Designing Liposomes To Suppress Extracellular Matrix Expression To

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Designing Liposomes to Suppress Extracellular Matrix Expression to Enhance Drug Penetration and Pancreatic Tumor Therapy Tianjiao Ji, Jiayan Lang, Jing Wang, Rong Cai, Yinlong Zhang, Feifei Qi, Lijing Zhang, Xiao Zhao, Wenjing Wu, Jihui Hao, Zhihai Qin, Ying Zhao, and Guangjun Nie ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b01026 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 14, 2017

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Designing Liposomes to Suppress Extracellular Matrix Expression to Enhance Drug Penetration and Pancreatic Tumor Therapy

Tianjiao Ji,#Ψ† Jiayan Lang,#†§ Jing Wang,#† Rong Cai,† Yinlong Zhang,†‡ Feifei Qi,† Lijing Zhang,Ψ Xiao Zhao,† Wenjing Wu†‡, Jihui Hao,ξ Zhihai Qin,*Ψ Ying Zhao,*† and Guangjun Nie*†

Ψ The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China † CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China; University of Chinese Academy of Sciences, Beijing 100049, China § Sino-Danish Center for Education and Research/ Sino-Danish College of UCAS, Beijing, 100190, China ξ Department of Pancreatic Carcinoma Tianjin Medical University Cancer Institute and Hospital National Clinical Research Center of Cancer Key Laboratory of Cancer Prevention and Therapy Tianjin, 300060, China ‡ College of Pharmaceutical Science, Jilin University, Changchun, 130021, China

#

These authors contributed equally to this work.

*Address correspondence to: Zhihai Qin The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China E-mail: [email protected] Ying Zhao and Guangjun Nie National Center for Nanoscience and Technology (NCNST), China; University of Chinese Academy of Sciences; Email: [email protected], [email protected] 1

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ABSTRACT During pancreatic tumor development, pancreatic stellate cells (PSCs) proliferate exuberantly to secrete extracellular matrix (ECM) in the tumor stroma, which presents major barriers for drug delivery and penetration in tumor tissue. Thus, down-regulating ECM levels via regulation of the PSCs may allow enhanced penetration of therapeutic drugs and thereby enhancing their therapeutic efficacy. To regulate the PSCs, a matrix metalloproteinase-2 (MMP-2) responsive peptide-hybrid liposome (MRPL) was constructed via co-assembly of a tailor-designed MMP-2 responsive amphiphilic peptide and phospholipids. By utilizing the MMP-2-rich pathological environment, the pirfenidone (PFD) loaded MRPL (MRPL-PFD) can specifically release PFD at the pancreatic tumor site and down-regulate the multiple components of ECM expressed by the PSCs. This resulted in significant increase in the penetration of gemcitabine into the tumor tissue, and enhanced the efficacy of gemcitabine for pancreatic tumor. Our design tailored for anti-fibrosis of pancreatic cancer may provide a practical approach to build functional liposomes through supramolecular assembly, and regulation of ECM may be a promising adjuvant therapeutic strategy for pancreatic and other ECM-rich tumors.

KEYWORD: peptide-hybrid liposome, MMP-2 responsive, pancreatic stellate cells, down-regulate ECM, enhanced pancreatic tumor therapy

Pancreatic tumors contain the highest amount of stroma in almost all solid tumors.1,2 The stroma contains multiple extracellular matrix (ECM) components and forms a physical barrier for the delivery of cytotoxic chemotherapeutics, molecular targeted biologics or nanomedicines to the tumoral milieu, thus diminishing their tumor-targeting and penetrating performance and antitumor efficacy.3,4 This is one of the major reasons leading to the treatment failure of pancreatic cancer, which is the fourth leading cause of cancer-related death in Europe and North America.5,6 Therefore, barrier penetration strategies are urgently needed to enhance drug perfusion and to improve therapeutic efficiency for pancreatic cancer.7-9 2


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In pancreatic cancer, pancreatic stellate cells (PSCs) are the major stromal cell type presenting in tumor tissues and the principal source of ECM production, responsible for stromal components up to 70% in some pancreatic tumors.10,11 The components of ECM interact closely with pancreatic tumor cells (PTCs) to create a tumor promoting microenvironment that facilitates and stimulates both local tumor growth and distant metastasis.12,13 Among PSC-specific ECM components, collagen I and fibronectin are the key components of stroma in both primary and metastatic sites, helping to shape the emerging neo-organ and promote the survival of tumor cells;14-17 tenascin C can enhance pancreatic cancer cell growth and motility through the activation of integrin intracellular signaling pathway;18,19 versican, a large proteoglycan in extracellular peri-tumoral, facilitates tumor invasion and metastasis by decreasing cell-ECM adhesion.20,21 In addition, these ECM components form physical barriers to inhibit the penetration and distribution of agents for tumor imaging and therapy.22 Consequently, regulating the activities of PSCs in pancreatic cancer may be a specific and efficient method to inhibit the secretion of ECM, which may provide an efficient adjuvant strategy for chemotherapy enhancement. Reviewing the ECM construction, both PTCs and PSCs secrete multiple matrix metalloproteinases (MMPs),23,24 which in turn provides opportunities to design responsive materials for pancreatic tumor imaging and drug delivery. Liposome-drug complex is one of the most popular formulations for cancer therapy and several liposomal formulations have been approved by US Food and Drug Administration (FDA), such as liposomal doxorubicin (Doxil, Janssen Biotech, Inc.) and liposomal irinotecan (Onivyde, Merrimack) and others under active clinical trials.25-27 Thus, developing improved functionality of liposomes may hold great potential for clinical applications. Some well-modified liposomal-chemotherapeutic drug formulations with targeting or penetrating functionalities have been developed, achieving improved therapy efficacy.28-30 However, most functional liposomes needs chemical conjugation of lipids with functional molecules,31,32 and some responsive liposomes need additional triggers such as light,33,34 heat35-37 and ultrasound,38-40 which is not benefit to mass production or clinical application. In addition, 3


