Codelivery Nanosystem Targeting the Deep Microenvironment of

Subscriber access provided by UNIV OF SOUTHERN QUEENSLAND

Communication

A codelivery nanosystem targeting the deep microenvironment of pancreatic cancer Xinli Chen, Wenxi Zhou, Qinjun Chen, Tao Sun, Yifei Lu, Yujie Zhang, Qin Guo, Chao Li, Yu Zhang, Chen Liang, Si Shi, Xianjun Yu, and Chen Jiang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00374 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

A codelivery nanosystem targeting the deep microenvironment of pancreatic cancer

Xinli Chen†, Wenxi Zhou†, Qinjun Chen†, Tao Sun†, Yifei Lu†, Yujie Zhang†, Qin Guo†, Chao Li†, Yu Zhang†, Chen Liang∥, ∇, Si Shi∥, ∇, Xianjun Yu∥, ∇,Chen Jiang†* † Key

Laboratory of Smart Drug Delivery

Ministry of Education State Key Laboratory of Medical Neurobiology Research Center on Aging and Medicine Department of Pharmaceutics School of Pharmacy Fudan University Shanghai 201203, China E-mail: [email protected] ∥Department

of Pancreatic and Hepatobiliary Surgery,

Fudan University Shanghai Cancer Center, 270 Dongan Road, Shanghai 200032, China ∇Department

of Oncology,

Shanghai Medical College, Fudan University, Shanghai 200032, China

Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered as one of the most aggressive malignancies due to its unique microenvironment of which the cardinal histopathological feature is the remarkable desmoplasia of the stroma, taking up about 80% of the tumor mass. The desmoplastic stroma negatively affects drug diffusion and the infiltration of T cells, leading to an immunosuppressive microenvironment. However, this unique microenvironment can limit the physical spread of pancreatic cancer via a neighbor suppression effect. Here, a tumor central stroma targeting and microenvironment responsive strategy was applied to generate a nanoparticle coloading paclitaxel and phosphorylated gemcitabine. The designed nanoparticle disrupted the central stroma while preserving the external stroma, thereby promoting the anti-tumor effectiveness of chemotherapeutics. 1 ACS Paragon Plus Environment

Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Additionally, the resulting nanoparticle can modulate the tumor immunosuppressive microenvironment by augmenting the number of cytotoxic T cells and restraining the percentage of T regulatory cells. The relatively intact external stroma can effectively maintain the neighbor suppression effect and prevent tumor metastasis. Combining stroma targeting with the delivery of stimuli-responsive polymeric nanoparticles embodies an effective tumor-tailored drug delivery system.

Key words: Tumor microenvironment, Stroma disruption, Codelivery, Immune therapy, Tumor metastasis

