeled PEG-PCL micelles and conventional liposomes, which was uptake into tumor cells based on the endocytosis, used as a control as shown in Figure S5. Taken together, these results indicate that MFLp can fused with the cell membrane. A statistic of co-location ratio of FITC with lysosome was carried out by counting pixels of yellow (merged signals of green 7



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with red) and red (signals presented lyso tracker) in each picture and was calculated as co-location ration (%) = (Counts yellow/(Counts red))×100%. As shown in Fgure S6, the co-location ratio in the cell incubated with MFLp was greatly lower than PEG-PCL micelles, indicating that the cellular endocytosis of MFLp was different from the uptake of PEG-PCL micelles. As shown in Figure S7, the result also could be found in MDA-MB-231 cells. Furthermore, the free FITC was incubated with 4T1 for 1 h as shown in Figure S8. The CLSM images indicated that the free FITC was also merged with the lysosome rather than in the membrane. The speedy cellular uptake and intracellular release of DOX. The speedy intracellular release profiles of DOX loaded MFLp were investigated in 4T1 cells using confocal laser scanning microscope (CLSM). Here, the red fluorescence signal from the DOX was used for the fluorescence imaging of cells. After 5 min incubation, DOX fluorescence appeared in 4T1 cells for DOX loaded MFLp (Figure 3b). However, compared with the free DOX, there was little fluorescence in 4T1 cells as shown in Figure S9. Meantime, with the 12 h incubation, the fluorescence of FMLp-FITC was almost on the membrane of 4T1 cells. Futhermore, real-time visualization of spatiotemporal DOX release indicated that the process of speedy internalization of DOX about 30 s after 4T1 cells incubated with FMLp/DOX as shown in Figure S10 and supporting information movie 1. Compared with the real-time visualization of free DOX into cells (Figure S11 and supporting information movie 2), the speed of DOX loaded FMLp was markedly quickly. Importantly, the cell uptake of S-mAb was studied by immunofluorescence. As shown in Figure 4, the MFLp/S-mAb and MFLp/DOX+S-mAb showed stronger signals than the naked S-mAb group, indicating that the S-mAb can be delivery into tumor cell through MFLp liposome. Semi-quantitative intensity of 4T1 cells were also evaluated to study the intracellular delivery antibody successful as 8


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shown in Figure S12 and S13. The effect of cell motility and cytoskeletal arrangement by targeting incellular S100A4. S100A4 has been shown to be able to regulate cell shape and motility by binding to cytoskeletal proteins resulting cancer cells migration.24 Membrane fluidity is a major requirement for cell motility to across the vascular barrier into the vascular system.43 Firstly, we evaluated the expression levels of S100A4 after 4T1 incubated with MFLp/mAb through western blot. As shown in Figure S14, after 4T1 cells incubated with S-mAb and MFLp/S-mAb, the expression levels of S100A4 in the MFLp/S-mAb group is significantly decreased than the free S-mAb group. This result indicated that the intracellular delivery S-mAb could efficiently neutralize S100A4. However, there was almost no influence about the expression of S100A4 in the free S-mAb group because free antibody can’t cross the plasma membrane by passive transport. Combined with result of cellular uptake and intracellular release in Figure 3, it confirmed that the S100A4 antibody could be delivered into cell cytoplasm and neutralize the intracellular S100A4. We proceeded to test some cytoskeletal proteins markers with western blot and found decreased expression of S100A4 after targeting intracellular S100A4 with MFLp/S-mAb correlated with repressed expression of E-cadherin and ZO-1 as shown in Figure 5a. In the membrane, a significant metastasis suppressor protein is the E-cadherin.44,

45

Cancer

progression often paralleled by the inactivation of E-cadherin by methylation of its promoter. The expression of cytoskeletal proteins of 4T1 cells incubated with MFLp/S-mAb was greatly increased compared with other groups, indicating that targeted intracellular S100A4 by anti-S100A4 neutralizing monoclonal antibody could suppress cell motility and cytoskeletal rearrangement. Some results above were further confirmed by confocal microscopy examination in 4T1 cells as shown in Figure 5b. However, the expression of E-cadherin proteins of 4T1 cells incubated with DOX and 9