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considering the rigid desmoplasia of pancreatic tumor tissues, it is extremely difficult for nanocarriers to penetrate the tumor stroma to target PTCs. Therefore, pathological and/or environmental stimulus triggered responsive release and regulation of pancreatic tumor microenvironment in the tumor stroma may be a suitable strategy for improved therapeutic efficacy. In this work, in contrast to functionalizing liposomes with chemical conjugation, we constructed a matrix metalloproteinase-2 (MMP-2) responsive peptide-hybrid liposome (MRPL) by co-assembly of a tailor-designed MMP-2 responsive amphiphilic peptide (MRP) with a phospholipid (L-α-phosphatidylcholine) with environmental responsive feature (Scheme 1). MRPL delivers the soluble anti-fibrosis drug pirfenidone (PFD) and specifically releases it at the tumor site, responsive to the extensive expression of MMP-2 by both PSCs and tumor cells (but not by normal tissues).24,41 The released PFD down-regulated the expression of multiple ECM components in tumors, with subsequent decrease in the desmoplasia and enhanced the perfusion of therapeutics (including chemotherapy drugs) into the deep tumor tissue. Given its easy operation and tumor-specific drug delivery capacity, this co-assembly strategy may provide opportunities to build up functional liposomes for regulating the stromal ECM.

Scheme 1. Proposed mechanism of MMP-2 responsive peptide hybrid liposome (MRPL) for down-regulation of ECM in pancreatic tumors. Pancreatic tumors possess an ECM-enriched microenvironment (left), and the MRPL can deliver the 4


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anti-fibrosis agent pirfenidone (PFD) to the tumor site and achieve a tumor specific release via MMP-2 cleavage in tumor stroma. The released PFD down-regulates multiple components of ECM expressed by PSCs (middle), and increases the perfusion of small molecules (probes and chemotherapeutics) in the tumor tissue (right).

RESULTS AND DISCUSSION MRP

design

and

MMP-2

responsiveness.

The

MRP

(SDK(C18)SGPLG-IAGQSK(C18)DS) is a key element of the MRPL. The hydrophilic domain of the amphiphilic peptide contains an MMP-2-specific cleavable sequence (GPLGIAGQ),31,42 and two other hydrophilic amino acids aspartic acid (D) and serine (S), containing carboxyl and hydroxyl groups, were included to increase the hydrophilicity. In addition, two octadecanoic acid chains were linked to the side-chains of the lysine residues to serve as the hydrophobic domain, which can interact with the tails of the phospholipid through hydrophobic force (Figure S1A). The purity of MRP was over 95% as shown in Figure S1B. We also synthesized an MMP-2-non-responsive peptide (MNP, DSK(C18)DGSALGQIGPDSK(C18)DS) with a structure and sequence similar to that of MRP as a negative control (Figure S2). The responsiveness of MRP and MNP towards MMP-2 was assessed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After incubation with MMP-2 for 3 h, the peak of MRP (M = 2079.8) (Figure S3A) disappeared with the emergence of two fragment peaks (M1 = 1025.9 and M2 = 1071.0), whose sizes corresponded to the two predicted digested segments (Figure S3B). By contrast, no responsiveness of MNP (M = 2309.1) towards MMP-2 was observed (Figure S3C, D).

MRP self-assembly and hybrid-liposome construction. The MRP tends to self-assemble to micrometer-long nanofibers (Figure 1). When MRP was mixed with 5


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the phospholipid at peptide/lipid (P:L) ratios (mass/mass) of 1:10, 1:8 or 1:5, the co-assembly products transformed into stable hybrid liposomal nanospheres with uniform size distributions (Figure 1 and Table S1). If the ratio of peptide/lipid was further changed to 1:3, fiber-like structures emerged in the solution, supposedly resulting from the self-assembly of the excess MRP. This result indicated that the insertion of MRP into hybrid liposomes reached saturation at the ratio of 1:3 (P:L), and continuous increase in MRP may decrease the stability and uniformity of the hybrid liposomes. To assess the stability of MRPLs and MNPLs (MMP-2 non-responsive peptide-hybrid liposomes) with different hybrid ratios (1:10, 1:8, 1:5 or 1:3), we investigated the PFD release profiles at 37ºC in PBS. The MRPL showed a MMP-2 responsive fast drug release (Figure 2A), while MNPL with P:L = 1:3 spontaneously released drug due to instability (Figure S4), while the other three groups did not exhibit significantly different at drug release rate compared to the liposomes without peptide component. Consequently, the ratios of P:L below 1:3 were considered as relatively stable formulations. However, to achieve a high MMP-2 responsive profile, the component of MRP needed to be increased. For the hybrid liposomes with low MRP proportion (P:L = 1:10 and 1:8), they exhibited limited MMP-2 responsive behavior and their PFD release efficacy was not significantly increased compared with the pure liposomes and MNPL (Figure S5, S6). When the P:L ratio was increased to 1:5, over 80% of encapsulated PFD was released promptly and adequately within 180 min in the presence of MMP-2 (Figure 2B). Based on these results, we chose the P:L = 1:5 MRPL as the optimal carrier for subsequent experiments (Table S2). The transmission electron microscopy (TEM) morphology changes may also be an evidence of the MRPL’s MMP-2 responsiveness. In the presence of MMP-2, approximately 90% liposomal vesicles were not smooth anymore, most likely due to the cleavage of MMP-2 responsive peptides (MRPs) (Figure 2C, Figure S7). In contrast, the morphology of MNPL did not exhibit any observable changes (Figure 2C, Figure S7). These results suggested that the MRPL can release the drug via a membrane disturbance when the MRPs were cleaved by MMP-2. This specific drug 6


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release behavior is supposed to increase the intratumoral drug concentration and therefore improve the efficacy.