2 ACS Paragon Plus Environment

Page 2 of 17

Page 3 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate of 3-8%. Gemcitabine (GEM) is the broadly applied therapeutic agent, with the median survival of only approximately 5.7 months.1 The poor outcome of patients with PDAC is largely due to its unique microenvironment of which the cardinal histopathological feature is the remarkable desmoplasia of the stroma, takeing up roughly 80% of the tumor mass.2 The massive stroma surrounding the local tumor distorts the normal architecture of pancreatic tissues leading to poor vascularization and high intratumoral pressure with the reduction in drug diffusion.3 In addition, desmoplastic stroma also exert pressure on tumor blood vessels and impair the excessive metabolic demand leading to lack of nutrition, hypoxia and acidity in the microenvironment. Pancreatic cancer cells can gradually adapt to the hypoxic environment and undergo additional genetic changes followed by the development of stem-cell-like properties which can make pancreatic cancer cells less sensitive to chemo- or radio-therapy.4 Compared with that of GEM alone, the therapeutic effect of the coadministration of GEM with albuminpaclitaxel (HSA-PTX; nab-paclitaxel) is significantly enhanced.1 HSA-PTX is reported to exhibit a stromal remodeling effect that remarkably alters collagen architecture and eliminates cancer-associated fibroblasts (CAF). Moreover, HSA-PTX leads to the spatial promotion of GEM penetration and intratumoral accumulation.5 In addition, the spatial stroma ablation of HSA-PTX can augment the cytotoxic T cell response resulting in the immunogenicity of tumor cells, upregulation of CD8+ T cell function and more cytotoxic T cell infiltration. 6, 7 Regulatory T cells (Tregs), the main tumor immunosuppressive cells, are also decreased by paclitaxel in a TLR4independent fashion.8 However, the stroma remodeling effect of paclitaxel also introduces risks. The pancreatic stroma is considered to be a “guardian” of tumor metastasis as well as a “fortress” fencing off tumor cells from chemo-therapy.9 According to recent experimental evidence, at least some stromal constituents can suppress tumor progression, rather than contributing to this process.10 By dissolving the matrix, paclitaxel increases tumor vascular permeability, thereby promoting dissemination and metastasis of cancer cell.11 One of the major drawbacks of the traditional nab-paclitaxel and GEM administration approach is that it cannot ensure the spatial and temporal uniformity of the two drugs when entering the tumor. Clinically, HSA-PTX is administered prior to GEM to dissolve the tumor stroma.12 If GEM fails to enter the tumor after the disruption of the stroma by paclitaxel, there is a risk to initiate the unbound tumor cells escaping into the systemic circulation. Fortunately, the unique microenvironment of pancreatic tumor can be utilized to overcome these disadvantages. The compressed blood vessels reduce the concentration of oxygen in pancreatic cancer, which transfer the adenosine triphosphate (ATP) production from oxidative phosphorylation to anaerobic glycolysis to meet the energy needs. Anaerobic glycolysis leads to the formation of lactic acid, which further reduces the pH in pancreatic tumor microenvironment.13 In addition, this transfer has been shown to coincide with the oxygen gradient around blood vessels.14 Thus, the external stroma, relatively closer to the blood vessel, where the oxygen is enough for the oxidative phosphorylation exhibits higher pH nearly 7.4. To the contrary, the tumor core is far away from the blood vessel. Anaerobic glycolysis dominates in this area that leads to the lower pH nearly 6.5. 15


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

This phenomenon inspired the design of a microenvironmental pH-sensitive codelivery system that can transport and release paclitaxel/GEM simultaneously to the core of the tumor.16 Herein, we developed monophosphorylated GEM (p-GEM, the intermediate active product of GEM) and paclitaxel codelivery micelles based on a polyethylene glycol-polyarginine-polylysine (PEG-pArg-pLys) platform.17 Paclitaxel was linked through pH-sensitive molecule (2-propionic-3-methylmaleic anhydride, CDM) to the lysine residue of PEGpArg-pLys. p-GEM was more cytotoxic than gemcitabine in resistant cancer cells (MiaPaCa-2 and PANC02 cells).17, 18 In our design, p-GEM can be carried by the guanidine groups in the side chains of pArg via electrostatic interactions. The micelle can remain stable in the outer layer of the tumor due to the higher pH. Upon entering the tumoral core, the pH of the tumor microenvironment gradually decreases, and the rupture of CDM can be triggered by the lower pH leading to micelle disintegration.19 Subsequently, PTX and p-GEM are released simultaneously from within, followed by the disruption of the internal tumoral stroma and the death of the cancer cells. The drug-release profile confirms that the stroma of the outer layer is relatively integrated, thereby reducing the possibility of tumor cell metastasis. The AE105 peptide (C-D-Cha-F-s-r-Y-L-W-S) has been demonstrated to specifically bind to urokinase type plasminogen activator receptor (uPAR), a receptor over-expressed on the surface of tumor and stroma cells with a high-affinity binding constant (Kd ≈ 0.4 nM), and can be applied in most uPAR-targeted imaging and therapy studies.20 The AE105 peptide was anchored to the micelle surface to promote the targeting effect.21


Page 4 of 17

Page 5 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Scheme 1. Illustration of the tumor central microenvironment targeting strategy and T-RKP codelivery induced chemotherapy and immunotherapy. The T-RKP micelles was found to be able to target the uPAR expressed in pancreatic tumor by the modification of AE105. The micelles remained stable in the “external” stroma of the tumor due to the higher pH in the microenvironment. After entering into the core of tumor, the whole micelle was disrupted which was triggered by the low pH in the deep tumor microenvironment. The released PTX disrupted the “internal” tumoral stroma while preserved the “external” stroma, thus preventing the tumor cells from metastasis. The p-GEM was transported into the tumor cells by the PEG-pArg-pLys. The death of tumor cells induced by p-GEM further led to the activation of T cells killing more tumor cells.

