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Biological and Medical Applications of Materials and Interfaces

Collagenase Encapsulated pH-Responsive Nanoscale Coordination Polymers for Tumor Microenvironment Modulation and Enhanced Photodynamic Nanomedicine Jingjing Liu, Longlong Tian, Rui Zhang, Ziliang Dong, Hairong Wang, and Zhuang Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17684 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 23, 2018

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ACS Applied Materials & Interfaces

Collagenase Encapsulated pH-Responsive Nanoscale Coordination Polymers for Tumor Microenvironment Modulation and Enhanced Photodynamic Nanomedicine

Jingjing Liu, Longlong Tian, Rui Zhang, Ziliang Dong, Hairong Wang, Zhuang Liu* Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China. * Email: [email protected]

Abstract The abundant tumor extracellular matrix (ECM) could result in the insufficient tumor retention and ineffective intra-tumor penetration of therapeutic agents, as well as acidic & hypoxic tumor microenvironment (TME), further leading to the unsatisfactory therapeutic outcomes for many types of therapies. Therefore, developing strategies to modulate the TME by selectively degrading the condensed ECM may be helpful to improve existing cancer therapies. Herein, collagenase (CLG) encapsulated nanoscale coordination polymers (NCPs) are synthesized based on Mn2+ and an acidsensitive benzoic-imine organic linker, and then modified by polyethylene glycol (PEG). Upon intravenous (i.v.) injection, these CLG@NCP-PEG nanoparticles show efficient accumulation within the tumor, in which CLG would be released due to the collapse of NCP structures within the acidic TME. The released CLG enzyme could then specifically degrade collagens, the major component of ECM, leading to loosened ECM structure, enhanced tumor perfusion and relieved hypoxia. As the results, the second-wave of nanoparticles, chlorin e6 (Ce6)-loaded liposomes (Liposome@Ce6), would exhibit enhanced retention and penetration within the tumor. Such phenomena together with relieved tumor hypoxia could then lead to greatly enhanced photodynamic therapeutic effect of Liposome@Ce6 for mice pretreated with CLG@NCP-PEG. Our work thus presents a unique strategy for TME modulation using pH-responsive NCPs as smart enzyme carriers.

Keywords: Tumor microenvironment, Extracellular matrix, Photodynamic therapy, Nanoscale coordination polymers, Collagenase,

Introduction

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The condensed tumor extracellular matrix (ECM) that is composed by cross-linked fibrousforming proteins (elastins, collagens, laminins and fibronectins), associated large glycoproteins and proteoglycans (hyaluronic acid, chondroitin sulphate and keratin sulphate), could provide support for tumor growth and progression.1-2 As an important component of tumor microenvironment (TME), ECM is highly interconnected with hypoxia of TME, together leading to the therapeutic resistance for those tumors.3-4 For instance, hypoxia could promote ECM remodeling to facilitate metastases by increasing the expression of collagen and collagen-modifying enzymes. On the other side, collagencontaining ECM could act as a natural barrier to prevent effective diffusion of therapeutic agents and oxygen into the internal part of a large solid tumor owing to the high interstitial flow pressure (IFP) within those tumors.5 Thus, the therapeutic outcome of many types of cancer treatment methods would be limited owing to the obstruction by the condensed tumor ECM.6 It has been proposed that the TME characteristics could be modulated by manipulating the structure of ECM to favor different types of antitumor therapies.7 Up to now, there are a number of small molecules that could effectively inhibit the production of ECM components and decompress the blood vessels, so as to enhance the penetration of therapeutic agents or even drug-loaded nanoparticles within the tumor for improved cancer treatment.8-10 On the other hand, owing to the highly susceptible of ECM to the action of proteases, several specific proteases have also been explored to selectively degrade the main components of ECM for increased diffusion and enhanced therapeutic efficacy of nanotherapeutics.11-13 For instance, recombinant human hyaluronidase enzyme (PH20), a genetically engineered soluble hyaluronidase approved by the U.S. Food and Drug Administration (FDA) could serve as an adjuvant to increase the absorption and dispersion of other drugs in clinic.14-16 However, while local administration of those enzymes into tumors may have less significant clinical value, systemic injection of exogenous enzymes also has its limitations such as limited tumor uptake, potential immunogenicity and other possible side effects.17-18 Thus, developing smart nano-carriers for delivery and tumor-specific release of ECM-decomposing enzymes may be an attractive approach for TME modulation and enhanced cancer treatment. Nanoscale coordination polymers (NCPs) are a class of nanocomposite fabricated through the coordination of metal ions or clusters and organic ligands.19-20 Owing to the natural biodegradability and diverse compositions by using different metal ions and organic ligands, NCPs with different morphologies and functionalities could be synthesized for various applications in biomedicine.21-27


