Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide

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Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide Donor to Induce Tumor Collagen Depletion Xiao Dong, Hai-Jun Liu, Hai-Yi Feng, Si-Cong Yang, Xue-Liang Liu, Xing Lai, Qin Lu, Jonathan F Lovell, Hong-zhuan Chen, and Chao Fang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide Donor to Induce Tumor Collagen Depletion Xiao Dong,† Hai-Jun Liu,† Hai-Yi Feng,† Si-Cong Yang,† Xue-Liang Liu,† Xing Lai,† Qin Lu,† Jonathan F. Lovell, ‡ Hong-Zhuan Chen,† Chao Fang†* †

Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Department of

Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine (SJTU-SM), 280 South Chongqing Road, Shanghai 200025, China ‡

Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo,

NY 14260, USA ABSTRACT: Delivery of therapeutics into the solid tumor microenvironment is a major challenge for cancer nanomedicine. Administration of certain exogenous enzymes which deplete tumor stromal components has been proposed as a method to improve drug delivery. Here we present a protein-free collagen depletion strategy for drug delivery into solid tumors, based on activating endogenous matrix metalloproteinases (MMP-1 and -2) using nitric oxide (NO). Mesoporous silica nanoparticles (MSN) were loaded with a chemotherapeutic agent, doxorubicin (DOX) as well as a NO donor (S-nitrosothiol) to create DN@MSN. The loaded NO results in activation of MMPs which degrade collagen in the tumor extracellular matrix. Administration of DN@MSN resulted in enhanced tumor penetration of both the nanovehicle and cargo (DOX), leading to significantly improved antitumor efficacy with no overt 1

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toxicity observed. KEYWORS: Nitric oxide, nanoparticles, tumor penetration, collagen depletion, matrix metalloproteinase

The integration of nanotechnology in cancer therapy has led to the successful translation of several nanomedicines in cancer clinic, such as Doxil (liposomal doxorubicin), Onivyde (liposomal irinotecan), Abraxane (albumin-bound paclitaxel), and Genexol-PM (polymeric micelle paclitaxel).1 However, the clinical performance of these nanomedicines leaves much room for improvement to prolong patient survival.1, 2 Except the newly approved Vyxeos (liposome-encapsulated daunorubicin-cytarabine) for hematological malignancy (acute myeloid leukemia) with a 3.7 months overall survival (OS) improvement, other marketed liposome-based nanomedicines for solid tumors do not contribute an significant OS benefit compared to the conventional parent drug.1 Poor nanoparticle penetration in solid tumors is thought to be a major factor which limits the clinical benefits of nanomedicines.3-5 Nanoparticles accumulate in tumor sites through the enhanced permeation and retention (EPR) effect.6-8 However, their distribution is usually confined to perivascular areas, with limited ability to penetrate into deeper tumor parenchyma due to the dense interstitial matrix, which comprises major components of collagen and hyaluronan.8 A representative study indicated that a large proportion of the Doxil extravasated from the tumor blood vessels only located around the blood vessel walls of tumors, but did not penetrate into the deep tumor tissue.9 Therefore this apparent targeted macrodistribution in tumors through EPR mechanism conceals microdistribution shortcoming which 2


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result in insufficient drug exposure towards deeper cancer cells.8 The exposure of sublethal drug concentrations can even lead cancer cells to develop a drug-resistant phenotype.3 Many strategies have been explored to improve nanoparticle penetration.10, 11 The modification of penetration-promoting ligands (typically iRGD) on the nanoparticles is widely reported for enhanced penetration. This method relies on the accessibility of the related receptor in both tumor endothelium and tumor cells.12, 13 Moreover, the effectiveness of iRGD is facing debate.14 The smaller sized nanoparticles can rapidly diffuse throughout the tumor matrix, compared to those larger ones that only stay near the vasculature.15, 16 Size switchable nanoparticles, which shrink into smaller sized particles (< 50 nm) in response to endogenous stimuli in tumor microenvironment (matrix metalloproteinase (MMP)3, 17-19 and low pH20) or external stimulus (light)21,