Figure 1. The morphology of MPR self-assembly, liposomes and MRPL with different peptide and lipid ratios, characterized by TEM. Scale bar, 200 nm.

Figure 2. The fine-tuned drug release profiles of the MRPL and MNPL. (A) The drug release curves of MRPL-PFD with different P:L ratios. The P:L = 1:3 group spontaneously leaked drug at a rapid rate, indicating instability of the nanocarrier. (B) 7


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The drug release profiles of MRPL-PFD (with different P:L ratios) incubated with MMP-2. A favorable responsive release profile was exhibited by the P:L = 1:5 group of MRPL. (C) TEM images of MRPL-PFD and MNPL-PFD (with P:L = 1:5) before and after incubated with MMP-2. The red boxed region is shown magnified in the insert. The scale bar in the top and middle rows, 200 nm; in the bottom row, 50 nm.

Extracellular release behavior of MRPL. Due to the high interstitial fluid pressure of pancreatic tumors43,44 most nanomaterials are not able to penetrate the tumor stroma; therefore the drugs need to be released when the nanocarriers reach the stroma, so that the released drug can be promptly taken up by PSCs. To confirm the specific expression of MMP-2 in tumor tissues, MMP-2 activity in PSCs and Mia-paca-2 (a pancreatic tumor cell line), and MMP-2 expression in the major organs in the PSC and Mia-paca-2 co-implanted tumor bearing mice were examined (Figure S8). The enzyme activity of MMP2 in PSCs was even higher than the tumor cells (Figure S8A), and the tumor tissue exhibited overexpression of MMP-2 compared to other organs (Figure S8B). This result allowed us to conclude that sufficient amount MMP-2 is active in the tumor tissue to trigger the release of PFD from MRPL-PFD. Next, we used a small molecular dye, rhodamine (Rhd) as a model drug to examine the extracellular release of MRPL carrier and the small molecule uptake behavior of PSC. The Rhd uptake by PSCs in the Rhd-encapsulated MRPL (MRPL-Rhd) group showed a fluorescence intensity similar with the free Rhd group, measured by laser confocal microscopy and flow cytometry (Figure S9), indicating the sufficient release of Rhd by MRPL-Rhd. In contrast, the MNPL-Rhd cannot efficiently release its Rhd content due to its non-responsiveness to MMP-2. Only weak Rhd signal likely derived from slight uptake of MNPL-Rhd by PSCs was measured. In PSCs treated with an MMP-2 inhibitor (ARP100) before the incubation with cells, the MRPL-Rhd was not cleaved, and the fluorescence signal was similar to the MNPL-Rhd group (Figure S9A, B). These results led us conclude that only when the MRPL nanocarriers encountered MMP-2, efficient cargo release was be triggered, resulting in subsequent enhanced cellular uptake of loaded small molecules. These factors ensured the specificity and 8


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efficiency of drug release at the tumor site.

ECM down-regulation of MRPL-PFD in vitro and in vivo. Collagen I and fibronectin are the key stromal components secreted by PSCs,14-17 and tenascin C and versican are also important PSCs-specific ECM components,18-20 thereby we chose these four components to evaluate the ECM regulation efficacy of each PFD formulation in cell culture and tumor tissues. After incubation with different formulations (MRPL, free PFD, MNPL-PFD or MRPL-PFD) (PFD concentration: 0.3 mg/mL) for 48 h, the PSCs were fixed and each ECM component was detected by immunocytochemistry. ECM expression in the blank MRPL treated group was not significantly different compared with the control group (Figure 3A), indicating no ECM regulatory effect from the nanocarrier itself. However, all four ECM components were significantly down-regulated by MRPL-PFD, with 28.1 ± 1.2% of Collagen I, 27.4 ± 1.5% of fibronectin, 51.7 ± 3.2% of versican and 13.4 ± 1.7% of tenascin C, compared to the control group (100%), a similar efficacy to that of free PFD (Figure 3B, Table S3). However, MNPL-PFD down-regulated each ECM component to a very limited degree, probably due to ineffective PFD release and limited cell uptake as demonstrated previously. These results demonstrated that MRPL-PFD could efficiently release the loaded PFD in response to MMP-2, which enabled efficient PFD-mediated down-regulation of ECM expression. To further explore the ECM suppression efficacy of the nanoformulations in vivo, PBS, MRPL, free PFD, MNPL-PFD or MRPL-PFD were intravenously injected into tumor bearing mice every two days (PFD dose: 30 mg/kg). Tumors were removed after two weeks for immunohistochemistry (IHC) analysis (Figure 3A,B). IHC images showed obvious down-regulation of the expression of all the four major ECM components in MRPL-PFD treated tumors compared with other groups (Figure 3A), with collagen I down-regulated to 19.2 ± 1.0%, fibronectin to 15.3 ± 1.4%, versican to 36.3 ± 2.6% and tenascin C to 38.5 ± 2.2%, compared to the control group. Although free PFD also down-regulated ECM components to some degree, its efficacy was much more limited (65.3 ± 4.8% of collagen, 60.2 ± 4.7% of fibronectin, 85.3 ± 4.8% 9