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MFLp/DOX was slightly lower compared with the control group as shown in Figure 5c. This result indicated that the cell motility and cell motility could not been inhibited by DOX. Therefore, it’s necessary to discovery novel therapeutic approaches that are more specific in targeting tumor metastasis factors rather than only debulking the main tumor mass. As shown in Figure 5d, the semiquantitative fluorescence intensity of E-cadherin incubated with MFLp/S-mAb was higher than orther groups indicating that the MFLp can delivery S-mAb into the 4T1 and neutralize intracellular S100A4 to suppress cell motility. These results were confirmed by facial action coding system (FACS) in 4T1 cells as shown in Figure 5e. Meanwhile, cell adhesion proteins ZO-1 were significantly increased after 4T1 cells were incubated with MFLp/S-mAb indicating that neutralizing intracellular S100A4 upregulated levels of epithelial markers as shown in Figure 5f and 5g. The effect of anti-S100A4 antibody on cell migration was assessed by wound healing assay. 4T1 cells had significantly speedyer closure of the wound area compared with cells incubated with MFLp/S-mAb (Figure S15). Moreover, 4T1 cells incubated with S-mAb and MFLp showed a greater degree of invasion compared with cells incubated with MFLp/S-mAb assessed by transwell assay as shown in Figure 5h and 5i and Figure S16. 4T1 cells incubated with MFLp/S-mAb showed a decreased degree of invasion through Matrigel as shown in Figure 5j and 5k. These results shown that targeted intracellular S100A4 by MFLp/S-mAb inhibited the cancer cells invasiveness. In vitro anticancer efficacy. The cytotoxicity of free DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb and MFLp/DOX+S-mAb was investigated in 4T1 cells by MTT assay as shown in Figure 6a. The cytotoxicity of S-mAb antibody and/or DOX-loaded MFLp was studied in 4T1 cells. It could be noted that MFLp/DOX+S-mAb had lower IC50 (half inhibitory concentration) values than DOX-loaded MFLp (4T1: 2.79 µg/mL for MFLp/DOX+S-mAb, 4.94 µg/mL for DOX-loaded 10


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MFLp and 2.49 µg/mL for DOX) as shown in Figure 6b. The higher cytotoxicity of MFLp/DOX+S-mAb was attributed to the anti-S100A4 neutralizing monoclonal antibody reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. As shown in Figure S17, the same result also could be found in MDA-MB-231 cells. As shown in Figure S18, the combination index (CI) values were < 1 in the effect range of the drugs (fractional effect: 0.3 - 0.8) indicating the synergistic effect of FMLp/DOX and FMLp/S-mAb. As shown in Figure 6c, the illustration of targeting intracellular S100A4 by anti-S100A4 antibody to reverse the effect of S100A4 on the sequestration of tumor suppressor. The levels of p53 in 4T1 cells incubated with DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb and MFLp/DOX+S-mAb were investigated as shown in Figure 6d. Compared with 4T1 cells incubated with DOX, S-mAb, MFLp/DOX groups, the p53 expression in cells incubated with MFLp/S-mAb and MFLp/DOX+S-mAb increased, indicating that the suppressed p53 could be reversed by S-mAb targeted intracellular S100A4. The initiating the cell apoptosis program attributed to the executioner protease caspase 3. Caspase 3 is activated into cleavage of procaspase 3 during apoptosis.46-48 As shown in Figure 6d, the expression of cleaved caspase 3 and caspase 9 protein in cells treated with the MFLp/DOX+S-mAb were obviously elevated compared with the control groups. The result indicated the reduction of S100A4 protein level by intracellular delivery of anti-S100A4 antibody accompanied with the activation of caspase 3 resulting in apoptosis of 4T1 cells. The effect of free DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb and MFLp/DOX+S-mAb on apoptosis was studied by PI and Annexin V-FITC double staining. As shown in Figure 6e, 4T1 cells exposed to blank S-mAb and MFLp/S-mAb shown little apoptosis after 48 h 11