PEG-pArg-pLys was synthesized through ring-opening polymerization.21 The preparation of the codelivery micelle is shown in Figure 1a and Figure S1. The mechanism of pH triggered release was illustrated in Figure S2. All polymers were characterized by 1H NMR spectroscopy (Figure S3-5). The molar ratios of PEG, pArg and pLys were set to 1:15:10 to fabricate a stable structure exhibiting relatively high ability to load paclitaxel (~ 26.3%) and p-GEM (~ 8.6%) (Table S1). PEG-pOrn15-pLys10-PTX4 micelles and AE105 modified PEG-pOrn15pLys10-PTX4 micelles were named RKP and T-RKP, respectively. The T-RKP had a hydrodynamic diameter of nearly 65.5 ± 4.3 nm and high monodispersity in aqueous media of pH 7.4 (Figure 1b). The morphology of TRKP consisted of uniform spheres as shown by TEM (Figure 1c). As the pH decreased from 7.4 to 6.5, the TRKP micelle cracked and showed a significant increase in particle size (Figure 1d and e). The release kinetics of both PTX and p-GEM were measured in a milieu simulating tumor acid microenvironment. When the pH varied from 7.4 to 6.5, T-RKP showed a significant rise in the accumulative release plateau of PTX (Figure 1f). However, only a slight increase in p-GEM was observed during the variation of pH (Figure 1g). In our design, the poly-lysine of PEG-pArg-pLys was exposed after the releasing of PTX (Figure S2). The positive charge of polylysine can facilitate the transportation of p-GEM into the tumor cells. As shown in Figure S6, p-GEM was transported into the tumor cells by the PEG-pArg-pLys during nearly 100 min. To investigate the in vitro tumor cell targeting ability, BODIPY-loaded formulations with different AE-105 modification rates (0, 10, 20, 40, and 80%) were prepared. The result showed that the optimized modification rate was nearly 40%, as increased modification did not further improve the uptake efficiency (Figure S7 and S8). Based on the in vitro uptake results in tumor cells, we further explored the tumor-targeting ability of T-RKP (40% modification rate) and RKP in vivo. D-luciferin was applied to indicate the tumor location (Figure 1h). More significant tumor accumulation was found in the T-RKP-treated group than in the RKP-treated group, which proved that the considerable active


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

targeting effect of T-RKP endowed by AE105 peptide modification (Figure 1i). The major organs (e.g., heart, liver, spleen, lung and kidney) and the whole tumor tissue were collected, showing similar distribution tendencies (Figure 1j, k and Figure S9). The tumor sections were stained with an uPAR antibody (green) to locate the expression of uPAR. Bodipy (red) enabled the tacking of the internal nanoparticles in tumor tissues (Figure 1i). Higher tumor accumulation and colocation of uPAR and nanoparticles were clearly found in the T-RKP treated groups than those in the RKP group (Figure 1m). In addition, the similar pharmacokinetics of PTX and p-GEM were observed in the T-RKP treated groups, exhibiting excellent stability during the systemic circulation (Figure 1n, o, Figure S10, Table S2 and S3).

Figure 1. Micelle construction and in vitro targeting effect. (a) Scheme of the construction and release mechanism of T-RKP micelle. (b) Size of T-RKP micelles measured at pH = 7.4 by dynamic light scattering (DLS). (c) Morphology of T-RKP micelles measured at pH = 7.4 by TEM (scale bar: 100 nm). (d) Size of T-RKP micelles measured at pH = 6.5 by dynamic light scattering (DLS). (e) Morphology of