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have been engineered. The need for an external stimulus

restricts applications to local tumors and hinders wide application. Recently, the direct use of enzymes to deplete tumor matrix (collagen, hyaluronic acid) for facilitated nanoparticle penetration was developed. Bromelain23 or hyaluronidase24 were modified on the nanoparticle surface, and the enzymatic hydrolysis of collagen and hyaluronic acid swept away the penetrating barrier of nanoparticles. However, the surface modification of high molecular weight enzymatic proteins increases the complexity of single nanoparticle fabrication. It is also noted that PEGylated recombinant human hyaluronidase (PEGPH20) combined with nab-paclitaxel and gemcitabine is undergoing phase III trial in patients with hyaluronic acid-high pancreatic ductal adenocarcinoma.25, 26 Recently, losartan, an approved angiotensin inhibitor, was found to inhibit collagen I production and improve the penetration and efficacy of nanomedicines.17, 27-29

Nitric oxide (NO) is an endogenous free radical with multiple biological activities, such as 3


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vasodilation, inhibition of platelet aggregation, anti-inflammation, and immune defense.30 Under pathological conditions, NO rapidly reacts with the superoxide anion (O2•-) to form the more powerful oxidant peroxynitrite (ONOO-), which is a key mediator of lipid peroxidation, protein nitration and oxidation, DNA oxidative damage.30, 31 Peroxynitrite-mediated cysteine oxidation activates pro-matrix metalloproteinases (pro-MMP) into active MMPs, which has been observed in inflammation condition (MMP-1, -8 and -9)32 and tumor tissues (MMP-2 and -9).33 MMPs are a family of zinc-dependent endopeptidases produced and secreted by tumor cells and stromal cells such as carcinoma-associated fibroblasts.34, 35 MMPs can degrade almost every component of the extracellular matrix (ECM), and their activities and activation are regulated by tissue inhibitors of metalloproteinases (TIMPs, the endogenous inhibitors). Although the MMP activity is usually higher in tumors than that in normal tissues, there is still a dense ECM in tumors which hinders the penetration of nanoparticles following extravasation from blood vessels. For this reason, many recent efforts have examined ECM degradation strategies to improved nanoparticle penetration.23, 24, 27, 28 Based on these previous findings, we hypothesize that the activated MMPs induced by NO may functionally disintegrate the tumor matrix collagen, and facilitate the penetration of nanoparticles. In this work, we confirm this hypothesis by using an engineered NO donor-assisted, doxorubicin (DOX)-loaded PEGylated mesoporous silica nanoparticles (DN@MSN) in the orthotopic 4T1 breast cancer model. Notably, NO has previously been explored as an EPR-enhancer to increase nanoparticle accumulation in tumors.36-42 The mechanism is claimed to be the NO-induced relaxation of smooth muscle cells (also popularly named pericytes as for those in tumors)43-46 to dilate vessels through the cyclic GMP signaling pathway, presenting a pericyte-dependent vasodilating modality.31 However, to our knowledge, this work 4


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is the first report revealing the role of NO in depleting tumor matrix collagen for enhanced nanoparticle penetration. Also, the depletion of tumor collagen can, in a pericyte-independent way, help decompress or dilate the interior blood vessels to facilitate the perfusion of nanoparticles into tumor deep region and promote EPR effect. The main steps for the preparation of DN@MSN is illustrated in Scheme 1A. Thiol groups were introduced on the nanoparticle surface through the modification of 3-mercaptopropyltrimethoxysilane (MPTMS). Then, cetrimonium bromide (CTAB, the surfactant template) was removed to generate the empty nanoparticles with the surface modified with SH (MSN-SH). Then, part of the thiol groups were reacted with Methoxy-PEG5000-malemide to confer PEGylation of the nanoparticle surface. The residual thiols were transformed into S-nitrosothiols (a common NO donor) by reaction with NaNO247-49 to produce S-nitrosothiol-loaded nanoparticles (N@MSN). Lastly, DOX was loaded into the N@MSN to generate DN@MSN. The nanoparticles penetrate deeply into tumor parenchyma via NO-mediated collagen depletion for enhanced antitumor efficacy (Scheme 1B). Distinct from previous strategies which directly using whole active enzymes to degrade matrix, this study presents an enzyme-activating, matrix depleting strategy to improve nanoparticle penetration.

