stealth magneto-nanomicelles for theranostics combining efficient MRI

Subscriber access provided by Eastern Michigan University | Bruce T. Halle Library

Article

Bioinspired “active” stealth magneto-nanomicelles for theranostics combining efficient MRI and enhanced drug delivery Kai-Long Zhang, Jie Zhou, Hong Zhou, Ying Wu, Rui Liu, LiLi Wang, Wei-Wen Lin, Guoming Huang, and Huang-Hao Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10086 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 24

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 Applied Materials & Interfaces

Bioinspired “active” stealth magneto-nanomicelles for theranostics combining efficient MRI and enhanced drug delivery Kai-Long Zhang,† Jie Zhou,† Hong Zhou,‡ Ying Wu,† Rui Liu,‡ Li-Li Wang,§ Wei-Wen Lin,§ Guoming Huang,*‡ and Huang-Hao Yang*† †

MOE Key Laboratory for Analytical Science of Food Safety and Biology, State Key Laboratory

of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China. E-mail: [email protected] ‡

College of Biological Science and Engineering, Fuzhou University, Fuzhou 350116, P. R.

China. E-mail: [email protected] §

Department of Diagnostic Radiology, Union Hospital, Fujian Medical University, Fuzhou

350001, P. R. China KEYWORDS: stealth, long-circulating, PLGA-PEG, MRI, drug delivery

ABSTRACT: The mononuclear phagocyte system (MPS), with key roles in recognition and clearance of foreign particles, is a major constraint to nanoparticle-based delivery systems. The desire to improve the delivery efficiency has prompted the search for stealthy long-circulating

ACS Paragon Plus Environment

1


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 24

nanoplatforms. Herein, we design an antiphagocytic delivery system with “active” stealth behavior for cancer theranostics combining efficient MRI and enhanced drug delivery. We modify self-peptide, a synthetic peptide for active immunomodulation, to biodegradable poly (lactide-glycolide)-poly (ethylene glycol) (PLGA–PEG), then utilize the self-assembly properties of PLGA–PEG to form nanomicelles that encapsulating iron oxide (IO) nanoparticles and anticancer drug paclitaxel (PTX). Through the interaction of self-peptide with the receptor SIRPα, which is expressed on phagocytes, the as-prepared nanomicelles can disguise as “self” to avoid being recognized as foreign particles by MPS, leading to improved blood circulation time and delivery efficiency. Compared to the “passive” stealth effect generating by PEG or zwitterionic polymers that only passively delay the physisorption of serum proteins to nanocarriers, the “active self” nanomicelles can more efficiently inhibit the MPS-mediated immune clearance and reduce “accelerated blood clearance (ABC)” phenomenon. Furthermore, this one-step clustering of IO nanoparticles and loading of PTX endow the resulted magnetonanomicelles with enhanced T2 MRI contrast performance and anti-tumor effect. We believe that this study provides a novel approach in designing of efficient stealth antiphagocytic delivery systems that resisting the MPS-mediated clearance for cancer theranostics.

1. Introduction The design and development of nanostructure-based delivery systems working to deliver therapeutic and diagnostic agents in a safer and more efficient manner for cancer treatment has boomed in the last decade.1-4 Nanocarriers are expected to circulate in the blood for long time, accumulate in tumor sites with leaky vasculature via the enhanced permeability and retention (EPR) effect.5-7 The mononuclear phagocyte system (MPS) that consists of the phagocytic cells