Page 6 of 17

Nano Letters

Page 7 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

T-RKP micelles measured at pH = 6.5 by TEM (scale bar: 100 nm). (f) The kinetics of paclitaxel release in phosphate buffer saline (PBS) at pH 7.4 or 6.5. (g) The kinetics of p-GEM release in PBS at pH 7.4 or 6.5. (h) The bioluminescence of MiaPaCa-2 cells in vivo; RKP (left) or T-RKP (right). (i) The in vivo imaging of mice after treatment with RKP (left) or T-RKP (right). (red: BODIPY) (j) The relative signal intensity of different organs after treatment with T-RKP or RKP. (k) The relative signal intensity of RKP and T-RKP in the tumor. (l) Fluorescence images of micelles and uPAR after T-RKP or RKP treatment. Green: uPAR, Red: Bodipy (m) The relative signal intensity of RKP and T-RKP in the tumor sections (n). Pharmacokinetic profiles of PTX in HSA-PTX plus GEM, RKP and T-RKP treated group. (o) Pharmacokinetic profiles of GEM or p-GEM in the HSA-PTX plus GEM, RKP or T-RKP treated group. Data represent the means ± s.d. (n = 6). **P < 0.01. n.s. refers to nonsignificance. Inspired by the active targeting effects of AE105, we conducted an MTT assay to further evaluate the in vitro cytotoxicity of T-RKP in MiaPaCa-2 (human pancreatic cancer cells, higher invasiveness) and PANC02 (mouse pancreatic cancer cells, lower invasiveness) cells.22 HSA-PTX and GEM, the clinically effective drugs, were set as the positive control groups. For a comprehensive study of antitumor efficacy, MiaPaCa-2 and PANC02 cells were treated with PBS, HSA-PTX, GEM, HSA-PTX plus GEM, RKP and T-RKP. The cell viability and IC50 values suggested that the AE105 modification significantly increased the toxicity of RKP in both cell lines (Figure 2a, b, Table S4 and S5). The antitumor efficacy was further elucidated using annexin V-FITC and PI assays, the annexin-V (green) signal represented the everted phosphatidylserine during early apoptosis, while the PI (red) label indicated membrane-permeable cells in late apoptosis. Compared with other groups, the T-RKP group demonstrated particularly enhanced antitumor ability because of its ability to facilitate cellular internalization (Figure 2c, d and Figure S11). The cytotoxic effect of polymer alone was further investigated in Figure S12 and S13. The polymer basically has a relatively low effect on tumor growth at concentration below 1 μM. Thus the anti-tumor effect of polymer alone treatment may be negligible. Hopefully this explain can satisfy the reviewer and editor.


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. In vitro cytotoxicity in pancreatic cancer cells. (a) MTT assays in Miapaca-2 cells and PANC02 cells (different PTX concentrations) in the HSA-PTX, HSA-PTX plus GEM, RKP or T-RKP treated group. (b) MTT assays in Miapaca-2 cells and PANC-02 cells (different GEM concentrations) in the GEM, HSA-PTX plus GEM, RKP or T-RKP treated group. (c) Flow cytometry of annexin-V/PI assay in PANC-02 cells after different treatments. (d) Annexin-V/PI assay in PANC-02 cells after different treatments. The green signal indicates the annexin-V signal, and the red signal indicates the PI signal. Original magnification = 200. Data represent the means ± s.d. (n = 6). Based on the in vitro cytotoxicity results in the tumor cells, we further explored the in vivo antitumor effects of the various formulations in a Balb/c orthotopic pancreatic cancer tumor model (PANC02) by intravenous administration every 3 days for 6 times. The tumoral bioluminescence was recorded per week to evaluate the antitumor efficacy. The T-RKP-treated group showed a significant reduction in the signal intensity of the tumor (Figure 3b). Furthermore, the longest life span was observed in mice treated with T-RKP, as anticipated (Figure 3c). T-RKP exhibited higher antitumor efficiency activity in vivo while maintaining excellent biocompatibility with the stable weight of mice (Figure 3d). It has been reported that PTX loaded nanoparticles that slowly release PTX in tumors can effectively awaken immune cells and enhance the antitumor potential of the immune system associated with the increased tumor infiltration by the CD4+ and CD8+ T cells.23 Thus, the ability of different


Page 8 of 17

Page 9 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

treatments to elicit tumor immune responses were further tested using T cell-based assays. Treatment with TRKP resulted in high percentages of cytotoxic T cells (CD45+ CD4- CD8+) and T helper cells (CD45+ CD4+ CD8-) (Figure 3e, f and Figure S14). Moreover, a significantly low percentage of Tregs cells (CD45+ CD4+ CD25+ FoxP3+) was found in the T-RKP treatment group (Figure 3g and h). These results indicated that T-RKP can arouse cytotoxic T cells while eliminating Tregs that suppress antitumor immunity. Interferon-γ (IFN-γ, antitumor cytokines), secreted by CD8-positive T cells, was also elevated in the T-RKP group (Figure S15). These results were further verified by immunofluorescence staining, which showed that the maximum CD8-positive cells and the minimum FoxP3 cells were in the T-RKP groups (Figure 3i-l).