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NCPs have been investigated as nanocarriers for loading of various therapeutic or imaging agents by utilizing the large surface area and porous structure of this type of nanomaterials.28-34 Recently, stimuliresponsive carriers based on NCPs have also been designed for controllable drug release under specific stimuli such as redox,28, 35 pH30, 36 and singlet oxygen.34 Furthermore, NCPs could serve as protective coatings for biomacromolecules including proteins, enzymes and DNA through biomineralization process, resulting in unique bio-functional nanocomposites with greatly retained bioactivity.37-38 Therefore, it would be interesting to encapsulate functional enzymes that are able to degrade the tumor ECM component into TME-responsive NCP-based delivery system, so as to achieve effective and specific TME modulation and cancer treatment enhancement. Herein, a new type NCPs with plate-like morphology is synthesized based on manganese ions (Mn2+) and a pH-responsive benzoic-imine linker (BI-linker) in the aqueous solution. This structure could be utilized to encapsulate collagenase (CLG), an enzyme that can break the peptide bonds specifically in collagen to destruct the condensed tumor ECM structures. After modification with polyethylene glycol (PEG), CLG@NCP-PEG showed great stability and efficient tumor uptake after intravenous (i.v.) injected into tumor bearing mice as revealed by in vivo magnetic resonance (MR) imaging and ex vivo biodistribution measurement. Notably, owing to the acidic-pH-sensitive cleavable of the BI-linker, such enzyme-loaded NCPs would be decomposed under the TME pH, releasing CLG specifically within the tumor. As the results of CLG-triggered degradation of collagen within the tumor ECM, greatly enhanced tumor perfusion and remarkably reduced tumor hypoxia are observed after i.v. injection of CLG@NCP-PEG. Furthermore, the tumor uptake of the second-wave nanoparticles, photosensitizer chlorin e6 (Ce6)-loaded liposomes (Liposome@Ce6), could be enhanced by ~1.6 fold. Additionally, those Liposome@Ce6 nanoparticles also showed remarkably improved intratumoral penetration into locations far from blood vessels owing to the partly destroyed ECM structure. Taking these advantages, the photodynamic therapeutic effect of Liposome@Ce6 on tumor bearing mice could dramatically enhanced with the assistant of CLG@NCP-PEG, which in the meanwhile resulted in no notable systemic toxicity to the treated healthy mice.

Results and Discussion The scheme for the fabrication of pH sensitive NCPs, CLG encapsulation and following modification is shown in Figure 1a. Firstly, the acidic-pH-sensitive BI-linker was prepared according



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to our previous paper.36 Then the aqueous solution containing 10 mmol L-1 BI-linker and 20 mmol L1 MnCl was reacted under the atmosphere of nitrogen overnight, obtaining nanosheet-structured NCPs, 2

as shown in the transmission electron microscope (TEM) images (Figure 1b). The hydrodynamic size of NCPs determined by dynamic light scattering (DLS) was about 165 nm (Supporting Information S1a). Furthermore, in the presence of CLG and stabilizing agent polyvinyl pyrrolidone (PVP), CLGencapsulated NCP (CLG@NCP) nanostructures could be formed (Figure 1c) with loading efficiency of CLG about 44.9 %. Next, the as-made CLG@NCP nanoparticles were modified by PEG via a lipid shell method, by mixing 1, 2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, Mn2+(CLG@NCP) and

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene

glycol)-5000)

(DSPE-PEG5k) at a mole ratio of 5:2.5:10:0.5 in aqueous solution. By utilizing the commonly used method for liposome preparation,39 a clear lipid shell could be formed on the surface of CLG@NCP nanoparticles and the morphology of nanoparticles was not changed after surface modification (Figure 1d). The process of PEG modification was conducted by extrusion through a 400 nm polycarbonate filters for 40 times, which could lead to the compacted structure of CLG@NCP-PEG nanoparticles with smaller size than NCPs. The obtained CLG@NCP-PEG nanoparticles showed great stability in different solutions with the hydrodynamic size at about 106 nm (Supporting Information S1a). The similar modification process was also carried out on the bare NCPs nanoparticles (no CLG loading) and the formed NCP-PEG nanoparticles showed similar morphology and size (Supporting Information S1b). Considering the acid-triggered hydrolysis of the BI-linker,36, 40 we next studied the pH responsive behaviors of CLG@NCP-PEG. Firstly, the hydrodynamic sizes of CLG@NCP-PEG nanoparticles dissolved in solutions with different pH values were measured. As shown in Figure 1e and Supporting Information S1c, when dispersed in neutral solution at pH 7.4, the hydrodynamic diameter of CLG@NCP-PEG nanoparticles kept largely constant within 7 days, indicating the stability of those nanoparticles under neutral condition. In contrast, the hydrodynamic sizes of CLG@NCP-PEG nanoparticles decreased over time in the solution at pH 6.5, indicating the gradual dissociation of those nanoparticles. The TEM images of nanoparticles incubated at different pHs for 6 h (Figure 1f & 1g) further demonstrated the structural integrity of those nanoparticles at pH 7.4 and the collapse of NCP structure at pH 6.5.