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incubation. In the DOX group, there were only 24.4% apoptotic. The percentage of early and late apoptosis of MFLp/DOX treated cells was 2.8% and 13.2%, respectively. However, MFLp/DOX+S-mAb induced more apoptosis about 32%. In this work, the MFLp/DOX+S-mAb group displayed a high apoptotic rate in 4T1 cells which is significantly higher than all control groups. These results indicated that the inhibition effect could be reversed by neutralizing the S100A4. Therefore, speedy delivery anti-S100A4 antibody and chemotherapy drug based on membrane fusogenic liposome was developed as an innovative approach for breast cancer treatment through targeting intracellular S100A4 by anti-S100A4 neutralizing monoclonal antibody to reverse the effect of S100A4 on the sequestration of tumor suppressor. In vivo anticancer and anti-metastasis. The ability of MFLp/DOX+S-mAb to inhibit the growth of the primary tumor was studied in 4T1 tumor-bearing Balb-c mice. Balb-c mice were injected with 150 µL of 4T1 cell suspension (1×106 cells) on the armpit. The mice were randomly separated into groups ( n = 10) one week later, and in vivo injected with PBS, free DOX, MFLp/DOX, MFLp/S-mAb and MFLp/DOX+S-mAb at an identical DOX dose of 1 mg/kg and S-mAb dose of 1 mg/kg as shown in Figure 7. Meantime, in order to simulate malignant tumor metastasia, we also injected with PBS, free DOX, MFLp/DOX, MFLp/S-mAb and MFLp/DOX+S-mAb after three weeks later as shown in Figure 8. PBS treatment did not have any substantial effect on the tumor growth as shown in Figure 7a. However, DOX had a very weak antitumor efficacy with about 40.88% reduction of the tumor growth rate compared to PBS group. MFLp/DOX group shown antitumor ability with 51.95% reduction compared with that PBS group. As shown in Figure S19, free S-mAb treatment in vein and enterocoelia did not have effect on the tumor growth. However, MFLp/S-mAb groups exhibited slightly reduction of the tumor growth rate which was in agreement 12


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with the result of anticancer efficacy as shown in Figure 6. Notably, the tumor volume treated with MFLp/DOX+S-mAb significantly decreased with 88.57% reduction compared with that PBS group. This result suggested that targeting intracellular S100A4 by anti-S100A4 neutralizing monoclonal antibody was a novel method to enhance the anti-cancer effect of DOX by reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. The body weight of mice was measured to evaluate the cytotoxicity of MFLp. As shown in Figure 7b, the MFLp/DOX+S-mAb treatment resulted in almost no difference in the physical activity level and body weight after 20 days. As shown in Figure 7c, the photographs of tumor, liver, lung and spleen from each group were excised on day 21. The spleen of mice injected with PBS, DOX, and MFLp/DOX appeared to have a swelling phenomenon indicating that the metastatic tumor cells had invaded the spleen. However, the groups treated with MFLp/S-mAb and MFLp/DOX+S-mAb shown no spleen swelling. As shown in Figure 7d, the expression of cleaved caspase-3 and caspase 9 protein in cells treated with the MFLp/DOX+S-mAb was obviously elevated which agreed with the result in Figure 5f, indicating that reduction of S100A4 protein level by intracellular delivery of anti-S100A4 antibody was accompanied with the activation of caspase 3 resulting apoptosis of 4T1 cells. The 4T1 tumor model is an established liver metastasis breast tumor model. As shown in Figure 7e and 7g, the PBS group showed an extensive tumor burden in liver DOX and MFLp/DOX groups exhibited similar tumor burden compared with the saline group, indicating weak anti-metastasis capacity. As shown in Figure 7f and 7h, the H&E of representative liver staining pictures indicated the inhibition liver metastasis of FMLp/S-mAb and FMLp/S-mAb+DOX. As shown in Figure S20, the representative liver staining pictures injected free S-mAb in vein and enterocoelia shown large tumor burden. The metastasis of MFLp/S-mAb and MFLp/DOX+S-mAb groups were significant 13