Figure 3. T-RKP improved chemo-therapy and increased the infiltration of immune cells in PANC-02 bearing mice. (a) Time schedule of drug treatment, pathological monitoring and therapeutic evaluation. (b) Statistic bioluminescence, (c) survival rate and (d) body weight of the mice during the 4-week treatment course. (e) T-RKP increased the infiltration of CD4+ and CD8+ positive T cells in pancreatic tumors. (f) Statistic results of CD8+ positive T cells in different groups. (g) T-RKP decreased the percentage of Tregs cells in pancreatic tumors. (h) Statistic results of Tregs cells in different groups. (i) Immunofluorescence of CD8 (red) in tumor sections after different treatments. (j) Statistic results of


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 17

positive CD8 signaling in tumor sections. (k) Immunofluorescence of FoxP3 in tumor sections after different treatments. (l) Statistical results of positive FoxP3 signal of tumor sections. Original magnification = 100. Data represent the mean ± s.d. (n = 6). *P < 0.05, **P < 0.01. The ability of PTX to disrupt the tumoral stroma has been reported in various articles.24 In most cases, this ability contributes to the intratumoral accumulation of antitumor chemotherapeutics. However, as previously reported, the biophysical function of the stroma appears to serve as a physical barrier, neither favorable nor detrimental.25 This function is primarily determined by the stroma-tumor balance and the microenvironment. Once the balance is interrupted, the change in the heterogeneous cellular constituents will cause the metastasis of cancer cells (Figure 4a). The prognosis of patients who chose stromal depletion approaches may be favorable for tumor aggressiveness and spread.26 In our design, CDM is used to selectively release PTX in the core of the tumor to prevent the escape of cancer cells after the external matrix is depleted. To verify our prospective design, the metastasis of pancreatic cancer was investigated in the MiaPaCa-2 (with higher invasiveness) tumor orthotopic model after different treatments. Mice receiving the T-RKP therapy benefited greatly in terms of survival rate and tumor burden (Figure 4b and c). After the treatment of each group, tumor sections from the mice in each group were stained for α-SMA and TUNEL to detect tissue-level stroma and apoptosis (Figure 4d and e). Highly continuous α-SMA expression was observed in groups without PTX loading (control and GEM groups), while the expression of α-SMA was fractured in the groups containing PTX (HSA-PTX, HSA-PTX plus GEM and T-RKP groups). Little α-SMA was eliminated in the RKP treatment group because of the poor targeting ability of RKP. In the HSA-PTX and HSA-PTX plus GEM groups, α-SMA was randomly disrupted in the tumor section. During the T-RKP treatment group, however, the disruption of α-SMA and the generation of TUNEL-positive signaling simultaneously occurred in the core of the tumor section. In addition, the α-SMA expression in the outer layer of the T-RKP treated tumor was relatively complete (Figure 4d, e). This result revealed that our nanoparticle design is capable of selectively disrupting the stroma and killing cancer cells in the tumoral core area while maintaining the relatively complete stroma expression in the outer layer of tumor. The distribution of T-RKP was detected by fluorescence microscope. A large amount of T-RKP was observed in the core area of the tumor (Figure S16). This is likely because uPAR was hypoxia inducible,

more uPAR is expressed in the center of the tumor. 27 AE105 peptide can enhance the accumulation of TRKP in the core region by binding to uPAR. The internal stroma disruption effect of T-RKP can also be confirmed by Col-Ⅰ expression (Figure S17). A few metastatic foci were observed in the liver of the control group and the HSA-PTX plus GEM group, whereas considerable metastatic foci were detected in the liver treated with HSA-PTX. No metastatic foci were found in the GEM, RKP and T-RKP group (Figure 4f and g), suggesting that the intact outer matrix of the tumor can prevent the metastasis of pancreatic tumor cells. Metastasis was also confirmed

by

hematoxylin

and

eosin

(H&E)

staining,

Masson


staining,

α-SMA

and

CK-19

Page 11 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

immunohistochemistry (Figure 4h and Figure S18). After different treatments, no metastatic foci were observed in the heart, spleen, lung and kidney (Figure S19). Paclitaxel can induce the influx of macrophages into the primary tumor, which is required for the assembly and function of the tumor microenvironment of metastasis (TMEM) with the expression of chemotactic prometastatic mammalian-enabled (MENA) isoforms, such as the MenaINV isoform, and relatively fewer amounts of the antimetastatic MENA isoform, MENA11a.28, 29 MenaINV is switched on in invasive tumor cells by NOTCHmediated macrophage contact and signaling.30 Accumulating evidence indicates that chemotherapy evokes a host repair response, during which bone marrow-derived cells (BMDCs) infiltrate the primary tumor microenvironment and boost neoangiogenesis and tumor regrowth.29 The recruitment of