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of versican, and 77.4 ± 5.9% of tenascin C, respectively, compared to the control group) (Figure 3B, Table S4). This occurred probably due to the short circulation time of free PFD in vivo and the low tumor accumulation efficiency. MNPL-PFD showed slightly superior efficacy than free PFD, which was different from the in vitro results, and may be attributed to its prolonged circulation time compared to free PFD in vivo, as well as its accumulation as whole particles at the tumor site via the enhanced access of tumor vasculature.45,46 However, without MMP-2 responsive property, even though the MNPL accumulated similar amount as of MRPL, MNPL nanoformulation cannot efficiently release the loaded cargos, which limited cargo perfusion in the tumor tissue (Figure S10). Based on these results, the MRPL-PFD mediated PFD in vivo delivery and tumor release by responding to the MMP-2 at the tumor site and exhibited the highest efficacy of the ECM down-regulation in vivo.

10


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Figure 3. The effects of MRPL-PFD on the key compotents of ECM in vitro and in vivo. (A) Expression levels of multiple ECM components (collagen I, fibronectin, 11


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versican and tenascin C) after incubation with various PFD formulations (PFD concentration: 0.3 mg/mL) detected by immunocytochemistry measurement for in vitro samples (upper panel, 48 h treatment) and immunohistochemistry measurment for in vivo tissue samples (lower panel, 2 week treatment, PFD dose: 30 mg/kg). MRPL-PFD significantly down-regulated the levels of four key components of ECM in both in vitro and in vivo samples. The scale bar, 100 µm. (B) Quantificative analysis of positive staining areas treated by the various formulations based on immunocytochemistry

measurement

(left

panel)

and

immunohistochemistry

measurement (right panel). Five random fields were observed for each group. The brown stained areas were taken into account. Data are presented as mean ± S.D. **p< 0.01, *p< 0.05. The detailed data were presented in Table S3 and Table S4.

Enhanced penetration and therapeutic efficacy. The overexpression of ECM is a major barrier to drug delivery, therefore the inhibition of ECM expression may increase the perfusion of therapeutics in the tumor. To verify this hypothesis, free Rhd was chosen to visualize the perfusion efficiency of small molecules at the tumor site after different treatments of PFD formulations for 2 weeks. One hour after injection, in vivo images showed that the strongest fluorescence signal in the MRPL-PFD treated group (Figure S11A), which was about 5.3 times stronger than the PBS and MRPL treated groups, 2.8 and 2.5 times stronger than the PFD and MNPL-PFD groups, respectively (Figure S11B). All these results indicate the maximum tumor perfusion of Rhd in the MRPL-PFD treated mice. Confocal microscopy images showed a gradient fluorescence of Rhd from the tumor edge to the core. Importantly, the fluorescence intensity of Rhd was much wider and stronger in MRPL-PFD-treated tumors compared with MNPL-PFD, free PFD, blank MRPL or control groups (Figure 4A). Further analysis of the average penetration depth of Rhd in each group showed that the penetration depth in MRPL-PFD treated group was 972.2 ± 28.3 µm, which was approximately 5.3, 3.0 and 9.2 times deeper than free PFD (182.3 ± 14.7 µm), MNPL-PFD (323.6 ± 21.5 µm), control (105.3 ± 12.1 µm) and MRPL (104.7 ± 11.2 µm) groups, respectively (Figure 4B). Taken together, these data demonstrated that we 12


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had successfully constructed a nanocarrier MRPL-PFD for down-regulating ECM and increasing the perfusion of small molecules in the pancreatic tumors. Since the down-regulation of ECM can increase the perfusion of small molecules, efficacy of chemotherapy drugs may also be enhanced. To explore the impact of ECM regulation on chemotherapeutic efficacy, PSCs/Mia-paca-2 pancreatic tumor bearing mice were administered (i.v.) PBS, PFD, MNPL-PFD or MRPL-PFD (n = 8) when tumor volume reached approximately 50 mm3. A dose of 30 mg/kg PFD in each formulation was given every two days for a total of seven injections (Day 3, 5, 7, 9, 11, 13 and 15). The results showed that the average tumor volume in the PFD treated groups was slightly smaller, but not significant, than that of the control group (Figure 4C), which may be due to fast growth of the tumors regulated by some autocrine regulation pathways in this tumor model.47-49 A dose of 20 mg/kg gemcitabine was given at Day 17, 19 and 21. Further monitoring of tumor growth showed that the tumors continued growing in the control group, indicating that gemcitabine did not effectively inhibit the fast-growing tumors, neither did the free PFD and MNPL-PFD groups (Figure 4C). However, the size of tumors in the MRPL-PFD group decreased significantly after initiation of gemcitabine treatment (**p< 0.01), compared to PBS, PFD and MNPL-PFD groups. Mice were sacrificed 7 days after gemcitabine treatment because of the excess large tumor volume in the control group and the serious side effects of gemcitabine (Figure S12). H&E stained tumor tissues in the MRPL-PFD treated group exhibited larger areas of apoptosis compared to other groups (Figure 4D). The percentage of apoptotic cells in each group was further determined by TUNEL immunohistochemical staining, and the TUNEL-positive cells in MRPL-PFD group were 59.7 ± 5.7%, while other groups were much lower (control: 9.8 ± 2.4%, PFD: 17.3 ± 3.4%, MNPL-PFD: 28.4 ± 4.5%) (Figure 4D, Figure S13). To directly measure drug perfusion and accumulation, the sum of the concentrations of gemcitabine and its metabolite, 2`,2`-difluorodeoxyuridine (dFdU)50 in tumor tissue detected by high performance liquid chromatography (HPLC) was used to evaluate the drug perfusion efficiency. The tumor drug concentration in MRPL-PFD (6.2 ± 0.8 pmol/mg protein) was three times higher than control group (2.1 ± 0.4 13