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The release of CLG embedded in those nanoparticles was then measured. Cyanine 5.5 conjugated CLG (CLG-Cy5.5) was prepared to determine the quantity of CLG based on Cy5.5 fluorescence.41-42 The released CLG-Cy5.5 from CLG@NCP-PEG nanoparticles under different pHs was collected by ultrafiltration and recorded by fluorescence spectroscopy (Figure 1h). It was found that CLG-Cy5.5 could be gradually released from CLG@NCP-PEG nanoparticles under acidic pH (6.5), and nearly 90 % of CLG-Cy5.5 was released after 24 h of incubation. In contrast, only a small amount of CLG-Cy5.5 (6.3 %) was released at pH 7.4 within 24 h, owing to the stable encapsulation of CLG enzyme by those nanoparticles. Furthermore, the activity of CLG was also determined before and after structural destruction of NCPs (Figure 1i). After being encapsulated within NCPs, CLG within CLG@NCPPEG would largely lose its enzyme activity (pH 7.4). In contrast, the CLG released from CLG@NCPPEG nanoparticles at pH 6.5 showed ~80 % of enzyme activity compared to free CLG at the same concentration. Therefore, our pH responsive NCPs could act as a smart enzyme carrier that could protect the enzyme under normal physiological pH and release the enzyme under a specific condition (e.g. reduced pH). Additionally, x-ray photoelectron spectroscopy (XPS) was used to determine the chemical state of manganese element in CLG@NCP-PEG nanoparticles (Figure 1j). The three characteristic peaks at 652.9 eV, 645.9 eV and 641.1 eV corresponding to the Mn (II) 2p1/2 and Mn (II) 2p3/2 spin-orbit peaks, revealed the existence of Mn2+ in CLG@NCP-PEG nanoparticles.43 Owing to the facts that Mn2+ would also be released from CLG@NCP-PEG nanoparticles in the acidic buffer due to the destruction of NCP nanostructure, and Mn2+ with five unpaired 3d electrons shows strong paramagnetism for T1 magnetic resonance (MR) imaging,44 we speculated that CLG@NCP-PEG may show pH-responsive contrast under MR imaging. Therefore, T1 MR imaging of CLG@NCP-PEG nanoparticles incubated under different pHs for 6 h was conducted. As shown in Figure 1k, CLG@NCP-PEG nanoparticles under pH 6.5 showed enhanced concentration-dependent brightening effect and the relaxivity (r1) was about 9.633 mM-1s-1, while the r1 of CLG@NCP-PEG nanoparticles under pH 7.4 was as low as 3.718 mM-1s-1. The obviously enhanced T1 relaxivity of nanoparticles in the acidic buffer would likely be owing to the increased number of Mn2+-coordinated water molecules after structural degradation, leading to the decrease of longitudinal relaxation time.45 Therefore, CLG@NCP-PEG may as a pH-responsive protein carrier and MR imaging contrast agent simultaneously.



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Then, the blood circulation behaviors of CLG@NCP-PEG nanoparticles with CLG-Cy5.5 encapsulation and free CLG-Cy5.5 were also examined by utilizing fluorescence spectrometer (Figure 2a). By determining CLG concentrations in the blood samples obtained from mice at different time points post i.v. injection of CLG-Cy5.5@NCP-PEG nanoparticles, we discovered that the blood circulation of CLG@NCP-PEG showed a two phase model with half-lives at t1/2(α) of 0.058 ± 0.02 h and t1/2(β) of 22.38 ± 1.60 h. In contrast, free CLG showed a very short half-life after i.v. injection and the signals of Cy5.5-CLG disappeared after 1 h, owing to the rapid clearance of free enzyme from the body. Considering the pH-responsive MR contrasting ability of such Mn2+-containing CLG@NCP-PEG nanoparticles, we thus used MR imaging to study the in vivo behaviors of CLG@NCP-PEG nanoparticles (Figure 2b, c) in mice bearing 4T1 murine breast tumors upon i.v. injection. It was found that the MR signal intensity at tumor site was obviously increased at 24 h post injection (p.i.), indicating the high tumor accumulation of nanoparticles through the enhanced permeability and retention (EPR) and efficient Mn2+ release from nanoparticles under the acidic TME. Furthermore, the biodistribution of CLG@NCP-PEG was also determined by measuring the concentration of Mn2+ in main tissues and organs of mice via inductively coupled plasma-mass spectrometry (ICP-MS) (Figure 2d).