perivascular

TIEhi/VEGFhi macrophages and intravasation of paclitaxel-induced TMEM-dependent tumor cells were investigated by the immunofluorescence and real-time PCR. The maximum number of TIEhi/VEGFhi macrophages was observed in the HSA-PTX group; however, the smallest number was found in the T-RKP group (Figure 4i and j).The expressions of PanMena, MENA11a, and MenaINV were measured by real-time PCR. The mice treated with paclitaxel and paclitaxel plus GEM expressed more PanMena and MenaINV but less MENA11a than did the control mice (Figure 4k-m). This result suggested that HSA-PTX may augment metastatic pancreatic cancer cells by increasing the MenaINV expression and augmenting the TIEhi/VEGFhi macrophages, which showed no significant change in the T-RKP treated group. (Figure 4l and m). WNT16b is expressed ubiquitously with highest levels in adult kidney, placenta, brain, heart, and spleen, whereas WNT16a is expressed at significant levels only in the pancreas.31 The WNT16a can bind to frizzled and LRP receptors, which, in turn, inactivate the degradation complex consisting of AXIN, DVL, and GSK3B.32 It prevents the phosphorylation of β-catenin by GSK3B, promotes its binding to the nuclear transcription factor TCF7L2, leading to the activation of different genes involved in tumor growth regulation. A higher level of TCF7L2 expression indicates a worse prognosis of pancreatic cancer. 33 It has been reported that the genotoxic damage to TAF can induce the expression of WNT16b, which can promote tumor growth through paracrine signaling and attenuate the effect of cytotoxic therapy.34, 35 The suppressed WNT signaling by nanoparticle can improve therapeutic treatment for desmoplastic tumors.36 However, the effect of chemotherapy of Wnt16a has not been reported yet. So we measured the expressions of WNT16a and TCF7L2 expressions in tumor outer tissue and tumor core, respectively. The results were shown in Figure S20-23. T-RKP can be gradually released by low pH in the tumor core (Figure 1f). The creeping release of PTX can activate tumor immune cells and eventually cause tumor cells lysis (Figure 1f and 3e). The immune anti-tumor mechanism does not increase the expressions of WNT16a and TCF7L2. Therefore, low expressions of WNT16a and TCF7L2 was observed in TRKP treated group. It can also explain the less metastasis of T-RKP treated group.


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. T-RKP disrupted the stroma in the core of the tumor and prevent tumor cells metastasis. (a) Illustration of PTX induced tumor metastasis mechanism. (b) Survival rate and (c) statistic bioluminescence of the mice during the 4-week treatment course. (d) Representative α-SMA images of the mice after different treatments. White dots indicate the disruptive area of tumor stroma. (e) Representative TUNEL-positive images of the mice after different treatments. White dots indicate the apoptosis area of tumor stroma. (f) Images of the livers of mice treated with different formulations for one month (g) Statistical results of metastatic foci in the livers. (h) H&E staining images of the livers after one month of treatment. Original magnification = 100. (i) IBA-1/VEGF immunofluorescence images of the tumor sections after one month of treatment. Red: IBA1, green: VEGF. Original


Page 12 of 17

Page 13 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

magnification = 100. (j) Statistical results of the IBA-1/VEGF positive signals of different treatment groups. (k) mRNA expression of PanMENA after different treatments. (l) mRNA expression of MENA

11a after different treatments. (m) mRNA expression of MENAINV after different treatments. Data represent the mean ± s.d. (n = 6). *P < 0.05, **P < 0.01.

In summary, we have developed a paclitaxel and p-GEM codelivery nanoparticle delivery system in response to pH differences in the tumor microenvironment. The resulting formulation (T-RKP) exhibited excellent tumortargeting ability in systemic circulation and high biocompatibility its PEG coating. Based on the unique releasing property of the CDM modification in the tumor core further endowed the formulation with tuned drug-release kinetics responsive to pH stimuli in the tumoral microenvironment. The combination of CDM and the AE105 peptide is capable of selectively disrupting the internal tumor stroma while preserving the outer layer of the stroma. This design enables the prevention of tumor cell metastasis while killing tumor cells.

Supporting Information. Detailed synthesis route and methods, characterization of polymers, pharmacokinetic profiles of control formulations and polymers, evaluation of tumor metastasis of liver and expressions of WNT16a and TCF7L2.