2

Page 3 of 24

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


with the capacity to uptake foreign particles, is the major constraint of nanocarriers to reach the target.8 When a nanomaterial enters a physiological environment, its surface is immediately covered by a layer of blood proteins, such as IgG and complement proteins, forming what is known as the protein corona.9-12 The formation of protein corona labels the nanocarriers as foreign material, and promote the phagocytosis of nanocarriers by MPS.9, 10 Much research has been devoted to fabricating delivery systems with “stealth” behavior that could avoid the immune clearance by MPS.13 Polyethylene glycol (PEG) is the most widely used stealth polymer in the nanomedicine field, due to its long history of safety in humans.14 PEGylated drugs and nanocarriers show longer circulation time in the bloodstream and less nonspecific uptake compared to unmodified drugs.15 Other coating materials such as zwitterionic polymers are also emerging as attractive alternatives in resisting nonspecific protein adsorption.16 However, whether PEG or zwitterionic polymers generating the stealth effects are “passive” that only passively delay the physisorption of serum proteins to nanocarriers, cannot efficiently inhibit the immune clearance by MPS. Based on many recent researches, the repeated administration of PEG could cause an unexpected immunogenic response known as the “accelerated blood clearance (ABC)” phenomenon.17, 18 The host immune system can recognize PEGylated particles after repeated injections, resulting in the increased clearance and reduced efficacy of PEGylated nanocarriers. Recent advances in cellular and molecular biology have revealed that phagocytosis relies on a balance between prophagocytic and antiphagocytic signals on target cells.19, 20 CD47 is a cell surface protein in the immunoglobulin superfamily, and has been implicated in diverse physiologic processes including phagocytes.21, 22 CD47 is capable of interacting with its receptor signal regulatory protein alpha (SIRPα), which is expressed on phagocytes, to negatively


3


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 24

regulate phagocytosis.23 In this way, CD47 serves as an antiphagocytic “don’t eat me” signal and a marker of self.24 For example, CD47 functions as a marker of self on red blood cells (RBC), enabling the cells circulate through the body without being recognized as foreign particles.25 Inspired by biomimetics, the utilization of natural RBC membranes for nanoparticle coating has emerged as a top-down approach to enhance the delivery of therapeutic agents.26 Recently, a synthetic 21-amino acid self-peptide was computationally designed from human CD47 and was demonstrated that can minimize the phagocytic uptake of nanoparticles,27 indicating its great potential in engineering antiphagocytic biomimetic nanocarriers. Herein, we modify the selfpeptide to poly (lactide-glycolide)-poly (ethylene glycol) (PLGA–PEG), a FDA-approved biodegradable polymer, and construct an “active stealth” theranostic nanoplatform for the first time (Figure 1). Compared to conventional PEGylated delivery systems that just passively reduce protein adsorption to increase the blood circulation time, our nanoplatform was designed to in the disguise of “self” to avoid being recognized as foreign particles, thus is an active immunoincompetent delivery system with improved circulation time and reduced “ABC” phenomenon. Moreover, the self-peptide modified PLGA-PEG (PLGA-PEG-peptide) was readily self-assembled into nanomicelles in aqueous solution because of their amphiphilicity, and the clustering of iron oxide (IO) nanoparticles and encapsulation of hydrophobic drug paclitaxel (PTX) can be achieved in such a one-step process. The resulting IO/PTX encapsulated PLGAPEG-peptide (IO/PTX@PLGA-PEG-peptide) nanomicelles present a significant increase in T2 MRI contrast enhancement and can effectively inhibit the growth of solid tumor. Therefore, it is a novel long-circulating delivery nanoplatform holding great promise in cancer imaging and therapy.


4

Page 5 of 24

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 1. Schematic illustration of the construction of IO/PTX@PLGA-PEG-peptide nanomicelles as an “active stealth” theranostic nanoplatform. 2. Results and discussion 2.1. Synthesis and characterization We first synthesized PLGA-PEG-COOH copolymer by direct conjugation of PLGA–COOH to NH2–PEG–COOH via a conventional EDC/NHS technique. The chemical composition of the assynthesized PLGA-PEG-COOH was confirmed by 1H NMR (Supporting Information, Figure S1). Then, self-peptide and PLGA-PEG-peptide copolymer were synthesized by standard solid phase methods. The successful combination of self-peptide and PLGA-PEG-COOH was confirmed by MALDI-TOF mass spectrum (Figure S2), that the characteristic peak at 25022.6445 m/z could be assigned to PLGA-PEG-peptide (the relative molecular mass of selfpeptide and PLGA-PEG-COOH were about 2000 and 23000, respectively). We then prepared IO/PTX@PLGA-PEG-peptide nanomicelles via an emulsion/solvent evaporation method. In aqueous solution, PLGA-PEG-Peptide readily self-assembled into spherical nanomicelles, and 6