Although

CLG@NCP-PEG

nanoparticles

showed

significant

accumulation

in

the

reticuloendothelial systems (RES) such as spleen and liver, a high level of nanoparticles was also observed in the tumor. As mentioned above, CLG would be released from CLG@NCP-PEG nanoparticles under the acidic TME owing to the pH-responsive degradation of nanostructure. Collagen as an important component of tumorous extracellular matrix (ECM) plays a great role in tumor stiffness and fibrosis, and further results in the high IFP, interruptive blood flow and insufficient drug delivery towards tumor cells.46-47 It has been demonstrated that fibrillar collagen could be effectively degraded by exogenous collagenase, which would then result in increased tumor distribution and enhanced therapeutic efficacy of nanoagents.48 Therefore, in our system, we first tested the collagen degradation effect induced by CLG@NCP-PEG. Collagen immunofluorescence staining assay was conducted on tumor slices from mice at 24 h post injection of different agents (Figure 3a). Besides collagen staining with anti-collagen antibody (green), blood vessels and cell nuclei were stained with anti-CD31 antibody (red) and 2-(4amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (blue), respectively. Compared with


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untreated and NCP-PEG treated tumors, in which abundant collagen signals were observed, tumors on mice treated with CLG@NCP-PEG exhibited apparently decreased collagen signals, indicating significant degradation of collagen in tumors on mice after i.v. injection of CLG@NCP-PEG. It has been proven that by destructing the ECM structure in the tumor, the IFP of the tumor could be reduced and tumor vasculatures could be effectively decompressed.49-52 Then the blood perfusion assay of mice was carried out on mice after being treated with different nanoparticles by ultrasound imaging using a commercial microbubble contrast agent (Figure 3b). Untreated tumors and tumors from mice treated with blank NCP-PEG without CLG loading showed increased ultrasound signals only in the tumor boundary regions but not in the central regions of those tumors (at 24 p.i.). In marked contrast, strong ultrasound signals appeared throughout the entire tumors were observed for mice treated with CLG@NCP-PEG nanoparticles at 24 p.i. Thus, CLG@NCP-PEG nanoparticles could effectively remodel the tumor ECM by degrading the collagen, so as increase blood flow perfusion in the tumor by decreasing tumor IFP.51 It has been demonstrated that the tumor hypoxia could be effectively alleviated after degradation of ECM to improve tumor perfusion.11 Photoacoustic (PA) imaging was then applied to measure tumor oxygenation through the absorbance spectra shift of deoxygenated and oxygenated hemoglobin at 750 nm and 850 nm, respectively.53-54 From the PA imaging data (Figure 3c), the tumor oxygenation level defined as vascular saturated O2 (sO2) was greatly increased over time in the tumors treated with CLG@NCP-PEG, while no significant signal change was found in the untreated tumors as well as tumors on mice treated with bare NCP-PEG (no CLG loading). Additionally, hypoxia-inducible factor (HIF)-1α immunofluorescence staining assay of tumor slices was also conducted on the mice at 24 h p.i. to verify the hypoxia status in the tumor. As shown in Figure 3d, we found that the hypoxia signals in tumors could be significantly reduced after treatment with CLG@NCP-PEG nanoparticles, in remarkable contrast to the strong hypoxia signals in untreated tumors and NCP-PEG treated ones. These results further verified the ability of CLG@NCP-PEG nanoparticles to effectively alleviate hypoxia through increasing the intra-tumoral blood flow. Next, we wondered whether the improved tumor perfusion and destroyed tumor ECM after treatment with CLG@NCP-PEG nanoparticles would enhance the accumulation and diffusion of the second-wave of nanoparticles. Chlorin e6 (Ce6), a commonly used photosensitizer, after being modified by hexyl amine to increase its hydrophobicity was then successfully loaded into the