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pmol/mg protein), two times higher than PFD group (3.0 ± 0.3 pmol/mg protein) and 1.5 times higher than MNPL-PFD group (4.3 ± 0.8 pmol/mg protein) (Figure S14). These results indicated that MRPL-PFD significantly enhanced the therapeutic efficacy of gemcitabine by increasing the perfusion of gemcitabine.

Figure 4. Evaluation of Rhd penetration and distribution and therapeutic efficacy of gemcitabine in tumors. (A) Rhd penetration and distribution in pancreatic tumor (PSCs/Mia-paca-2 co-implanted) tissues after two weeks’ treatment of the different PFD formulations. Frozen tumor sections were stained with DAPI 14


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(blue) to label nuclei. Red: Rhd. Dotted lines show the border between edge and core of tumor tissue. Scale bar, 100 µm. (B) Quantification of the depth of Rhd penetration in tumors treated by the PFD formulations. Five random fields were observed for each group under confocal microscopy. Data are presented as mean ± S.D. **p< 0.01. (C) The growth curves of PSCs and Mia-paca-2 co-implanted pancreatic tumors in mice treated by the different PFD formulations. The dose of PFD was 30 mg/kg in all groups at the Day 3, 5, 7, 9, 11, 13, 15 (hollow arrows). At the Day 17, 19 and 21 (solid arrows), the various PFD treatments were terminated and gemcitabine was injected (gemcitabine dose: 20 mg/kg). Data are presented as mean ± S.D. (n = 8). **p< 0.01. (D) Histological analysis of tumor slices of each group after treated with gemcitabine. H&E staining, the top row; TUNEL immunohistochemical staining, the bottom row. Scale bar, 100 µm. The tumor tissue of MRPL-PFD pre-treated group exhibited the largest apoptosis areas and the highest TUNEL-positive cells among all groups.

CONCLUSION In conclusion, considering the pathological environment of pancreatic tumors, via carefully engineering molecular self-assemblies, we have constructed an MMP-2 responsive liposome (MRPL) through co-assembly of functional peptide and phospholipid. The designed hybrid nanosystem achieved tumor specific delivery and release of PFD at the PSCs-enriched pancreatic tumors. The released PFD down-regulates expression of multiple ECM components of PSCs, which increase the perfusion of small molecules in the tumor, thereby enhancing therapeutic efficacy of gemcitabine. Due to the ease of production and tumor-specific drug delivery, the peptide-hybrid liposomal nanoformulations may provide an attractive approach to build stroma responsive liposomes with the functionality of regulating the tumor stroma and enhancing drug penetration into deep tumor tissue. Regulation of ECM may become an efficient adjuvant strategy to improve the efficacy of pancreatic tumor therapy. 15


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EXPERIMENTAL SECTION Enzyme-responsiveness of MRP and MNP. Recombinant human MMP-2 (rhMMP-2, R&D system, USA) was diluted to 0.2 µg/mL in the assay buffer (50.0 mM Tris, 10.0 mM CaCl2, 150.0 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5). After 50.0 µL rhMMP-2 (0.2 µg/mL) was loaded into a plate, the reaction was started by adding 50.0 µL of 100.0 µM MRP (dissolved in assay buffer). After 3 h, the reaction product was analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (Bruker, Germany). The MS detection range was 600-2200 Da for MRP and 600-2500 for MNP. Self-assembly of MRP and MNP. 0.05 mg MRP or MNP was dissolved in 5.0 µL dimethyl sulfoxide (DMSO) (Sigma Aldrich, USA), and then added into 1.0 mL distilled water to be ultrasonicated (KQ2200E, China) at 100 W for 1 min. The resulted nanostructures was characterized by transmission electron microscopy (TEM, HT7700, HITACHI, Japan) using a negative staining method with phosphotungstic acid at different times of incubation. Preparation and Characterization of Liposomes, MNPL and MRPL. Liposomes were prepared by a thin film hydration method followed by membrane extrusion.35 Briefly, 1.0 mg L-α-Phosphatidylcholine (soybean lecithin, J&K Scientific, China), 0.02

mg

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2 000] (DSPE-PEG2000) (Nanocs, USA), and 0.25 mg cholesterol (J&K Scientific, China) were dissolved using 10.0 mL methylene chloride in a round-bottom flask and incubated for 5 min. Then a phospholipid film was formed through a rotary evaporator under reduced pressure. 1.0 mL PBS was added into the round-bottom flask with phospholipid film for hydration for 1 h. After hydration, closely graded unilamellar liposomes were obtained by 11 times extrusion through 200 nm and 100 nm membranes using an extruder (LiposoFast-basic. Avestin, CA). To prepare the MRPL, MRP was dissolved in methanol at first, the lecithin, 0.02 mg PEG-DSPE and 0.25 mg cholesterol were dissolved using 10.0 mL methylene chloride, the total mass of the MRP and lecithin was 1.0 mg but with the different ratios of P:L (such as 1:10, 16