5


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 24

nm IO nanoparticles (Figure S3) and anticancer drug PTX were simultaneously encapsulated inside the core. To better demonstrate the active stealth property of IO/PTX@PLGA-PEGpeptide nanomicelles, we also synthesized IO/PTX@PLGA-PEG nanomicelles as a control sample. The transmission electron microscopy (TEM) images showed that the IO/PTX@PLGAPEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles had similar size, which was about 90-100 nm (Figure 2a and Figure S4). Dynamic light scattering (DLS) measurements revealed that the hydrodynamic diameter of IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles were 106 ± 1.23 and 93 ± 1.16 nm, respectively (Figure 2b). The increase in hydrodynamic diameter of IO/PTX@PLGA-PEG-peptide nanomicelles was induced by the modification of self-peptide to PLGA-PEG. The IO/PTX@PLGA-PEG-peptide nanomicelles were negatively charged with a zeta potential of −58 ± 2.08 mV, which was much more negative compared to that of −16 ± 1.47 mV for PTX/IO@PLGA-PEG nanomicelles (Figure 2b), owing to the more negative potential of self-peptide. Both the IO/PTX@PLGAPEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles displayed excellent stability in phosphate buffer solution (PBS) and serum without observation in significant change of hydrodynamic diameters over at least 3 days (Figure S5). Afterwards, we investigate the MRI performance of the IO/PTX@PLGA-PEG-peptide nanomicelles by phantom imaging and relaxivity measurements. IO/PTX@PLGA-PEG-peptide nanomicelles exhibited a significantly stronger negative contrast effect (darker signal) than the control monodisperse IO nanoparticle at the same Fe ion concentration (Figure 2c). This could be attributed to the clustering of IO nanoparticles in PLGA-PEG-peptide nanomicelles, which can significantly enhance the T2 contrast effect of IO nanoparticles.28-30 Moreover, the transverse relaxivity (r2) value of IO/PTX@PLGA-PEG-peptide nanomicelles was determined to be 340.6


6

Page 7 of 24

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


± 7.4 mM−1 s−1, which was about twice as high as that of 181.1 ± 10.2 mM−1 s−1 for IO nanoparticles (Figure 2d and Figure S5), further confirming that the clustered IO nanoparticles on PLGA-PEG-peptide nanomicelles core can afford more effective magnetic relaxations to water protons, resulting in greater T2 relaxivity. These results suggested that IO/PTX@PLGAPEG-peptide nanomicelles could be a good MRI contrast agent. We then determined the drug encapsulation efficiency (EE), loading capacity (LC), and in vitro drug release efficiency of IO/PTX@PLGA-PEG-peptide nanomicelles. The EE and LC of optimized IO/PTX@PLGAPEG-peptide nanomicelles were 42.6 ± 2.6% and 1.08 ± 0.05%, respectively. The release profiles of PTX from IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles were investigated in PBS of pH 7.4 at 37 oC. Two nanomicelles presented similar release profiles in PBS (Figure 2e). A fast release of PTX was observed in the first 24 h (48.4 ± 2.7% and 53.0 ± 7.2% for IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGAPEG nanomicelles, respectively). In the following 48 h, the cumulative release reached 60.8 ± 2.1% and 66.9 ± 4.5% for IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGAPEG nanomicelles, respectively. After that, both IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles exhibited sustained release with the total release reached 86.4 ± 3.5% and 91.2 ± 4.8%, respectively, at 12 days. The biocompatibility of nanomaterials is a primary concern for biomedical applications. We used CCK-8 assay to evaluate the cytotoxicity of IO@PLGA-PEG-peptide nanomicelles on MCF-7 cancer cells. With the concentration of Fe ranging from 0 to 320 µg mL−1, all of the cells retained over 98% viability (Figure S6), confirming that IO@PLGA-PEG-peptide nanomicelles possess low cell cytotoxicity and good biocompatibility. We then evaluated the in vitro cytotoxicity of Taxol® (free

PTX),

IO/PTX@PLGA-PEG

nanomicelles,

and

IO/PTX@PLGA-PEG-peptide


7


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 24

nanomicelles. All Taxol®, IO/PTX@PLGA-PEG nanomicelles, and IO/PTX@PLGA-PEGpeptide nanomicelles exhibited dose-dependent cytotoxicity, could effectively inhibit the growth of cancer cells (Figure 2f).