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hydrophobic lipid bilayer of PEGylated liposomes (Liposome@Ce6) following our reported procedure (Figure 4a).55 As reported previously, upon i.v. injection of these Liposome@Ce6 nanoparticles into tumor bearing mice, these nanoparticles showed excellent tumor uptake via the EPR effect and efficient singlet oxygen production under light irradiation. Additionally, Ce6 with great performance in chelating many metal ions (Mn2+, 64Cu2+, 99mTc4+) could be used as efficient chelating agent of 99mTc4+ for single photon emission computed tomography (SPECT) imaging, a powerful molecular imaging approach for diagnostic clinical studies with great sensitivity and high spatial resolution. Thus, 99mTc4+ labeled Liposome@Ce6 (Liposome@Ce6-99mTc) with great stability was prepared to investigate the uptake and distribution of nanoparticles in tumor bearing mice with or without the pre-treatment of CLG@NCP-PEG by SPECT imaging (Supporting Information S2). We found that the accumulation of Liposome@Ce6-99mTc in the tumor regions increased along time (Figure 4b). Notably, Liposome@Ce6-99mTc nanoparticles were mainly located at the peripheral site of tumors on mice without any pretreatment. In contrast, for mice with pre-treatment of CLG@NCP-PEG given 24 h prior to the injection of the second wave of nanoparticles, those Liposome@Ce6-99mTc nanoparticles could enter into the interior parts of tumors. The quantitative biodistribution of Liposome@Ce6-99mTc nanoparticles in the mice at 24 p.i. was determined by gamma counter. Notably, for mice with pretreatment of CLG@NCP-PEG, their tumor uptake of Liposome@Ce6-99mTc was increased by ~1.6 fold compared to those without CLG@NCP-PEG pre-treatment (Figure 4c). To understand the intra-tumoral distribution of the second-wave of nanoparticles, we also carried out immunofluorescence staining assay for tumor slices on mice post injection of Liposome@Ce6 nanoparticles (Figure 4d). In tumors from mice without pre-treatment by CLG@NCP-PEG after i.v. injection with Liposome@Ce6 nanoparticles, the fluorescence signals of Ce6 (green) were mostly colocalized with tumor blood vessels (red), suggesting the limited extravascular penetration of nanoparticles within those solid tumors. However, in tumors from mice pretreated with CLG@NCPPEG, significant Ce6 fluorescence signals appeared far from the blood vessels, indicating that these nanoparticles could leak out from blood vessels and deeply penetrated into the central part of a tumor. The possible mechanism for the enhanced tumor uptake and penetration of nanoparticles in our experiments could be attributed to the lowered collagen content and the reduced IFP after destruction of collagen by CLG delivered into and released inside the tumor by CLG@NCP-PEG. As discussed above, the oxygenation level and accumulation of photosensitizer (PS) in the tumor


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as two important factors of PDT could be significantly improved with the help of CLG@NCP-PEG by degrading the collagen within the tumor ECM. Then PDT based on Liposome@Ce6 was carried out on 4T1 tumor bearing mice. In our experiments, the mice were randomly divided into five groups (five mice in each group) when the tumor size reached to ~100 mm3: (1) Control; (2) Liposome@Ce6 ([Ce6]: 6 mg kg-1); (3) CLG@NCP-PEG ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1); (4) Liposome@Ce6 ([Ce6]: 6 mg kg-1) + Light; (5) CLG@NCP-PEG ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1) and Liposome@Ce6 ([Ce6]: 6 mg kg-1) + Light. The mice in group 5 were i.v, injected Liposome@Ce6 nanoparticles at 24 h post injection of CLG@NCP-PEG. The PDT of mice in group 4 and 5 were carried out at 24 h post injection of Liposome@Ce6 under 660 nm light irradiation (5 mW cm-2, 60 min). The tumor sizes and body weights of mice were recorded every the other day after receiving treatment (Figure 5a, Supporting Information S3a). Additionally, all the mice were sacrificed on the 12th day and then the tumors were extracted for weighing and photographing (Supporting Information S3b, Figure 5b). We observed that although Liposome@Ce6-based PDT could delay the growth of tumors in the first few days post light irradiation, tumors in this group grew quite fast later on. Importantly, although CLG@NCP-PEG treatment alone showed no obvious tumor growth inhibition effect, pretreatment of mice with CLG@NCP-PEG could dramatically enhance the therapeutic effect of PDT by Liposome@Ce6 nanoparticles (Group 6) to effectively inhibit the tumor growth for those mice. As revealed by hematoxylin and eosin (H&E) staining of tumor slices (Figure 5e), more severe PDT-induced tumor cell destruction was observed for mice in group 6, in which Liposome@Ce6-based PDT was conducted after pre-treatment of tumor-bearing mice with CLG@NCP-PEG. Finally, we also tested the long-term toxicology of CLG@NCP-PEG ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1) in healthy nude mice. At 1, 7, 14 days post i.v. injection of CLG@NCP-PEG nanoparticles, the histological examination of H&E stained main organs from mice showed no significant toxic side effect in our experiments (Supporting Figure S4). In the meanwhile, the serum biochemistry assay and complete blood panel test were also conducted at the same time points. Notably, all measured parameters fell within normal ranges and appeared to be similar compared to the control group (Table 1). Our results therefore demonstrated that i.v. injected CLG@NCP-PEG nanoparticles showed no significant acute toxicity to mice at our tested dosage within 14 days. Considering the inherent biodegradability of those NCPs, we conclude that CLG@NCP-PEG may be a safe enzyme



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delivery system promising for further in vivo use.