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Scheme 1. Schematic representation of DN@MSN preparation (A) and the proposed mechanism for NO donor-mediated collagen depletion and improved nanoparticle penetration in tumors (B). DN@MSN were spherical with a diameter of ~100 nm in the observation using transmission electron microscopy (TEM) (Figure 1A), which was consistent with the observation of its hydrodynamic size (107.5 nm) determined by DLS (Figure 1B). The introduction of S-nitrosothiols conferred a pink color to the nanoparticle and a specific shoulder peak at ~335 nm as shown in the UV-Vis spectrum 6


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(Figure S1).50 DOX loading resulted in red nanoparticles, identified by a ~ 488 nm absorbance peak (Figure S1). Compared to the D@MSN with zeta potential of -24.8 mV, the inclusion of S-nitrosothiols (DN@MSN) led to a more negative surface charge of -40.9 mV (Figure 1C). Colloidal stability of the nanoparticles (N@MSN, D@MSN, and DN@MSN) in 10% FBS supplemented PBS at 37 °C were well maintained for 48 h at least (Figure 1D). The loading of NO (converted from S-nitrosothiol) and DOX in DN@MSN were 3.6 μg/mg and 33.4 μg/mg, respectively. DOX release exhibited a pH-dependent profile, suggesting a faster release when the nanoparticles are internalized in the acid lysosomes of tumor cells (Figure 1E). The release half-lives of NO were shorter in acidic conditions (15.1 h for pH 5.5 and 18.3 h for pH 6.8) than that in neutral environment (22.2 h for pH 7.4). This observation is consistent with previous report showing that NO release from Snitrosoglutathione (a kind of S-nitrosothiol) was accelerated under acidic conditions.51 Thus NO release form the nanoparticles in this study is expected to be accelerated in tumor sites, which may be favorable for MMP activation (Figure 1F). The observation of a long release half-life from S-nitrosothiol on silica nanoparticles is consistent with the other reported studies.52 Compared to other NO donors such as diazeniumdiolate with a very short NO release half-life (Figure S2),53 the sustained NO release conferred by S-nitrosothiol in this study might consistently help the nanoparticles hydrolyze and deplete collagen during the process of penetrating tumor tissue, forming a positive feedback cascade (Scheme 1B). We then investigated the cytotoxicity of the nanoparticles in 4T1 cells, the model cancer cell in this study. The NO donor alone (N@MSN) only induced moderate toxicity to the cells at the tested concentration, nor did its combination with DOX (DN@MSN) lead to increased cell toxicity compared to DOX alone (D@MSN) (Figure 1G). This result was confirmed in a dynamic cell proliferation assay 7


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(Figure S3). It is known that NO can act synergistically with anticancer drugs through several mechanisms, such as induction of DNA strand breaks, severe depletion of cellular ATP and NAD+, upregulation of p53 genes, blocking of P-glycoproteins,38, 54 and these effects are thought to be also partially exerted by peroxynitrite (ONOO-), the key mediator generated from NO and O2.-.30 The low cytotoxic results in our study may be ascribed to the relatively low S-nitrosothiol dose tested.55 NO release in both cells and tumors was identified using the DAF-FM DA fluorescent probe.38 Using this approach, it was observed that NO levels increased 5.4-fold in 4T1 cells and 4.3-fold in 4T1 tumors treated by N@MSN compared to those treated with empty MSN (Figure 1H, I, J).