Figure

2.

(a)

TEM

images

of


nanomicelles.

Inset:

IO/PTX@PLGA-PEG-peptide nanomicelles were negatively stained with 3% phosphotungstic acid. (b) DLS measured hydrodynamic diameter and zeta potential of IO/PTX@PLGA-PEGpeptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles, respectively. (c) T2-weighted phantom images and (d) r2 values of the IO/PTX@PLGA-PEG-peptide nanomicelles and IO nanoparticles in aqueous solution with different Fe concentrations, respectively. (e) Release profiles of PTX from IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG


8

Page 9 of 24

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


nanomicelles in PBS of pH 7.4 at 37 oC, respectively (n = 3). (f) Cell viability of MCF-7 cells after incubated with Taxol®, IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEGpeptide nanomicelles with different PTX concentrations at 37 oC for 24 h, respectively. 2.2. Pharmacokinetics and biodistribution studies We first verify the capability of self-peptide for helping the nanomicelles to escape from the macrophage phagocytosis by evaluating the intracellular uptake of nanomicelles in the mouse macrophage, RAW264.7 cells. As shown in Figure 3, both the confocal fluorescence microscope and flow cytometry results showed that IO@PLGA-PEG-peptide nanomicelles exhibited a lower uptake by RAW264.7 cells compared to IO@PLGA-PEG nanomicelles, confirming that the selfpeptide could suppress the macrophage phagocytosis. Encouraged by these in vitro results, we then performed pharmacokinetics studies using mice as models to assess whether IO/PTX@PLGA-PEG-peptide nanomicelles capture the advantage of long circulation time from self-peptide. In brief, twenty-four mice were randomly divided into three groups (n = 8 per group). In the first administration, Taxol®, IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEG-peptide nanomicelles were respectively injected through the tail vein at the dose of 3 mg kg−1 PTX to all eight mice. Following 7 days’ intervals after the first injection, four mice of each group were selected to inject the test dose again (the second administration group). At different time points after the administration, 50 µL of blood were collected from mouse tail vein for followed PTX content determination by tandem mass spectrometry (LCMS/MS). The results showed that Taxol® was rapidly cleared from the systemic circulation, while the IO/PTX@PLGA-PEG-peptide nanomicelles consistently exhibit significantly enhanced blood retention over a span of 24 h, compared to the IO/PTX@PLGA-PEG nanomicelles (Figure 4a). We then compared the pharmacokinetic profiles of three groups


9


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 24

between the first administration group (Figure 4a) and the second administration group (Figure 4b), and we found that they are significant different. The clearance of IO/PTX@PLGA-PEG nanomicelles in the second administration was obviously faster than that in the first administration, but the clearance of IO/PTX@PLGA-PEG-peptide nanomicelles and Taxol® had no obvious change. We speculated that this mainly resulted from the “ABC” phenomenon in PEG. Previous studies have demonstrated that when the PEGylated nanocarriers were repeatedly applied to the same animal, the immune responses occurred. The first injection of PEGylated nanoparticles triggered a reduction in the circulation time and an increase in hepatic and splenic accumulation of the second administration of PEGylated nanocarriers in a time-interval.18

Figure 3. (a) Confocal fluorescence microscope images of RAW264.7 cells after incubated with IO@PLGA-PEG nanomicelles and IO@PLGA-PEG-peptide nanomicelles, respectively, for 1 h. The nucleus of cells was stained with Hoechst 33342 (blue). The nanomicelles were loaded with DiD (red). (b) Quantitative analyses of intracellular DiD fluorescence intensity by flow cytometry after 1 h incubation.