Conclusion In conclusion, we have successfully prepared a unique type of pH sensitive NCPs with CLG encapsulation to enhance the nanomedicine-based PDT treatment of cancer via multiple mechanisms. Owing to the acidity-triggered breaking of the organic BI-linker, our pH sensitive NCPs while being stable under neutral pH could be rapidly dissociated within the acidic TME. Therefore, CLG stably encapsulated within the NCPs during blood circulation could be released specifically within the tumor in response to the reduced tumoral pH, and then enable the effective degradation of collagen to destruct the condensed tumor ECM. As the results, greatly enhanced tumor perfusion, effectively relieved tumor hypoxia, as well as remarkably improved tumor retention and intra-tumoral penetration of the second-wave of therapeutic nanoparticles, Liposome@Ce6 as a model in our study, could be realized. All those effects acting together would lead to the synergistically enhanced nanomedicine-based PDT therapeutic outcome for mice pretreated with CLG@NCP-PEG. In addition, no noticeable systemic side effect was observed in mice upon receiving such combination therapy. Our work shows the great promises of employing NCPs as a new type of smart enzyme carrier to realize tumor-targeted delivery and specific release. Moreover, we demonstrate that modulating ECM within the TME by enzymeloading nanoparticles may be an effective approach to improve existing cancer therapies, not only for nanomedicine-based PDT as illustrated in this work, but also for other types of therapies based on nanoscale therapeutics, such as chemotherapy-drug-loaded nanomedicine, or even immune checkpoint blockade antibodies.

Methods Materials Collagenase (CLG), hexylamine, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Chlorin e6 (Ce6) and cholesterol were obtained from J&K Scientific Ltd. The EnzChek Gelatinase/Collagenase Assay Kit was purchased from Molecular Probes. BCA Protein Assay Kit was obtained from Thermo Scientific. Other chemical reagents were obtained from China National Pharmaceutical Group Corporation.


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Characterization The hydrodynamic sizes of nanoparticles were measured by a Zetasizer Nano-ZS (Malvern Instruments, UK). Transmission electron microscopy (TEM) images were obtained by utilizing a Philips CM300 transmission electron microscope operating at an acceleration voltage of 200 kV. The concentration of Mn ions was measured by inductively coupled plasma -mass spectrometry (ICP-MS).

Synthesis of NCPs and CLG@NCP nanoparticles The solution containing 6.1 mg MnCl2 and 7.9 mg BI-linker was reacted under the atmosphere of nitrogen overnight. The obtained light yellow solution was washed with water for three times. Then, the purified NCPs nanoparticles were re-dispersed in water for further use. For the fabrication of CLG@NCP nanoparticles, 7.9 mg BI-linker, 5.7 mg CLG and 30 mg polyvinylpyrrolidone (PVP) were dissolved in water, then a solution containing 6.1 mg MnCl2 was added drop by drop under the atmosphere of nitrogen overnight. The obtained solution was washed with water for three times. Then, the purified CLG@NCP nanoparticles were re-dispersed in water for further use.

PEGylation of NCPs and CLG@NCP nanoparticles Firstly, 15 mg DPPC, 5 mg cholesterol and 10 mg DSPE-mPEG5k were dissolved in the solution of CHCl3, then the solution was vortically evaporated under 40 oC to remove the solvent. Then, the dry lipid film was obtained at the wall of the round-bottom flask. The formed dry lipid film was hydrated by the solutions of NCPs and CLG@NCP nanoparticles under stirring, then rehydrated with five repeated freezing and thawing cycles. The obtained liposome suspensions were extruded through a 400 nm polycarbonate filters at 43 oC for 40 times and formed NCP-PEG and CLG@NCP-PEG nanoparticles for further use. The preparation of Liposome@Ce6 nanoparticles according to our previous article.55

pH responsive degradation and CLG release To study the degradation behavior, the hydrodynamic diameters of CLG@NCP-PEG (1 mg mL1)

in pH 6.5 and 7.4 PBS solution at different time points were tested. In the meanwhile, the released

CLG removed by ultra-filtration was determined by fluorescence spectrophotometer through



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measuring the florescence intensity of dye conjugated on CLG.