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less than that of the PBS group indicating that intracellular targeting delivery anti-S100A4 antibody into cell to neutralize S100A4 was effective way to suppress the tumor metastasis. As shown in Figure S21, the expression of cytoskeletal proteins E-cadherin and ZO-1 of the groups injected with MFLp/S-mAb and MFLp/DOX+S-mAb was greatly increased compared with other groups, indicating that targeted intracellular S100A4 by anti-S100A4 neutralizing monoclonal antibody could suppress cell motility and cytoskeletal rearrangement. Angiogenesis also enhanced the tumor metastasis.51, 52 The MMP-9 and vascular endothelial growth factor (VEGF) played important role in angiogenesis. The expression of VEGF and MMP-9 protein in tumor tissues were evaluated. As shown in Figure S20, the expression of VEGF and MMP-9 protein treated with the MFLp/DOX+S-mAb was obviously decreased compared to the control groups indicating that targeted intracellular S100A4 by anti-S100A4 neutralizing monoclonal antibody could suppress tumor angiogenesis. Furthermore, the tumor vessel stained by the mouse enhothelia cell marker CD31. As shown in Figure S21, the vessel density in the MFLp/DOX+S-mAb group was lower than control groups. To evaluate tissue distributions, fluorescence labeled S-mAb was used, free S-mAb and/or DOX and DOX and/or S-mAb loaded MFLp at the same DOX dose (1 mg/kg) and S-mAb dose (1 mg/kg) were intravenously injected into 4T1 tumor-bearing mice. The time dependent tumor accumulation and tissue distribution were observed via fluorescence imaging of DOX and S-mAb as shown in Figure 7i. After injection, the major organs (heart, liver, spleen, lung, and kidney,) and tumor were isolated for observation of ex vivo images and the fluorescence intensity was quantitatively analyzed. Furthermore, the same result also observed in the malignant tumor metastasia model as shown in Figure 8.

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CONCLUSION In summary, a tumor microenvironment activated membrane fusogenic liposome was prepared to speedy deliver anti-S100A4 antibody and DOX into the cytoplasm directly in order to bypass the cellular endocytosis to avoid the inefficient escape and degradation in acidic endosome. The real-time visualization of spatiotemporal DOX release indicated that the process of speedy internalization of DOX after 4T1 cells incubated with FMLp/DOX. After intracellular S100A4 blockage with anti-S100A4 antibody, the cytoskeleton of breast cancer 4T1 cells was rearranged and cell motility was suppressed. Meantime, the anti-tumor effect of doxorubicin was enormously enhanced by reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. Speedy delivery antibody and doxorubicin into cytoplasm based on a new membrane fusogenic liposome was an innovative approach for breast cancer treatment through targeting intracellular S100A4 to suppress primary tumor metastasis and enhance the inhibition of cancer cell proliferation by reversing the effect of S100A4 on the sequestration of tumor-suppressor protein p53. MATERIALS AND METHODS Materials. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were purchased from Avanti Polar Liposome (Alabaster, AL). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-Hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Milwaukee, USA). Doxorubicin hydrochloride (DOX·HCl) was purchased from Wuhan Hezhong Biochemical in manufacturing co.,Ltd (China, Wuhan). Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM and bovine serum (FBS) were purchased from Invitrogen Corporation (Carlsbad, CA). Preparation of Methoxy Poly(ethylene glycol) Benzaldhyde mPEG (1 g, 0.5 mmol) in 15



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dichloromethane was added p-formylbenzoic acid (10 equiv) and DCC with DMAP. After 24 h, the mixture was filtered and concentrated. Then the compound was dissolved in isopropanol (10 mL). The resulting product (PEG-CHO) collected by filtration and washed with diethyl ether. Preparation of 3-phosphoethanolamine-N- benzaldhyde-[methoxy(polyethyleneglycol)-2000] (DSPE-Hy-PEG2k) PEG-CHO and DSPE were added into dichloromethane. The stirred mixture was heated at 40 °C for 6 h. The resulting product was dialyzed against water for 24 h and then freeze dried to obtain DSPE-Hy-PEG. Preparation and characterization of liposome carrying anti-S100A4 antibody and/or DOX liposome Liposome loaded with anti-S100A4 antibody and/or DOX were prepared based on a double emulsion-solvent evaporation method. Briefly, an aqueous solution of anti-S100A4 antibody (0.5 mg) in 15 µL of water was emulsified by sonication for 60 s in 0.2 mL of chloroform containing 0.5 mg of cholesterol over an ice bath, 15 mg of DOPC and 0.5 mg DSPE-Hy-PEG. This primary emulsion was further emulsified in 3 mL of water by sonication (70 W for 60 s) over an ice bath. The mixture solvent was concentrated under reduced pressure by a rotary evaporator to a volume of 1 mL. Preparation FITC labeled MFLp An aqueous solution in 15 µL of water was emulsified by sonication for 60 s in 0.2 mL of chloroform containing 0.5 mg of cholesterol over an ice bath, 15 mg of DOPC, 0.5 mg DSPE-Hy-PEG, and 0.1 mg FITC labled DSPE-PEG. This primary emulsion was further emulsified in 3 mL of water by sonication (70 W for 60 s) over an ice bath. The mixture solvent was concentrated under reduced pressure by a rotary evaporator to a volume of 1 mL. Bovine serum albumin (BSA) adsorption FMLp (1 mg/mL) was incubated with BSA (1.5 mg/mL) for 1, 2, 3, 4, 5 h at 37 oC. The mixtures were electrophoresed using Native-polyacrylamide gel 16