10

Page 11 of 24

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


We next calculated the pharmacokinetic parameters by Drug and Statistics software (DAS, version 3.2.2) using a two-compartment model to fit the pharmacokinetic profile of PTX. IO/PTX@PLGA-PEG-peptide nanomicelles showed significant longer elimination half-life (t1/2β), slower clearance rate (CL), smaller elimination rate constant (K10), and higher area under the curve (AUC) when compared with IO/PTX@PLGA-PEG nanomicelles and Taxol® (Table S1). The clearance rate of IO/PTX@PLGA-PEG-peptide nanomicelles was 0.299 L/h/kg, which was obviously slower than that of IO/PTX@PLGA-PEG nanomicelles (0.482 L/h/kg), and the AUC of IO/PTX@PLGA-PEG-peptide nanomicelles was 10647.1 µg/L*h, which was obviously bigger than that of IO/PTX@PLGA-PEG nanomicelles (6596.2 µg/L*h), demonstrating that IO/PTX@PLGA-PEG-peptide

nanomicelles

have

a

longer

circulation

lifetime

than

IO/PTX@PLGA-PEG nanomicelles. As we compared the differences in pharmacokinetic profiles between the first administration and the second administration, we found that the AUC of IO/PTX@PLGA-PEG nanomicelles of the second administration (3175.2 µg/L*h) was obviously smaller than that of the first administration (6596.2 µg/L*h), but the AUC of IO/PTX@PLGA-PEG-peptide nanomicelles had no obvious change, further confirmed the existence of ABC phenomenon in PEG and the protection of self-peptide.


11


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 12 of 24

Figure 4. (a) Blood retention profiles of PTX after intravenous administration of Taxol®, IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEG-peptide nanomicelles in mice at the dose of 3 mg kg−1 (n = 4). (b) Blood retention profiles of PTX collected at the second administration (following 7 days’ intervals after the first injection). (c, d) Biodistribution of PTX at (c) 1 h and (d) 8 h after intravenous administration of Taxol®, IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEG-peptide nanomicelles to S180 tumor bearing mice at the dose of 3.0 mg kg−1 (n = 4). (e, f) Biodistribution of PTX at (e) 1 h and (f) 8 h after the second administration (following 7 days intervals after the first injection with the same dose) (n = 4).


12

Page 13 of 24

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


Encouraged by the results that IO/PTX@PLGA-PEG-peptide nanomicelles showed enhanced blood retention and circulation time compared to IO/PTX@PLGA-PEG nanomicelles, to further address whether IO/PTX@PLGA-PEG-peptide nanomicelles exhibit improved biodistribution and enhanced tumor uptake when were systematically administered, we performed biodistribution studies. Briefly, forty-eight mice were randomly divided into three groups (n = 16 per group). In the first administration, Taxol®, IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEG-peptide nanomicelles were respectively injected through the tail vein at the dose of 3 mg kg−1 PTX, and in the second administration that following 7 days’ intervals after the first injection, eight mice of each groups were injected the test dose again. At 1 h and 8 h time points after the injection, four mice of each group were sacrificed, respectively, and the heart, liver, spleen, lung, kidney, and tumor were collected to analyze the content of PTX. Our results showed that PTX was widely distributed in various organs, such as heart, liver, spleen, kidney, lung and tumor after intravenous injection of the three samples (Figure 4c-f). At 1 h and 8 h after the first injection, the IO/PTX@PLGA-PEG-peptide nanomicelles had PTX tumor uptakes of 23 ± 3.2 ng/100 mg and 58 ± 11.4 ng/100 mg, respectively, much higher than that of IO/PTX@PLGA-PEG nanomicelles (19 ± 6.0 ng/100 mg and 24 ± 4.6 ng/100 mg, respectively) and Taxol® (16 ± 2.3 ng/100 mg and 17 ± 6.8 ng/100 mg, respectively), confirming the significant enhancement in tumor uptake of IO/PTX@PLGA-PEG-peptide nanomicelles. In the second administration, the PTX tumor uptakes of IO/PTX@PLGA-PEG-peptide nanomicelles (24 ± 3.1 ng/100 mg and 58 ± 14.2 ng/100 mg at 1 h and 8 h, respectively) maintained almost the same as that in the first administration. However, the PTX tumor uptakes of IO/PTX@PLGAPEG nanomicelles significantly decreased in the second administration (13 ± 6.2 ng/100 mg and 15 ± 6.4 ng/100 mg at 1 h and 8 h, respectively). The significant increase in tumor uptake for


