Targeting Tumor Microenvironment by Bioreduction-Activated

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

Targeting Tumor Microenvironment by BioreductionActivated Nanoparticles for Light-Triggered Virotherapy S.-Ja Tseng, Ivan M Kempson, Kuo-Yen Huang, Hsin-Jung Li, Yu-Chen Fa, Yi-Cheng Ho, Zi-Xian Liao, and Pan-Chyr Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02813 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 2, 2018

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 35 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

ACS Nano

Targeting Tumor Microenvironment by Bioreduction-Activated Nanoparticles for LightTriggered Virotherapy S.-Ja Tseng,†,‡,▼ Ivan M. Kempson,§,▼ Kuo-Yen Huang,▲, Hsin-Jung Li, ⊥ Yu-Chen Fa,# Yi∥

Cheng Ho,¶ Zi-Xian Liao,*,# and Pan-Chyr Yang*,▲,△ †

Graduate Institute of Oncology and △Department of Internal Medicine National Taiwan University College of Medicine, Taipei 10051, Taiwan ‡

§

National Taiwan University YongLin Scholar

Future Industries Institute, University of South Australia, Mawson Lakes, S.A. 5095, Australia

▲

Institute of Biomedical Sciences and ⊥Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, Taiwan

∥

Graduate Institute of Health Industry Technology and Research Center for Industry of Human

Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan #

Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

¶

Department of BioAgricultural Science, National ChiaYi University, Chiayi City 60004, Taiwan

ACS Paragon Plus Environment

1

ACS Nano 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 2 of 35

*E-mail: [email protected] (Z.-X. Liao). *E-mail: [email protected] (P.-C. Yang).


2

Page 3 of 35 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

ACS Nano

Abstract

Solid tumors characteristically display higher levels of lactate production due to anaerobic metabolism of glucose. Meanwhile, the US Food and Drug Administration (FDA) has approved virotherapy for use in cancer treatment, however systemic administration remains as a particular challenge. Here we report exploitation of tumor lactate production in designing a hypoxiaresponsive carrier, self-assembled from hyaluronic acid (HA) conjugated with 6-(2nitroimidazole)hexylamine, for localized release of recombinant adeno-associated virus serotype 2 (AAV2). The carrier is loaded with lactate oxidase (LOX) and is permeable to small molecules such as the lactate that accumulates in the tumor. Subsequently, LOX oxidizes the lactate to pyruvate inside the carrier, accompanied by internal lowering of oxygen partial pressure. Bioreduction of the 2-nitroimidazole of the HA conjugated with 6-(2-nitroimidazole)hexylamine converts it into a hydrophilic moiety and electrostatically dissociates the carrier and virus. Efficacious and specific delivery was proven by transduction of a photosensitive protein (KillerRed) enabling significant limitation in tumor growth in vivo with photodynamic therapy. An approximate 2.44-fold reduction in tumor weight was achieved after a 2-week course, compared with control groups. Furthermore, conjugation of the AAV2 with iron oxide nanoparticles (‘Magnetized’ AAV2) facilitated magnetic resonance imaging (MRI) tracking of the virus in vivo. Taken together, the solid tumor microenvironment promotes bioreduction of the lactate-responsive carrier, providing rapid and specific delivery of AAV2 for light-triggered virotherapy via systemic administration.

KEYWORDS: virotherapy, tumor microenvironment, nanoparticle, lactate oxidase, photodynamic therapy