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1:8, 1:5, 1:3), then mixed the methanol and methylene chloride solution and prepared the phospholipid film. The morphology of liposome and MRPL were characterized by TEM (HT7700, HITACHI, Japan) using a negative staining method. The size, polydispersity index, and zeta-potential of them were measured by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern, UK). The MNPLs were prepared in the same method of MRPLs. Preparation and Characterization of PFD Loaded Liposomes, MNPL and MRPL. After the phospholipid film was formed through a rotary evaporator under reduced pressure, 1.0 mL PFD-containing PBS was added into the round-bottom flask with phospholipid film for hydration for 1 h. After hydration, closely graded unilamellar liposomes were obtained by 11 times extrusion through 200 nm and 100 nm membranes using an extruder. Next, the liposome, MNPL or MRPL was collected after centrifugation at 10,000 g for 10 min at room temperature. For evaluating PFD encapsulation efficiency, lyophilized PFD-loaded liposome MNPL or MRPL was dissolved in DMSO and measured by UV-Vis spectrophotometer (LAMBDA650, PerkinElmer, USA) after making a standard curve with free PFD (the detected wavelength was 263 nm). The encapsulation efficiency (EE) was calculated according to the following formula: EE (%) = (mass of PFD encapsulated in nanoparticles/mass of PFD added) × 100% The in vitro PFD release kinetics of liposome-PFD, MNPL-PFD and MRPL-PFD was measured via dialysis. Briefly, 1.0 mL liposome-PFD, MNPL-PFD or MRPL-PFD solution was injected into dialysis cartridge with a molecular weight cut-off value of 2 kDa. The cartridge was dialyzed against 10.0 mL PBS (10.0% FBS) and shaken at 37ºC at 100 rpm with activated charcoal to create an ideal sink condition. The concentration of PFD remaining in the dialysis cartridge at different time points was measured by UV-Vis spectrophotometry. Preparation of Rhd Loaded MNPL and MRPL. After the phospholipid film was formed through a rotary evaporator under reduced pressure, 1.0 mL Rhodamine 6G (Rhd) (Aladdin Industrial Inc., China) containing PBS was added into the round-bottom flask with phospholipid film for hydration for 1 h. After hydration, 17


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closely graded unilamellar liposomes were obtained by 11 times extrusion through 200 nm and 100 nm membranes using an extruder. Next, the MNPL-PFD or MRPL was collected after centrifugation at 10,000 g for 10 min at room temperature. Cell Culture and Animals. We separated human Pancreatic stellate cells (PSCs) from pancreatic cancer surgical specimens using the outgrowth method described by Bachem and Apte,51,52 and all of the established PSCs were used between Passage 3 and 8. The use of pancreatic cancer surgical specimens was approved by the department of pancreatic carcinoma Tianjin Medical University, Cancer Institute and Hospital National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy (Tianjin, China), and was conducted according to the Ethical Guidelines for Human Genome/Gene Research enacted by the Chinese Government and the Helsinki Declaration. Mia-paca-2 (pancreatic cancer cell) cell line was purchased from the National Platform of Experimental Cell Resources for Sci-Tech (China), and the cell line was passaged in the laboratory for less than 3 months after resuscitation and maintained in a 37ºC/5% CO2 humidified chamber. Mia-paca-2 cells were grown in RPMI-1640 media (WISENT, Canada) with 10% FBS (WISENT, Canada); and PSCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (WISENT, Canada) with 10.0% FBS, 1.0% GlutaMax (Invitrogen, USA), 1.0% Na-pyruvate (Invitrogen, USA), 1.0% NEAA (Invitrogen, USA), 10.0 ng/mL bFGF (Sigma Aldrich, USA). BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. All animal protocols were approved by the Institutional Animal Care and Use Committee. In the pancreatic co-implanted tumor model, PSCs and Mia-paca-2 cells (2.5×106 each) were suspended in a 100.0 µL PBS and Matrigel mixture (1:1, v/v; BD, USA), and subcutaneously co-inoculated at the fore limb of each nude mouse (female, 6 weeks age, 15-17 g body weight). MMP2 Activity Detection. MMP2 expression and activity were determined by gelatin zymography assay. In brief, PSCs and Mia-paca-2 cells were cultured with the mentioned medium without serum after the cells growth reached approximately 80%. Twenty-four hours later, the conditioned medium was collected. 10.0 µL of 18