Catalytic activity of CLG The catalytic activity of free CLG and CLG@NCP-PEG could be tested according to the EnzChek Gelatinase/Collagenase Assay Kit following the vendor’s protocol (Molecular Probes).

Tumor models Nude mice purchased from Nanjing Peng Sheng Biological Technology Co. Ltd. were used under protocols approved by the Soochow University Laboratory Animal Center.

In vivo MR imaging and biodistribution of CLG@NCP-PEG nanoparticles 4T1 tumor-bearing nude mice were i.v. injected with CLG@NCP-PEG nanoparticles ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1) for in vivo MR imaging. For biodistribution, the tumor bearing mice at 24 h p.i. of CLG@NCP-PEG were executed and the main organs and tumor were collected. Then the quantity of Mn ions in each tissue was determined by ICP-MS according to our previous article.36

In vivo SPECT imaging and biodistribution of Liposome@Ce6 nanoparticles 1 ml Liposome@Ce6 nanoparticles (2 mg mL−1) were mixed with 500 µL stannous chloride (SnCl2, 2 mg mL−1) and ascorbic acid (2 mg mL−1). Then 1 mL radioactive technetium-99m (99mTc) precursor, Na99mTcO4, was added into the mixed solution and reduced from

99mTcVII

to

99mTcIV

by

SnCl2 at 37 oC for an hour. The obtained Liposome@Ce6-99mTc was collected to determine the labeling efficiency. After 24 h p.i. of NCP-PEG and CLG@NCP-PEG, the tumor bearing mouse was i.v. injected with Liposome@Ce6-99mTc (0.6 mg mL−1) for in vivo SPECT imaging. At 24 h p.i., the ex vivo biodistribution was determined according to our previous article.56

In vivo ultrasound imaging and PA imaging For in vivo ultrasound imaging, tumor bearing mice at 24 h p.i. of NCP-PEG and CLG@NCPPEG nanoparticles were further i.v. injected with a commercial microbubble ultrasound contrast agent


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(MB, Fujifilm) and imaged according to our previous article.54 For in vivo PA imaging, tumor bearing mice at different time intervals p.i. of NCP-PEG and CLG@NCP-PEG nanoparticles were imaged using the Visualsonic Vevo 2100 LAZER system. The tumor saturated O2 (sO2) indicated variations in tumor hypoxia was recorded according to previous paper.53

Immunofluorescence staining For collagen staining, mice bearing 4T1 tumors were sacrificed at 24 h p.i. of NCP-PEG and CLG@NCP-PEG nanoparticles and the tumors were dissected for making frozen sections. Then these sections were incubated with primary antibodies to collagen I (dilution 1:200, ab34710) and FITCconjugated goat-anti-rat secondary antibody (dilution 1:500, Jackson Inc.) according to the kit’s instructions. For HIF-1α staining and following blood vessels and nuclei staining were conducted according to our previous paper.35, 53

In vivo PDT Nude mice bearing 4T1 tumors (~100 mm3) were randomly divided into five groups: (1) Control; (2) Liposome@Ce6 ([Ce6]: 6 mg kg-1); (3) CLG@NCP-PEG ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1); (4) Liposome@Ce6 ([Ce6]: 6 mg kg-1) + Laser; (5) CLG@NCP-PEG ([Mn]: 6 mg kg−1, [CLG]: 6.73 mg kg−1) and Liposome@Ce6 ([Ce6]: 6 mg kg-1) + Laser. The tumor volumes were recorded every the other day.

Associated content Supporting Information Hydrodynamic size of NCP and CLG@NCP-PEG, TEM image of NCP-PEG, Size change of CLG@NCP-PEG within 7 days, Radiolabeling stability of Liposome@Ce6-99mTc4+, Body weight data of mice in different groups, Tumor mass data in different groups, H&E stained images of major organs obtained from CLG@NCP-PEG treated mice and healthy nude mice.