3

ACS Nano 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 4 of 35

A high rate of anaerobic glycolysis in solid tumors contributes to an acidification of pH to ~6.7–7.2 in the tumor microenvironment due to lactate accumulation.1-3 pH variations have been exploited to control the delivery of drugs in specific organs when subtle environmental changes are associated with pathological indications.4-6 The main existing strategies for pH-sensitive polymeric carriers are either use of polymers with ionizable groups that undergo conformational and/or solubility changes in response to environmental pH variation4,6 or polymeric systems with acid-sensitive bonds whose cleavage enables the release of molecules anchored to the polymer backbone.5 Efficient delivery systems sensitive to pH are required to provide a dramatic response to a subtle change in pH existing in the tumor extracellular microenvironment. Sufficient sensitivity of such systems to a discrete variation of pH is not a straightforward achievement, and challenges related to delivery beyond vascular periphery and extravasation deeper into the tumor mass need addressing. A particular opportunity exists for triggered release based on lactate accumulation in hypoxic tumor microenvironments for triggered drug release, rather than a direct pH-response. This conceptually facilitates greater penetration into tumor tissues and targets hypoxic regions that correlate with poorer prognosis.7,8 Tumor microenvironment stimulated release, optimized to correlate with the degree of hypoxia is thus alluring to achieve preferential therapeutic response in tumor volumes that lead to poor outcomes. 2-Nitroimidazole has been applied in bioimaging of tumor-associated hypoxia due to a high sensitivity to the hypoxic condition.9-11 The hydrophobic 2-nitroimidazole is reductively activated to hydrophilic 2-aminoimidazole12 and is a promising candidate for triggering a response in hypoxic tumor volumes. Additionally, virotherapy in itself is becoming a promising class of cancer therapeutics which has successfully advanced to clinical use.13 However, a major challenge is to achieve specificity and thus minimize undesirable ectopic transduction.


4

Page 5 of 35 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

ACS Nano

Recombinant adeno-associated virus serotype 2 (AAV2) has shown significant promise at both preclinical and clinical stages.14 We previously developed magnetically controllable AAV2 achieved via chemical conjugation with magnetic nanoparticles for micro-virotherapy.15,16 While the ‘magnetized’ virus can enable magnetic guidance and in-vivo tracking by magnetic resonance imaging (MRI),17 challenges remained with regard to the extent of extravasation and generating a three-dimensional (3-D) magnetic field conformal to the tumour volume.18 Furthermore, conventional techniques to assess viral biodistribution,19 such as polymerase chain reaction (PCR) and immunohistochemistry, although sensitive, are highly laborious and lack vital real-time, noninvasive information which is ideal for clinical development and translation. A solution to these challenges is encapsulation of virus in a material responsive to lactate in the tumor microenvironment. Upon the carrier reaching a tumor, a stimulus releases virus and confines viral infection within local tissue. Lactate oxidase (LOX) is useful for enzymatic catalysis of lactate oxidation, producing pyruvate and H2O2 as end products.20 Taken together, this literature collectively motivated the design of a lactate-activated hypoxia-responsive carrier (lactate-responsive nanoparticle) for tumor virotherapy, as reported here (figuratively represented in Figure 1A). The lactate-responsive nanoparticle, loaded with LOX and magnetized virus, was designed to provide specific transduction within hypoxic, lactate-rich, tumor microenvironments. The hydrophobic side-chains (2-nitroimidazole) of hyaluronic acid (HA) conjugated with 6-(2nitroimidazole)hexylamine are reduced into hydrophilic chains (2-aminoimidazole), resulting in disassembly of the nanoparticles, subsequently releasing AAV2. The validity of this concept was successfully tested with viral delivery of a photodynamic therapy (PDT) sensitizer, circumventing both ectopic transduction and side-effects due to the sensitizer and providing tumor bioreduction-activated light-triggered virotherapy.


5

ACS Nano 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 6 of 35

Results and Discussion Characterization of Lactate-responsive Nanoparticles in Acidic Environments. The incorporation of the hydrophobic group of 6-(2-nitroimidazole)hexylamine was accomplished with

conjugation

with

hyaluronic

acid

(HA)

through

1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysulfosuccinimide (SulfoNHS).12

The

6-(2-nitroimidazole)hexylamine

and

HA

conjugated

with

6-(2-

nitroimidazole)hexylamine were obtained and confirmed by 1H nuclear magnetic resonance (NMR) spectra based on DMSO-d6 (99.8%) and D2O (99.9%) as a solvent (Figure S1A, S1B, and S1C), respectively. Lactate-responsive nanoparticles were formed by self-assembly of HA conjugated with 6-(2-nitroimidazole)hexylamine, encapsulating magnetized recombinant adenoassociated virus serotype 2 (AAV2) (hydrodynamic diameter of 48 ± 3.6 nm)15 and lactate oxidase (LOX) in the aqueous solution. After incubation in respective aqueous solutions at pH 7.4 for 6 hr, the lactate-responsive nanoparticles had a hydrodynamic diameter of ca. 250 nm (Figure S2A). To evaluate the nanoparticles response to lactate, lactate-responsive nanoparticles was incubated in complete culture medium at pH 7.4 or pH 6.8 adjusted by lactate (1.0 M) or hydrochloric acid (1.0 N). The use of HCl for adjusting pH did not give rise to any change in hydrodynamic diameter (Figure S2B). When exposed to the culture medium at pH 6.8 adjusted by lactate, the lactate-responsive nanoparticles showed a hydrodynamic diameter of 85 ± 49 nm due to magnetized AAV2 aggregation. Transmission electron microscopy (TEM) of lactateresponsive nanoparticles and disassembled of the lactate-responsive nanoparticles showed diameters of ca. 220 nm and 40-80 nm, respectively (Figure S2C and S2D). To examine the