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supernatant was subjected to zymographic SDS-PAGE containing 0.1% gelatin (w/v). The gel was eluted in a eluting butter (2.5% Triton 100, 50.0 mM Tris-HCl, 5.0 mM CaCl2, pH 7.6) for 80 min to obtain active MMP2, and was then washed with washing buffer (50.0 mM Tris-HCl, 5.0 mM CaCl2, pH 7.6) for 40 min, and was further incubated in incubation buffer (50.0 mM Tris-HCl, 5.0 mM CaCl2) for 48 h. Lastly, the gel was stained with Coomassie brilliant blue and then destained. The zone of gelatinolytic activity was shown by negative staining. Confocal Microscopy and Flow Cytometry Detection of Rhd Uptake in PSCs. PSCs were seeded in confocal dishes (Nunc, USA). When the cells were approximately 70% confluence, they were incubated with Rhd, MNPL-Rhd or MRPL-Rhd for 5 h at 37ºC. All group carried equivalent concentration of 10.0 nM Rhd. In the MMP-2 inhibitor group, ARP 100 was incubated with cell at 50.0 nM for 2 h before MRPL-Rhd added. The nucleus were stained with HOECHST33342 (Invitrogen, USA) for 5 min. Cells were then washed 3 times with PBS to be observed by confocal microscopy (LSM710, Carl Zeiss, Germany). To quantitively measure the uptake of Rhd and the Rhd-loading nanoparticles, PSCs were seeded in 24 well plates. Cells were incubated with free Rhd, MNPL-Rhd and MRPL-Rhd in the present or absent of MMP-2 inhibitor for 5 h at 37ºC, respectively. All group carried equivalent concertation of 10 nM Rhd. In the MMP-2 inhibitor group, ARP 100 was incubated with cell at 50.0 nM for 2 h before MRPL-Rhd was added. Then the cells were washed 3 times with PBS and resuspended in PBS for the flow cytometric analysis (BD Accuri™ C6, BD, USA). A total of 10, 000 events were collected for each sample. Western Blot Analysis. Mice bearing ~200 mm3 PSCs/Mia-paca-2 tumors were dissected, and tumors and major normal organs (liver, spleen, lung and kidney) were grinded and lysed with RIPA buffer (Solarbio, China) containing 1.0 mM phenylmethanesulfonyl fluoride (PMSF) (Solarbio, China). Protein samples (40-80 µg) were electrophoresed on 10.0% sodium dodecyl sulfate–polyacrylamide gels. The proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane. Loading and transfer were then confirmed by Ponceau red staining. After 19


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pre-incubation in blocking solution at room temperature for 1 h, the PVDF membrane was incubated with mouse anti-human MMP-2 monoclonal antibody (1:1,000) (R&D, USA) for 2 h at room temperature. After being washed 3 times for 10 min with Tris-buffered saline (TBS) containing 0.5% Tween-20 (Solarbio, China), the membrane was incubated with a secondary antibody linked to horseradish peroxidase (goat anti-mouse IgG, 1:10,000) (Santa Cruz, USA) for 60 min at room temperature. Immunoreactive

proteins

were

visualized

using

SuperSignal

West

Pico

Chemiluminescent Substrate (Thermo Scientific, Rockford, USA). Immunocytochemistry (ICC) Analysis. For ICC, PSCs were plated on 24 well plates and incubated at 37ºC for at least 24 h to allow attachment. After incubation with different formulations (MRPL, free PFD, MNPL-PFD or MRPL-PFD) (PFD concentration was 0.3 mg/mL in each PFD containing formulation) for 48 h, cells were fixed with formalin for 15 min, and then washed with PBS. A two-step plus®Poly-HRP Anti Mouse/Rabbit IgG Detection System (GBI) was used to stain ECM protein. Briefly, cells were blocked with 1.0% bovine serum albumin/PBS and then labeled overnight at 4ºC with the antibodies against either collagen I (rabbit, Abcam, ab138492, 1:1000), fibronectin (mouse, R&D, MAB1918, 8.0 µg/mL), versican (rabbit, Gene Tex, GTX87516 1:100) or tenascin C (mouse, Abcam, ab58954, 1:50), respectively. Subsequently, cells were incubated with HPR anti-mouse/rabbit IgG for 30 min. DAB was used as a detection system (GBI) according to the manufacturer`s recommendations. Cells were counterstained with hematoxylin (Solarbio, China) for 5 min. The results were analyzed by using a light microscopy (AMG EVOS xl core, Life Technologies, USA). All stainings were quantified using Image J pro analysis software with the same threshold; results were expressed as percentage staining per visual field. Five fields were analyzed in each group. Tumor Immunohistochemistry (IHC) Analysis. For IHC, nude mice bearing the PSCs and Mia-paca-2 co-implanted tumors were treated with PBS, MRPL, PFD, Lipo-PFD or MRPL-PFD (PFD dose: 30.0 mg/kg) for two weeks (a total of 7 injections). Harvested tumor were formalin fixed prior with paraffin embedding. 20


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Sections 4.0 µm thick were deparaffinised, rehydrated, and boiled for 1 h on 10.0 mM citrate buffer at pH 6.0. Staining for ECM protein was processed using the 2-step plus®Poly-HRP Anti Mouse/Rabbit IgG Detection System (GBI). Briefly, the tissue sections were blocked with 1.0% BSA in PBS for 30 min prior to incubation with the primary antibody. Sections were then incubated with HPR anti-mouse/rabbit IgG for 30 min. DAB was used as a detection system (GBI) according to the manufacturer`s recommendations. The following primary antibodies were used: rabbit anti-collagen I (Abcam, UK, ab138492) 1:1000, mouse anti-fibronectin (R&D, USA, MAB1918) 8 µg/mL, rabbit anti-versican (Gene Tex, USA, GTX87516) 1:100, and mouse anti-tenascin C (Abcam, UK ab58954) 1:50. Sections were counterstained with hematoxylin (Solarbio, China) for 5 min, dehydrated in alcohols, cleared in xylene, and cover-spilled. The results were analyzed by using a light microscopy (AMG EVOS xl core, Life Technologies, USA). All staining was quantified using Image J pro analysis software with the same threshold; results were expressed as percentage staining per visual field. Bio-distribution of MNPL and MRPL, Rhd Signal Detection of MNPL-Rhd and MRPL-Rhd Treated Tumor Bearing Mice. and

MNPL,

0.5%

(in

mass)