Contribution Dr. Xinli Chen and Miss. Wenxi Zhou contribute equally to this study.

Acknowledgements

This work was supported from National Science Fund for Distinguished Young Scholars (grant No.81425023), National Natural Science Foundation of China (grant No. 81872808) and Program of Shanghai Academic Research Leader (18XD1400500). We acknowledge Prof. Zhigang Zhang at Shanghai Cancer Institute for providing the PANC02 cell lines.


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References: (1)

Von Hoff, DD.; Ramanathan, RK.; Borad, MJ.; Laheru, DA.; Smith, LS.; Wood, TE.; Korn, RL.;

Desai, N.; Trieu, V.; Iglesias, JL.; Zhang, H.; Soon-Shiong, P.; Shi, T.; Rajeshkumar, NV.; Maitra, A.; Hidalgo, M. J Clin Oncol. 2011, 29, 4548-54. (2)

Erkan, M.; Reisererkan, C.; Michalski, C. W.; Kleeff, J. Exp Oncol, 2010, 32, 128-131.

(3)

Von Hoff, DD.; Ramanathan, RK.; Borad, MJ.; Laheru, DA.; Smith, LS.; Wood, TE.; Korn, RL.;

Desai, N.; Trieu, V.; Iglesias, JL.; Hidalgo, M. Asco Meeting Abstracts. 2009, 27, 4525. (4)

Chang, Q.; Jurisica, I.; Do, T.; Hedley, DW. Cancer Res. 2011, 71, (8), 3110-20.

(5)

Alvarez, R.; Musteanu, M.; Garciagarcia, E.; Lopezcasas, P. P.; Megias, D.; Guerra, C.; Muñoz,

M.; Quijano, Y.; Cubillo, A.; Rodriguezpascual, J. Br J Cancer. 2013, 109, 926-33. (6)

Tang, W.; Yang, J.; Yuan, Y.; Zhao, Z.; Lian, Z.; Liang, G. Nanoscale. 2017, 9, 6529-36.

(7)

Ehrke, MJ.; Verstovsek, S.; Maccubbin, DL.; Ujházy, P.; Zaleskis, G.; Berleth, E.; Mihich, E. Int

J Cancer. 2000, 87, 101-9. (8)

Vicari, AP.; Luu, R.; Zhang, N.; Patel, S.; Makinen, SR.; Hanson, DC.; Weeratna, RD.; Krieg,

AM. Cancer Immunol Immunother. 2009, 58, 615-628. (9)

Liang, C.; Shi, S.; Meng, Q.; Liang, D.; Ji, S.; Zhang, B.; Qin, Y.; Xu, J.; Ni, Q.; Yu, X. Do anti-

stroma therapies improve extrinsic resistance to increase the efficacy of gemcitabine in pancreatic cancer? Cell Mol Life Sci. 2018, 75, 1001-12. (10)

Rhim, AD.; Oberstein, PE.; Thomas, DH.; Mirek, ET.; Palermo, CF.; Sastra, SA.; Dekleva, EN.;

Saunders, T.; Becerra, CP.; Tattersall, IW. Cancer Cell. 2014, 25, 735-747. (11)

Karagiannis, GS.; Pastoriza, JM.; Wang, Y.; Harney, AS.; Entenberg, D.; Pignatelli, J.; Sharma,

VP.; Xue, EA.; Cheng, E.; D'Alfonso, TM. Sci Transl Med. 2017, 9, 397.


Page 14 of 17

Page 15 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

(12)

Von Hoff, DD.; Ervin, T.; Arena, FP.; Chiorean, EG.; Infante, J.; Moore, M.; Seay, T.; Tjulandin,

SA.; Ma, WW.; Saleh, MN.; Harris, M.; Reni, M.; Dowden, S.; Laheru, D.; Bahary, N.; Ramanathan, RK.; Tabernero, J.; Hidalgo, M.; Goldstein, D.; Van Cutsem, E.; Wei, X.; Iglesias, J.; Renschler, MF. N

Engl J Med. 2013, 369, 1691-703. (13)

Tannock, IF.; Rotin, D. Cancer Res. 1989, 49, 4373-84.

(14)

Dewhirst, MW.; Walenta, S.; Snyder, S.; Mueller-Klieser, W.; Braun, R.; Chance, B. Int J Radiat

Oncol Biol Phys. 2001, 51, 840-848. (15)

Helmlinger, G.; Yuan, F.; Dellian, M.; Jain, RK. Nat Med. 1997, 3, 177-82.