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electrophoresis. Then gels were stained in 0.1% (w/v) R250 Coomassie Brilliant Blue and destained with a composite solution of 10% (v/v) ethanol and 10% (v/v) acetic acid. In vitro release of DOX or anti S100A4 antibody. In vitro DOX release profiles from liposomes were investigated in different media, i.e. phosphate buffered solution (PBS) (10 mM, pH 7.4), and PBS (10 mM, pH 6.0). 5 mL DOX-loaded MFLp (1 mg/mL) were sealed in a dialysis tube (Mn = 3500 Da) and incubated in 20 mL release media. The cumulative drug release percentage was calculated by the following equation: n −1

Er（%）=

Ve ∑ Ci + V 0Cn 1

m

× 100%------- [3]

DOX

Where mDOX represents the amount of DOX in the MFLp, V0 is the whole volume of the release media (V0 = 25 mL), Ve is the volume of the replaced media (Ve = 5 mL), and Cn represents the concentration of DOX in the sample. Anti S100A4 antibody profiles from liposomes were measured as in the above method. The content of antibody was detected by enzyme-linked immunosorbent assay (ELISA). Cell Viability. 4T1 or MDA-MB-231 cells were seeded in 96 well plates at a density of 4000 cells  L complete DMEM medium. Flowing incubated 12 h, DOX, MFLp/DOX and per well in 100

MFLp/DOX+S-mAb (S-mAb 1 µg/mL) with different concentration was added in to each well with 6 parallel samples. After 24 h incubation, 20 µL MTT solution (5 mg/mL) was added into each well. Then after 4 h, 100 µL of DMSO was added to dissolve the formazan crytals. The OD of solution was measured at 590 nm. Determination of the Synergistic Effect of DOX and S-mAb 4T1 or MDA-MB-231 cells were seeded at 4000 cells/well of a 96 well plate. Concentration ranges were chosen to span the complete 17



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dose-response range of DOX and S-mAb. Cell proliferation/viability was determined after 24 h by using the Luminescent Cell Viability Assay (Promega). Combination index (CI) was performed by using CalcuSyn software (Biosoft, Cambridge, United Kingdom).50, 51 Cell uptake studies. The 4T1 cells were seeded in 35 mm microscopy dishes at a density of 1×105 in 2 mL and cultured at 37 oC in a 5% CO2 atmosphere for 12 h. The cells were incubated with FITC labeled FMLp or FITC labeled PEG-PCL micelles in complete DMEM (pH 6.8) at 37 oC for additional 1 h. Finally, the lysosome was stained by Lyso Tracker Deep Red for 20 min. Then the cells was imaged by confocal laser scanning microscopy. Real-Time Evaluation of Delivery The 4T1 cells were seeded in 35 mm microscopy dishes at a density of 1×105 in 2 mL and cultured at 37 oC in a 5% CO2 atmosphere for 12 h. The 4T1 cells was wash by PBS. DOX loaded FITC-FMLp with 10 ug/ml DOX in complete DMEM (pH 6.8) was added. The microscopy dish was immediately fixed onto the observation platform of the CLSM instrument. The fluorescence variations were captured once every 30 s. Immunofluorescent staining 4T1 Cells were seeded in 6 well plates at 2 × 105 cells/well. S-mAb, FMLp, FMLp with S-mAb and FMLp/S-mAb (S-mAb: 1 µg/mL) was added and incubated with 4T1 cells. After 12 h, 4T1 cells were fixed in 4% formaldehyde. 4T1 cells were permeabilized using 0.1% Triton-X for 50 min. Cells were washed and incubated with primary antibody (1:100) at 37 oC for 1 h. After washing with PBS, 4T1 cells were incubated with secondary Alexa Fluor 488 antibody (1:100) at 37 oC for 1 h. Samples were imaged using a confocal fluorescence microscope (Olympus FV1000). Flow cytometry 4T1 cells were seeded in 6 well plates at 2 × 105 cells/well. S-mAb, FMLp, FMLp with S-mAb and FMLp/S-mAb (S-mAb: 1 µg/mL) in complete DMEM was added. After 12 h, 4T1 18