13


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 14 of 24

IO/PTX@PLGA-PEG-peptide nanomicelles suggested that the “active stealth” property of selfpeptide can endow IO/PTX@PLGA-PEG-peptide nanomicelles with superior circulation lifetime, resulting in greater EPR-mediated accumulation of nanomicelles in tumor. In addition to enhanced tumor uptake, the IO/PTX@PLGA-PEG-peptide nanomicelles also presented significantly decreased accumulations within certain major organs such as MPS organs liver and spleen comparing to the IO/PTX@PLGA-PEG nanomicelles whether in the first administration or in the second administration (Figure 4c-f). Moreover, the accumulations of PTX within liver, spleen, and kidney of IO/PTX@PLGA-PEG nanomicelles group in the second administration were higher than that in the first administration. But for IO/PTX@PLGA-PEG-peptide nanomicelles group, the PTX accumulations approximately keep the same between the first and the second administration. These results confirmed that IO/PTX@PLGA-PEG-peptide nanomicelles could effectively delay the phagocytic clearance by MPS and without the “ABC” phenomenon. SIRPα is a regulatory membrane glycoprotein from SIRP family expressed on phagocytes. Engagement of SIRPα by self-peptide provides a downregulatory signal that inhibits host cell phagocytosis, and self-peptide therefore functions as a “don’t-eat-me” signal.27 We further examined the expression of SIRPα in liver and spleen, which are two primary organs of the MPS. Immunofluorescence images showed that the expression of SIRPα both in the liver and spleen were higher in the IO/PTX@PLGA-PEG-peptide nanomicelles group than in the other groups (Figure 5). These results further revealed that IO/PTX@PLGA-PEG-peptide nanomicelles showed an efficient immune evasion capability relying on the SIRPα signaling pathway.


14

Page 15 of 24

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 5. Representative immunofluorescence images of (a) hepatic and (b) splenic sections, which were stained with DAPI (blue) and SIRPα (green). 2.3. In vivo MRI and therapy To further evaluate the ability of IO/PTX@PLGA-PEG-peptide nanomicelles for cancer imaging, we conducted the in vivo MRI on S180 tumor-bearing nude mice. We intravenously injected

the


nanomicelles

and

IO/PTX@PLGA-PEG

nanomicelles into the mice and scanned the mice on a 3 T MRI scanner at different time points. Both nanomicelles caused a gradual T2 contrast enhancement (i.e., darker) in the tumor and liver sites after the intravenous administration (Figure 6a). To quantify the contrast enhancement, we calculated the signal-to-noise ratio (SNR) by finely analyzing the area of tumor sites, and defined the contrast enhancement as the change of SNR, △SNR = |SNRpost - SNRpre|/SNRpre. IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles showed no obvious difference in △SNR at the first 2 h after the administration, with △SNR values of 13.54 ± 0.78% and 13.38 ± 0.91% at 2 h time point for IO/PTX@PLGA-PEG-peptide nanomicelles and IO/PTX@PLGA-PEG nanomicelles, respectively (Figure 6b). However, the △SNR of