6

Page 7 of 35 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

ACS Nano

lactate-responsive disassembly of lactate-responsive nanoparticles, release of virus and transduction, carrier formulations (encompassing the carrier along with various control formulations) were incubated in complete culture medium (w/o sodium bicarbonate) with H1975 cells at different pH values (7.4, 7.0, 6.8 or 6.5) adjusted by lactate (1.0 M) or hydrochloric acid (1.0 N). Lactate-responsive nanoparticles loaded with magnetized AAV2-green fluorescent protein (GFP) and LOX were assessed after 6 days for viral transduction of the GFP gene. Infection increased with both LOX content and decreasing pH when adjusted by lactate (Figure 1B). No such pH response was detected in any of the controls by using hydrochloric acid (P > 0.2, Figure 1C). Furthermore, the oxygen consumption caused by the oxidation of lactate catalyzed by LOX was evaluated by an oxygen-sensitive phosphorescent molecular probe.21 When exposed to the culture medium at pH 6.8, lactate-responsive nanoparticles had significantly greater oxygen consumption compared with the negative control and nanoparticles without LOX (Figure 1D). Overall, the lactate-responsive nanoparticles evidently responded to addition of lactate in the culture medium, which mimicked acidosis in a tumor microenvironment. Correspondingly, this implies a reduction in lactate concentration. Lactate modulates immune cell function and promotes tumor invasion and metastasis.22,23 An interesting extension of this concept, although not shown here, is that this process may offer further therapeutic benefit by reducing tumor aggressiveness. In Vitro Light-Triggered Virotherapy. AAV2 encoded to transduce cells to express the photosensitive KillerRed protein has been shown to effectively produce reactive oxygen species (ROS) under illumination that damage DNA and induce apoptosis.4,15,16 The lactate-responsive nanoparticle described herein was designed to disassemble under acidic conditions specifically induced by lactate, and subsequently release KillerRed-encoded AAV2. Figure S3A shows that


7

ACS Nano 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 8 of 35

acidification of the culture media with lactate does indeed lead to KillerRed transduction while comparable acidification with HCl does not (Figure S3B). Correspondingly, the percentage of apoptotic cells increased noticeably with decreasing pH, due to lactate, and the carriers’ LOX content (Figure 1E) as opposed to HCl (Figure 1F). Successful induction of cell death stained by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was also veriﬁed by Confocal microscopy which showed increased numbers of apoptotic cells with decreasing pH (Figure 1G and Figure S4). Molecular Dynamics Simulation of Lactate-Responsive Nanoparticles. Transmission electron microscopy (TEM) images and molecular dynamics (MD) simulation at various timepoints shows HA conjugated with 6-(2-nitroimidazole)hexylamine enables the self-assembly of the lactate-responsive nanoparticles by Van der Waal forces, and activation energy started from 164.78 ± 11.53 kcal mol-1 to -363.47 ± 25.00 kcal mol-1 (Figure 2A). 2-Nitroimidazole of HA conjugated with 6-(2-nitroimidazole)hexylamine provides a functional response to the lowering of oxygen concentration internally within the lactate-responsive nanoparticles. When the local, internal oxygen concentration lowers because of oxidation of lactate, catalyzed by LOX (Figure 1D), the nitroimidazole becomes water-soluble, leading to disassembly of the lactate-responsive nanoparticles (final activation energy: -719.75 ± 22.67 kcal mol-1) (Figure 2B). Disintegration of lactate-responsive nanoparticles was clearly observed with TEM in the presence of lactate and simultaneously predicted by MD simulation. In Vivo Tracing of Lactate Biodistribution. We further explored the lactate biodistribution in subcutaneous non-small cell lung cancer (NSCLC) H1975 xenografts. Firstly, glucose metabolism in tumor volumes was confirmed in mice performing intravenous delivery via tailvein injection of a XenoLight RediJect 2-DeoxyGlucosone (DG) 750 probe exhibiting an