For fluorescently labeling of MRPL of

Rhd-conjugated

(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine

rhodamine

lipid B

sulfonyl),ammonium salt (Avanti Polar Lipids, Inc., USA) was added when preparation of MNPL and MRPL nanoparticles. Nude mice bearing the PSCs and Mia-paca-2 co-implanted tumors were intravenously injected with the Rhd-labelled MNPL or MRPL (100.0 µL). At 12 h after injection, the major organs and tumors were excised for ex vivo imaging. The organs were scanned with a MaestroTM in vivo imaging system (CRI, Woburn, MA, USA). The excitation wavelength was 520-560 nm and the emission wavelength was 590 nm. Nude mice bearing the PSCs and Mia-paca-2 co-implanted tumors were intravenously injected with MNPL-Rhd or MRPL-Rhd (100.0 µL). At 12 h after injection, organs and tumors were excised for ex vivo imaging. The organs were 21


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scanned with a Maestro in vivo imaging system. The scanned excitation wavelength was 520-560 nm and the emission wavelength was 590 nm. Evaluation of Rhd Penetration in Tumors. Nude mice bearing the PSCs and Mia-paca-2 co-implanted tumors were treatment with PBS, MRPL, PFD, MNPL-PFD or MRPL-PFD (PFD dose: 30.0 mg/kg) for two weeks (a total of 7 injections). Then mice were injected (i.v.) free Rhd. After 1 h, mice were scanned with Maestro in-vivo imaging system, the wavelength of excitation light is 520-560 nm and the emission wavelength was 590 nm. Excised tumors were frozen in optimum cutting temperature (OCT) medium (Sakura Finetek, USA) at -80ºC. The corresponding slices (6.0 µm) were prepared, air dried for 10 min, and fixed with 4.0% paraformaldehyde (Solarbio, China) for 10 min. The nuclei were stained with DAPI (Invitrogen, USA). Sections were then washed three times with PBS and cover-spilled to be observed by confocal microscopy (LSM710, Carl Zeiss, Germany). Therapeutic

Studies.

For

the

subcutaneous

xenograft

mouse

model

of

PSCs/Mia-paca-2 pancreatic tumor model, treatment was initiated when tumor volume reached approximately 50 mm3. Mice were administered (i.v.) with PBS, PFD, MNPL-PFD or MRPL-PFD (n = 8). A dose of 30.0 mg/kg PFD was given every 2 days for a total of 7 injections (Day 3, 5, 7, 9, 11, 13 and 15). At the Day 17, a dose of 20.0 mg/kg gemcitabine was given (i.v.) every 2 days for a total 3 times. Tumor sizes were measured by a digital caliper, and tumor volume was calculated by the formula (L×W2)/2, where L is the longest and W is the shortest in tumor diameters (mm). Relative tumor volume (RTV) equals the tumor volume at a given time point divided by the tumor volume before treatment started. For humane reasons, animals were sacrificed at the Day 23 for the body weight decreased seriously because of the side-effect of gemcitabine. To evaluate necrosis, we stained tumor sections with H&E and analyzed them using a light microscopy (AMG EVOS xl core, Life Technologies). Tumor Gemcitabine Concentration Analysis by High Performance Liquid Chromatography (HPLC). PSCs/Mia-paca-2 pancreatic tumor bearing mice were administered (i.v.) with PBS, PFD, MNPL-PFD or MRPL-PFD (n = 5). A dose of 30 22


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mg/kg PFD was given every 2 days for a total of 7 injections (Day 3, 5, 7, 9, 11, 13 and 15). At the Day 17, a dose of 20.0 mg/kg gemcitabine was given (i.v.). After 1 h injection, tumors were harvested and homogenized in RIPA buffer (Solarbio, China). Liquid-liquid extraction, external standardization and HPLC was used to measure gemcitabine and its metabolite, 2`,2`-difluorodeoxyuridine (dFdU) in tumor tissue. Briefly, tumor homogenates in RIPA buffer were extracted with acetonitrile (ACN), and the extract was transferred to a different tube, evaporated to dry all ACN. Mobile phase (ACN:H2O = 73:27) was added to each tube, an aliquot was injected into the HPLC (Shimadzu, Kyoto, Japan), and the compounds were separated using a symmetry C18 reverse phase column (5 µm; 4.6 × 150 mm Thermo Scientific) and eluted using an acetonitrile 73.0%, water 27.0%, trifluoroacetic acid (0.1% v/v) gradient. The flow rate was 1.0 mL/min and the eluent was monitored at 270 nm for gemcitabine and dFdU. The sum concentration of gemcitabine and dFdU was used to evaluate the drug quantity in the tumors. All separations were performed at room temperature. Statistical Analysis. Statistical analysis was conducted by the Student t test for comparison of 2 groups, and one-way ANOVA for multiple groups, followed by Newman–Keuls test if overall p< 0.05. A p value of less than 0.05 was considered significant (*), while a p value of less than 0.01 was considered very significant (**).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Authors [email protected]; [email protected]; [email protected]

Author Contributions 23


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#

These authors contributed equally.

ACKNOWLEDGMENTS We thank Prof. Saraswati Sukumar from Johns Hopkins University School of Medicine for critical reading of the manuscript. This work was supported by the grants from MOST 973 (2013CB932701), NSFC (Grant No. 81630068, 21373067, 51673051, 31325010, 11621505 and 31571021), Beijing Nova Program (Grant No. Z171100001117010), Beijing Natural Science Foundation (Grant No. 7172164), Beijing Municipal Science & Technology Commission No. Z161100000116035, the Frontier Research Program of Chinese Academy of Sciences (Grant No. QYZDJ-SSW-SLH022) and Youth Innovation Promotion Association CAS (Grant No. 2017056).

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Designing Liposomes To Suppress Extracellular Matrix Expression To

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