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cells were harvested using Trypsin-EDTA. Then 4T1 cells were fixed in 4% formaldehyde and permeabilized using 0.1% Triton-X for 50 min. 4T1 cells were washed and incubated with primary antibody (1:100) at 37 oC for 1 h. After washing with PBS, 4T1 cells were incubated with secondary Alexa Fluor 488 antibody (1:100) at 37 oC for 1 h. Stained cells were analysed on a FACSCalibur cytometer (BD Biosciences). Transwell migration and invasion assay Cells migration assays were done using transwell chambers (8 µm pores, 6.5 mm diameter). 4T1 cells were seeded on the upper chamber. S-mAb, FMLp, FMLp with S-mAb and FMLp/S-mAb (S-mAb: 1 µg) were added and incubated with cells for 12 h, the cells migrated to the lower side were counted and stained with crystal violet staining solution. The intensity of violet staining was measured at 560 nm. For quantification purpose, 50% acid was added to dissolve crystal violet. Furthermore, a Matrigel-coated chamber was used to study the invasive potential of the cells as the similar method above. Cell apoptosis detection 4T1 Cells were seeded in 6 well plates at 2 × 105 cells/well. FMLp/DOX+S-mAb (DOX: 5µg/mL, S-mAb: 1 µg/mL) was added and incubated with cells. After the treatment, the 4T1 cells were cultured at 37 oC for 24 h. Then 4T1 cells were collected and stained with Annexin V-FITC/PI. Each sample was analyzed by a flow cytometry. Western blotting 4T1 Cells were seeded in 6 well plates at 2 × 105 cells/well and lysed using RIPA lysis buffer supplemented with complete protease inhibitor cocktail tablets. Twenty micrograms of proteins were separated by SDS-polyacrylamide gel electrophoresis. Then the protein were transferred onto polyvinylidene difluoride membranes. Then the membranes were blocked by 1.5% BSA for 1 h, incubated with the primary antibody overnight. Antimouse or antirabbit antibodies conjugated with horseradish peroxidase were used as secondary antibody. Membranes were 19



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visualized with a chemiluminescent substrate (SuperSignal, Pierce Chemical Co.). In vivo tumor inhibition study. The anti-tumor activity was conducted using 4T1 tumor-bearing Balb-c mice. When the tumor volume reached about 100 mm3, the mice were randomly divided into four groups (n = 10 per group) and treated with PBS, free DOX, FMLp/DOX, FMLp/S-mAb, and FMLp/DOX+S-mAb (DOX: 1 mg/kg, S-mAb: 1mg/kg). At designated times, tumor growth were measured using a caliper and calculated according to the formula (length × width2) × 1/2. The body weight of mice in each group were also monitored. All the animal experiments were performed in accordance with the protocol approved by Chinese Academy of Medical Science and Peking Union Medical College, and adhered to the Guiding Principles in the Care and Use of Animals of the American Physiological Society. Ex vivo distribution of DOX and antibody. Free DOX, PE labeled antibody and DOX and/or antibody laoded FMLp were injected to 4T1 tumor-bearing mice, respectively (DOX 1 mg/kg, PE-antibody 1 mg/kg). After treatment 12 h, the organs were imaged by IVIS Spectrum. Histomorphological analysis Tumor, heart, liver, spleen, lung and kidney were harvested and fixed in 4 % paraformaldehyde. After 24 h, tumor, heart, liver, spleen, lung and kidney were trimmed, embedded in paraffin, and cut into 8 µm thick sections for hematoxylin and eosin (H&E). The photos were observed by optical microscope. Statistical analysis All data are reported as mean ± standard deviation (SD) from at least three independent experiments. Significant differences were evaluated by Student’s unpaired t-test. Places needing multiple comparisons were evaluated by one-way ANOVA. ASSOCIATED CONTENT Supporting Information 20


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Synthesis routes of mPEG2k-CHO and DSPE-Hy-PEG2k. The