(16)

Li, H.; Fan, X.; Houghton, J. Tumor microenvironment: The role of the tumor stroma in cancer,

J Cell Biochem. 2007, 101, 805-15. (17)

Lansakara-P, DS.; Rodriguez, BL.; Cui, Z. Int J Pharm. 2012, 429, 123-134.

(18)

Grégoire, V.; Rosier, JF.; De Bast, M.; Bruniaux, M.; De, CB.; Octave-Prignot, M.; Scalliet, P.

Radiother Oncol. 2002, 63, 329-38. (19)

Sun, C. Y.; Liu, Y.; Du, J. Z.; Cao, Z. T.; Xu, C. F.; Wang, J. Angew Chem Int Ed Engl. 2016,

128, 1022-1026. (20)

Dan, L.; Liu, S.; Hong, S.; Peter, C.; Li, Z. Theranostics. 2013, 3, 507-515.

(21)

Guo, Y.; Zhang, Y.; Li, J.; Yu, Z.; Lu, Y.; Jiang, X.; Xi, H.; Ma, H.; An, S.; Chen, J. Acs Appl

Mater Interfaces. 2015, 7, 5444-53. (22)

Duxbury, MS.; Ito, H.; Zinner, MJ.; Ashley, SW.; Whang, EE. Oncogene. 2004, 23, 465-473.

(23)

Shurin, GV.; Tourkova, IL.; Shurin, MR. J Immunother. 2008, 31, 491-499.

(24)

Bouzin, C.; Feron, O. Drug Resist Updat. 2007, 10, 109-120.

(25)

Gore, J.; Korc, M. Cancer Cell. 2014, 25, 711-712.

(26)

Saif, MW.; Chakerian, P.; Kaley, K.; Lamb, L. JOP. 2010, 11, 273-274.

(27)

Leo C, Giaccia AJ, Denko NC. Semin Radiat Oncol. 2004, 14, 207-14.


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(28)

Roussos, ET.; Balsamo, M.; Alford, SK.; Wyckoff, JB.; Gligorijevic, B.; Wang, Y.; Pozzuto, M.;

Stobezki, R.; Goswami, S.; Segall, JE. J Cell Sci. 2011, 124, 2120-31. (29)

Pignatelli, J.; Bravocordero, JJ.; Rohjohnson, M.; Gandhi, SJ.; Wang, Y.; Chen, X.; Eddy, RJ.;

Xue, A.; Singer, RH.; Hodgson, L. Sci Rep. 2016, 6, 37874. (30)

Harney, AS.; Arwert, EN.; Entenberg, D.; Wang, Y.; Guo, P.; Qian, B. Z.; Oktay, MH.; Pollard,

JW.; Jones, JG.; Condeelis, J. S. Cancer Discov. 2015, 5, 932-43. (31)

Fear, MW.; Kelsell, DP.; Spurr, NK.; Barnes, MR. Biochem Biophys Res Commun. 2000, 278,

814-20. (32)

Howard, EW.; Been, LF.; Lerner, M.; Brackett, D.; Lightfoot, S.; Bullen, EC.; Sanghera, DK.,

BMC Genet. 2013, 14, 28. (33)

Xiang, J.; Hu, Q.; Qin, Y.; Ji, S.; Xu, W.; Liu, W.; Shi, S.; Liang, C.; Liu, J.; Meng, Q., Cell Death

Dis. 2018, 9, 321. (34)

Yu, S.; Judith, C.; Celestia, H.; Beer, TM.; Peggy, P.; Ilsa, C.; Lawrence, T.; Nelson, PS.Nat

Med. 2012, 18, 1359-1368. (35)

Miao, L.; Wang, Y.; Lin, C. M.; Xiong, Y.; Chen, N.; Zhang, L.; Kim, W. Y.; Huang, L. J Control

Release. 2015, 217, 27-41. (36)

Hu, K.; Miao, L.; Goodwin, T. J.; Li, J.; Liu, Q.; Huang, L. Acs Nano.2017, 11, 4916-4925.


Page 16 of 17

Page 17 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nano Letters

Illustration of the tumor central microenvironment targeting strategy and T-RKP codelivery induced chemotherapy and immunotherapy. 80x14mm (600 x 600 DPI)

ACS Paragon Plus Environment

Codelivery Nanosystem Targeting the Deep Microenvironment of

Recommend Documents