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Figure 1. DN@MSN characterization. (A) TEM image of DN@MSN. (B) Size and (C) Zeta potential determined by dynamic light scattering (DLS). (D) Colloid stability in 10% FBS supplemented PBS at 9


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37 °C during 48 h incubation. Cumulative DOX (E) and NO (F) release in PBS (pH 7.4, 6.8 and 5.5). (G) 4T1 cell viabilities after treatment with DN@MSN and the controls (empty MSN, N@MSN and D@MSN). Representative images showing NO in 4T1 cells (H) and tumors (I) detected using DAF-FM DA fluorescent probe. (J) Quantified fluorescence intensity in panel H and I. Values are expressed as mean ± s.d. (n = 3), ***p< 0.001. The formation of ONOO- is the first step for MMP production (Figure 2A). As it is difficult to directly determine the production of ONOO-, 3-nitrotyrosine (3-NT) in proteins is commonly used as a biomarker for ONOO- activity.56 Immunohistochemical staining against 3-NT was performed at 48 h after intravenous nanoparticle injection. Compared to saline, empty MSN alone could not induce more 3-NT in tumors (Figure S4). However, NO-releasing nanoparticles (N@MSN and DN@MSN) resulted in nearly 2 fold increased amount of 3-NT (Figure 2B, C). The ONOO- scavenger uric acid, administered orally (1 g/kg), could significantly suppress the 3-NT formation. Taken together, these data demonstrate that administration of N@MSN and DN@MSN increased NO and ONOO- levels in treated cells and tumors. Then we investigated if the increase in ONOO- could lead to increased expression of MMP enzymes. Two well-studied and representative MMPs (MMP-1 and -2)32, 33, from more than 20 MMPs,35 were selected for analysis. These two MMPs hydrolyze various types of collagen, are expressed in multiple types of cancer, and have been shown to be activated by peroxynitrite in inflammation condition and tumors.32, 33, 57, 58 MMP-1 and -2 detection significantly increased by up to 3.5 fold in tumors treated with NO-releasing nanoparticles (Figure 2D, E, F). The ONOO- scavenger uric acid clearly blocked this effect, indicating the relevance between ONOO- formation and increased MMP expression. The increased MMP 10


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levels correlated with enhanced enzymatic activity that was assessed with in situ zymography (Figure 2G, H). Again, low MMP activity was observed in the tumors treated with uric acid. Notably, clear depletion of collagen I, the main matrix component hindering nanoparticle penetration in various types of cancer,27 was observed in the tumors treated with NO-releasing nanoparticles (N@MSN, DN@MSN) (Figure 2I, J). Uric acid inhibition also hindered this change, and MSN (empty) did not affect the collagen content in tumors (Figure S5). Such decreased collagen may be ascribed to the induced, increased levels of MMP-1 and -2 in both content and activity (Figure 2BH).

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Figure 2. NO-releasing nanoparticles activate MMPs and degrade collagen through peroxynitrite (ONOO-) intermediates. (A) Schematic mechanism of ONOO--induced MMP production and collagen degradation. ONOO- oxidizes the sulphydryl moiety of the cysteinyl group coordinated to the Zn2+ ion of pro-MMP. Then the active pro-MMP undergos inter- and intramolecular proteolysis to yield active MMP without the propeptide domain. The active MMP is then secreted outside the cells for collagen 12


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depletion. (B) Immunohistochemical examination of 3-nitrotyrosine (3-NT, brown) in tumors. The cell nuclei (green) were stained with methyl green. (C) Quantitative assay of 3-NT intensity in panel B. (D) Western blot assay showed the increased production of MMP-1, and 2 induced by NO-releasing nanoparticles. (E, F) Statistical assay of MMP-1, and 2 contents. (G) In situ zymography assay of tumor cryosections (10 μm). Green, fluorescence signal resulted from the hydrolysis of DQ collagen (quenched fluorescence due to heavily conjugated fluorescein) by collagen enzymes; Blue, DAPI. The green fluorescent intensity reflects the collagenase activity. (H) Quantitative comparison of the green fluorescent intensity in panel G. (I) Collagen I immunofluorescent staining of tumors. Red, collagen I; Blue, DAPI. (J) Quantitative comparison of the collagen fluorescence intensity in panel I. Values are presented as mean ± s.d. (n = 3~5). ***p

Enhanced Drug Delivery by Nanoscale Integration of a Nitric Oxide

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