1

H-NMR of PEG-CHO,

DSPE-Hy-PEG, and DSPE-FITC. FITC fluorescence quenching of MFLp. In vitro release of DOX and S-mAb. Characteristics of FMLp/DOX+S-mAb. CLSM images of PEG-PCL and Lipososme. Western blot. CLSM images of MDA-MB-231 cells. The real-time visualization. Migration and invasion. Cytotoxicity. Combination index. The relative tumor volumes treated with free S-mAb in vein and enterocoelia. The representative liver staining pictures from mice injected free S-mAb. CLSM images tumors stained for CD31. AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

*

E-mail: [email protected]

ORCID Hongzhang Deng: 0000-0003-3637-8905 Jianhua Zhang: 0000-0001-7833-9715 Author Contributions ‡

H.D. and K.S. contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS

This research was financially supported by the National Natural Science Foundation of China (81630068, 31670881, 31470963 and 81371667), the Ministry of Science and Technology of China (2016YF1302305), and the National Major Scientific Instruments Development Project (91229203). 21



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Cancer Cells. Oncogene 2009, 28, 2940-2947. 46. Yen, Y.-P.; Tsai, K.-S.; Chen, Y.-W.; Huang, C.-F.; Yang, R.-S.; Liu, S.-H. Arsenic Induces Apoptosis in Myoblasts Through A Reactive Oxygen Species-induced Endoplasmic Reticulum Stress and Mitochondrial Dysfunction Pathway. Arch. Toxicol. 2012, 86, 923-933. 47. Shi, Y.; Yi, C.; Zhang, Z.; Zhang, H.; Li, M.; Yang, M.; Jiang, Q. Peptide-Bridged Assembly of Hybrid Nanomaterial and Its Application for Caspase-3 Detection. ACS Appl. Mater. Inter. 2013, 5, 6494-6501 48. Zhang, Y.; Hou, Z.; Ge, Y.; Deng, K.; Liu, B.; Li, X.; Li, Q.; Cheng, Z.; Ma, P. a.; Li, C.; Lin, J. DNA-Hybrid-Gated Photothermal Mesoporous Silica Nanoparticles for NIR-Responsive and Aptamer-Targeted Drug Delivery. ACS Appl. Mater. Inter. 2015, 7, 20696-20706. 49 Jia, W.; Gao, X.; Zhang, Z.; Yang, Z.; Zhang, G.; S100A4 silencing suppresses proliferation, angiogenesis and invasion of thyroid cancer cells through downregulation of MMP-9 and VEGF, Eur. Rev. Med. Pharmacol. Sci. 2013, 17, 1495-1508. 50 Moriyama-Kita, M.; Endo, Y.; Yonemura, Y.; Heizmann, C. W.; Miyamori, H.; Sato, H.; Yamamoto, E.; Sasaki, T., S100A4 regulates E-cadherin expression in oral squamous cell carcinoma. Cancer Lett. 2005, 230, 211-218. 51. Chou, T.; Talalay, P. Quantitative Analysis of Dose-effect Relationships: the Combined Effects of Multiple Drugs or Enzyme Inhibitors, Adv. Enzyme Regul. 1984, 22, 27-55. 52 Junttila, T.; Akita. R.; Parsons, K.; Fields, C.; Phillips, G.; Friedman, L.; Sampath, D.; Sliwkowski, M. Ligand-Independent HER2/HER3/PI3K Complex Is Disrupted by Trastuzumab and Is Effectively Inhibited by the PI3K Inhibitor GDC-094, Cancer Cell 2009, 15, 429-440.

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Figure 1. The illustration of membrane fusogenic FMLp speedy delivery antibody and DOX to suppress the tumor metastasis and enhance the anticancer efficiency of DOX.

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Figure 2. (a) The structure of MFLp loaded with S-mAb and/or DOX were characterized by TEM. The scale bare is 200 nm. (b) Size distribution were characterized by laser particle size analyzer. (c) The loading capacity of anti-S100A4 antibody loaded MFLp was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions (native PAGE). (d) The ability of BSA adsorption on MFLp was investigated by native PAGE, FMLp+BSA-1, FMLp+BSA-2, FMLp+BSA-3, FMLp+BSA-4, FMLp+BSA-5 represented FMLp incubated with BSA for 1, 2, 3, 4, 5 h at 37 oC. (e) The stability of the designed MFLp was studied by FRET (DiO, donor and DiI, acceptor). (f and g) S-mAb and DOX encapsulation efficiency was probed using a fluorescence quenching approach.