8

Page 9 of 35 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

ACS Nano

elevated glucose uptake rate in comparison to surrounding tissues/organs. Near-infrared fluorescence images showed clear targeting to H1975 xenografts (Figure 3A, lateral view, and Figure S5, posterior view). Glucose uptake is important for metabolic imaging in solid tumors24 and lactate is generally considered as a waste product from glucose conversion1, however this work indicates the utility of lactate as a primary target for functional materials and imaging. Furthermore, to assess accumulation of lactate associated with tumors, xenograft tumors and organs were harvested to determine lactate content. Qualitative analysis of the peripheral tumor mass revealed a greater lactate content compared to the inner volume of the tumor and other organs (Figure 3B). In Vivo Viral Biodistribution by Lactate-Responsive Nanoparticles. We assessed the specificity of tumor bioreduction-activated, magnetized virus (AAV2-Luciferase) release from lactate-responsive nanoparticles in subcutaneous NSCLC H1975 xenografts. Luciferase biodistribution was imaged with bioluminescence at Day 7 after administration. As anticipated, the liver exhibited bioluminescence consistent with the clearance pathway when mice were injected by viruses only (Figure 3C).15 Consistent with the observations of glucose uptake and lactate accumulation (Figure 3A and 3B), viral transduction infected by lactate-responsive nanoparticles was confirmed by significant bioluminescence in the tumor (Figure 3D). Threedimensional (3-D) bioluminescence imaging of mice with lactate-responsive nanoparticles further verified successful delivery (right panel, Figure 3D). We also tracked the magnetized AAV2 released from lactate-responsive nanoparticles by MRI due to contrast provided by the iron oxide nanoparticles.17 In T2-weighted images or T2*-weighted images in mice bearing H1975 xenografts in both Case 1 and Case 2, magnetized AAV2 was consistently observed in


9

ACS Nano 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 35

tumors and liver (Figure 3E or Figure S6). This reinforces the functional dependence upon lactate accumulation controlling specificity in virus delivery. Biosafety in Preclinical Studies. In general, larger nanocarriers (>200 nm) have been shown to accumulate in the liver.25-27 For systemically administered AAV2 or iron oxidase nanoparticles, the liver is the default destination.15,28 Consistent with the observations of bioluminescence (Figure 3C and 3D), both virus and nanoparticles were observed in the liver. Next, toxicity was evaluated in animals treated with lactate-responsive nanoparticles (magnetized AAV2-KillerRed) determined by levels of pyruvic oxallotransaminase (GPT), glutamic oxallotransaminase (GOT), total bilirubin (TBIL), and creatinine (CRE) which are indicators of liver and kidney function. These biochemical analyses showed no significant liver (Figure 3F) or kidney toxicity (Figure 3G). As compared with the control group, the magnetized AAV2 or lactate-responsive nanoparticle treated mice showed no obvious damage or inflammation in the major organs (i.e. heart, liver, lung, spleen, and kidney) (Figure S7). Furthermore, mice exposed to various treatments showed no statistically significant differences in body weight, indicating no notably serious lactate-responsive nanoparticles or light irradiation-related toxicities (Figure S8). In Vivo Light-Triggered Antitumor Activity. We then tested the potential use of the bioreduction-activated release concept in a therapeutic context using the NSCLC xenograft model with a treatment course as described in Figure 4A (upper panel). A single administration, via tail vein injection, of lactate-responsive nanoparticles resulted in significant suppression of tumor outgrowth in Day 3 to Day 6, however it was limited with respect to long-term suppression (lower panel, Figure 4A and Figure S9A). A double-course treatment further significantly increase tumor sensitivity to light-triggered virotherapy treatment. An approximate 2.68-fold or 2.44-fold reduction in tumour volumes (P < 0.005, Figure 4A) or tumor weight (P

Targeting Tumor Microenvironment by Bioreduction-Activated

Recommend Documents