Acknowledgement



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This work was partially supported by the National Basic Research Programs of China (973 Program) (2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041), a Collaborative Innovation Center of Suzhou Nano Science and Technology, a ‘111’ program from the Ministry of Education of China, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Figure 1. The synthesis and characterization of CLG@NCP-PEG nanoparticles. (a) The scheme for the preparation of NCP nanoparticles and CLG encapsulated CLG@NCP nanoparticles, surface modification and pH sensitive degradation. (b, c, d) TEM images of NCP, CLG@NCP and CLG@NCP-PEG nanoparticles. (e) The hydrodynamic diameter change of CLG@NCP-PEG nanoparticles dissolved in pH 7.4 and 6.5 buffer solutions. (f, g) TEM images of CLG@NCP-PEG nanoparticles incubated at pH 7.4 or 6.5 for 6 h. (h) The release of CLG from CLG@NCP-PEG nanoparticles dispersed in different buffer solutions. (i) The enzyme activity of CLG in CLG@NCPPEG nanoparticles before and after 24 h incubation with acidic buffer solution (pH 6.5), free CLG was used as control. (j) The XPS spectrum of CLG@NCP-PEG nanoparticles. (k) T1-weighted MR images of CLG@NCP-PEG nanoparticles under various concentrations. The samples were incubated in pH 7.4 and 6.5 buffer solutions for 6 h prior to MR imaging. The longitudinal relaxivities (r1) of CLG@NCP-PEG at pH 7.4 and 6.5 were determined to be 3.718 mM-1s-1 and 9.633 mM-1s-1, respectively.


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Figure 2. In vivo behaviors of CLG@NCP-PEG nanoparticles. (a) Blood circulation of CLG@NCPPEG and free CLG in mice by measuring the concentration of CLG in blood at different time points p.i.. (b) In vivo T1-weighted MR images of a tumor bearing mouse taken before and 24 h p.i. of CLG@NCP-PEG nanoparticles. (c) T1-weighted MR signals in the tumor before and 24 h p.i. of CLG@NCP-PEG. (d) Biodistribution of CLG@NCP-PEG in tumor bearing mice by measuring the Mn2+ ion levels in various organs and tissues (Li: Liver; Sp: Spleen; Ki: Kidney; H: Heart; Lu: Lung; St: Stomach; In: Intestine; T: Tumor).



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Figure 3. TME modulation with CLG@NCP-PEG by degrading the collagen of ECM. (a) Immunofluorescence images of tumor slices after being treated with NCP-PEG and CLG@NCP-PEG nanoparticles. Untreated tumor slices were served as the control. The nuclei of cells (blue), tumor blood vessels (red) and collagen (green) were stained with DAPI, anti-CD31 antibody, and an antibody specifically bind to collagen, respectively. The right column shows relative collagen positive areas as


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recorded from more than ten micrographs of each group. (b) Ultrasound images of tumor vascular perfusion after i.v. injection of microbubble contrast agent. The tumors were pretreated with NCPPEG and CLG@NCP-PEG nanoparticles. The right column shows contrast mean power intensity of tumor from different groups over time. (c) Representative 2D PA images of tumors on mice before and after i.v. injection of NCP-PEG and CLG@NCP-PEG nanoparticles. The right column shows quantification of the oxyhemoglobin saturation in the tumor from different groups over time. (d) Immunofluorescence images of tumor slices after hypoxia staining. The HIF-1α (green), cell nuclei (blue), and blood vessels (red) were stained by an antibody specifically bind to HIF-1α, DAPI and anti-CD31 antibody, respectively. The right column shows relative HIF-1α positive areas as recorded from more than ten micrographs of each group.



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Figure 4. Enhanced tumor accumulation and penetration of Liposome@Ce6 nanoparticles after treatment with CLG@NCP-PEG. (a) The structure of Liposome@Ce6-99mTc. (b) In vivo SPECT imaging of tumor bearing mice at 12 h and 24 h post i.v. injection of Liposome@Ce6-99mTc. Mice with (right) and without (left) pretreatment of CLG@NCP-PEG were included. (c) Biodistribution of Liposome@Ce6-99mTc determined at 24 h p.i. (d) Confocal fluorescence images of tumor slices from mice at 24 post i.v. injection of Liposome@Ce6. The green, blue and red signals were from the fluorescence of Ce6, DAPI stained nuclei and anti-CD31 stained blood vessels, respectively.


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Figure 5. In vivo PDT with Liposome@Ce6 enhanced after treatment with CLG@NCP-PEG. (a) Tumor growth curves of mice after receiving various treatments including: (1) Untreated control, (2) Liposome@Ce6 alone, (3) CLG@NCP-PEG alone, (4) Liposome@Ce6 + Laser, and (5) CLG@NCPPEG + Liposome@Ce6 + Laser (5 mW cm-2, 60 min). (b) The photo of tumors obtained from mice from various groups collected at day 12. (c) H&E stained images of tumor slice collected 24 h after laser irradiation (day 2).



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Table 1. Serum biochemistry assay and complete blood panel data for mice treated with CLG@NCPPEG determined at various time points. The measured parameters included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and blood urea nitrogen (BUN), hematocrit (HCT), blood levels of white blood cells (WBC), hemoglobin (Hgb), red blood cells (RBC), platelets (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Statistic was based on 5 mice per data point.


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