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Figure 3. (a) CLSM images of 4T1 cells cultured with FITC labled MFLp for 1 h. Lyso Tracker red dyed lysosomes are shown in red. Scale bars are 10 µm. (b) CLSM images of 4T1 cells incubated with MFLp-FITC/DOX (DOX 10 µg/mL) at different incubation time (scale bar, 25 µm).

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Figure 4. Representative CLSM images of 4T1 cells incubated with free DOX, DOX with S-mAb, DOX loaded MFLp, S-mAb loaded MFLp and DOX and S-mAb co-loaded MFLp (DOX 10 µg/mL) for 12 h (scale bar, 10 µm). The S-mAb was labelled by immunofluorescence; and the cell nuclei were stained with DAPI (blue).

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Figure 5. After 4T1 incubated with S-mAb, FMLp, FMLp+S-mAb, and FMLp/S-mAb, protein levels of E-cadherin and ZO-1 were detected by (a) western blot and (b) immunofluorescence staining. Scale bars are 20 µm. (c) After 4T1 incubated with DOX, FMLp/DOX, and FMLp/DOX+S-mAb, protein levels of E-cadherin and ZO-1 were detected by western blot. (d) Semi-quantitative E-cadherin intensity of 4T1 tumor cells. (e) 4T1 cells expressing E-cadherin were analysed by FACS. (f) Semi-quantitative ZO-1 intensity of 4T1 tumor cells. (g) 4T1 cells expressing ZO-1 were analysed by FACS. (h and i) Transwell migration and Matrigel invasion assays, quantification of migrated cells through the membrane and invaded cells through Matrigel of each cell line are shown as proportions of their vector controls. The intensity of violet staining was measured as absorbance at 560 nm, n=3. (j and k) For quantification purposes, 50% acetic acid was added to each well to dissolve crystal violet. The stain intensities were measured as absorbance under 560 nm. Data were expressed as mean ± SD (Standard deviation) from at least 3 independent experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 33



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Figure 6. (a) Cytotoxicity of free DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb, and MFLp/DOX+S-mAb in 4T1 cells and (b) IC50 (half inhibitory concentration) values. (c) The illustration of targeting intracellular S100A4 by antibody to reverse the effect of S100A4 on the sequestration of tumor suppressor. (d) Western blot analysis of expression levels of the p53 and caspase family members. (e) The effect on apoptosis was investigated by Annexin V-FITC and PI double staining and analyzed via flow cytometry. Data were expressed as mean ± SD (Standard deviation) from at least 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Figure 7. (a) The relative tumor volumes in tumor xenograft models treated with free DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb, and MFLp/DOX+S-mAb (DOX 1 mg/kg). (b) Body weight changes of mice bearing 4T1 metastatic tumor after treatment. (c) The photographs of tumors, liver, lung and spleen from each group excised on day 21. (d) Western blot analysis of expression levels of the p53 and caspase family members. (e) Lung were isolated and metastatic nodules in lung surface were macroscopically quantified, red arrow indicates the metastatic nodule on the lung. (f) 35



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H&E and representative lung staining pictures from mice, red arrow indicates the metastatic nodule on the lung. Scale bars are 500 µm. (g) Nodules rich in densely packed cells, as indicated by the red arrows, were quantified as tumor nodules. (h) The area of tumor nodules in H&E (%) was calculated as quantitative analysis of the pulmonary metastatic nodules. (i) Ex vivo fluorescence images of tissue distribution. Data were expressed as mean ± SD (Standard deviation) from at least 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8. (a) The relative tumor volumes in tumor xenograft metastasis models treated with free DOX, S-mAb, DOX with S-mAb, MFLp/DOX, MFLp/S-mAb, and MFLp/DOX+S-mAb (DOX 1 mg/kg). (b) Body weight changes of mice bearing 4T1 metastatic tumor after treatment. (c) The 36


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photographs of tumors, liver, lung and spleen from each group excised on day 32. (d) H&E and representative lung staining pictures from mice. Red arrow indicates the metastatic nodule on the lung. Scale bars are 500 µm. (e) Western blot analysis of expression levels of the p53 and caspase family members. (f) The area of tumor nodules in H&E (%) was calculated as quantitative analysis of the pulmonary metastatic nodules. Data were expressed as mean ± SD (Standard deviation) from at least 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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TOC Figure

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Tumor Microenvironment Activated Membrane Fusogenic Liposome

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