15


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 16 of 24

IO/PTX@PLGA-PEG-peptide nanomicelles increased significantly at later 6 h and 12 h (25.81 ± 0.55% and 32.26 ± 0.42%, respectively), while that of IO/PTX@PLGA-PEG nanomicelles exhibited little change (14.74 ± 0.34% and 15.87 ± 0.77%, respectively). These results indicated that IO/PTX@PLGA-PEG-peptide nanomicelles had longer blood circulation time, allowing greater tumor accumulation of nanomicelles, therefore can generate greater T2 contrast enhancement in tumor. We further calculated the contrast-to-noise ratio (CNR) of tumour-toliver contrast, where CNR = |SNRtumor - SNRliver|/SNRtumor, and △CNR = |SNRpost SNRpre|/SNRpre. Compared to IO/PTX@PLGA-PEG nanomicelles that exbihited small change of △CNR (39.84 ± 2.4%, 41.00 ± 1.3%, 43.23 ± 1.5% and 44.14 ± 2.7% for 1 h, 2 h, 6 h, and 12 h, respectively), IO/PTX@PLGA-PEG-peptide nanomicelles can produce a greater and gradually increased △CNR, with the △CNR values of 51.46 ± 1.5%, 54.63 ± 1.8%, 58.14 ± 2.5% and 63.03 ± 2.6% for 1 h, 2 h, 6 h, and 12 h, respectively (Figure 6c). The “active stealth” behavior enabled IO/PTX@PLGA-PEG-peptide nanomicelles to effectively delay the phagocytic clearance by liver, which promoted the accumulation of nanomicelles in tumor, therefore resulting in enhanced T2 contrast in tumor. We then examined the therapeutic performance of IO/PTX@PLGA-PEG-peptide nanomicelles. After the treatment, IO/PTX@PLGA-PEG-peptide nanomicelles, IO/PTX@PLGA-PEG nanomicelles, and Taxol® all showed an inhibitory effect on the growth of solid tumor as compare with the control group (Figure 6d, e). More importantly, the IO/PTX@PLGA-PEG nanomicelles treated group had better therapeutic efficacy compared with other three groups owing to more efficient delivery of PTX to tumor. Hematoxylin and eosin (H&E) staining of tumor sections revealed that tumor tissues in IO/PTX@PLGA-PEGpeptide nanomicelles group exhibited more obvious necrosis that tumor cells were more irregularly shaped with shrinking cell nucleus than that in Taxol® and IO/PTX@PLGA-PEG


16

Page 17 of 24

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


nanomicelles group (Figure 6f). However, in the control group, cancer cells maintained regular morphology with intact cell nucleus. Furthermore, the body weights of the mice of all groups showed no significant loss (Figure S8), and the H&E stained histological images of heart, liver, spleen, lung and kidney exhibited no noticeable organ damage or inflammatory lesion (Figure S9), suggesting the low toxicity of all treatments.

Figure 6. (a) T2-weighted in vivo MRI images of mice collected at different time points after intravenous injection of IO/PTX@PLGA-PEG nanomicelles and IO/PTX@PLGA-PEG-peptide nanomicelles, respectively. The red and blue arrows are pointing to tumor and liver, respectively. (b) Quantificational analysis of signal-to-noise ratio changes (△SNR) and (c) contrast-to-noise


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

Page 18 of 24

ratio changes (△CNR) at different time points after intravenous administration injection of IO/PTX@PLGA-PEG

nanomicelles

and


nanomicelles,

respectively (n = 3). (d) Tumor growth curves of S180 tumor-bearing mice after different treatments. (e) Representative photos of excised tumor, and (f) H&E stained histological images of tumor collected at 7 days after different treatments. 3. Conclusion In summary, using the antiphagocytic properties of self-peptide and self-assembly capabilities of PLGA-PEG, we developed an “active stealth” theranostic nanoplatform. The functionalization of self-peptide endowed the nanoplatform with “self” identity to avoid being recognized as foreign particles by MPS, leading to persistent circulation in blood and enhanced accumulation in tumor. Additionally, the “active stealth” nanoplatform showed a circumvent of the “ABC” phenomenon, an unexpected immunogenic response usually occur in PEGylated nanocarriers. Furthermore, the self-assembly of PLGA-PEG-peptide could easily encapsulate the IO nanoparticles and PTX, resulting in the clustering of IO nanoparticles and loading of PTX. Therefore, the as-prepared nanoplatform also exhibited an excellent T2 MRI contrast enhancement and could effectively inhibit the growth of solid tumor. This totally synthetic nanomicelle is easy to manufacture on a large scale. Our technology may therefore provide a practical, broadly applicable bottom-up strategy for the design of efficient antiphagocytic delivery systems. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Experimental details, supporting figures and table (PDF)


18

Page 19 of 24

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


AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. U1505221, 21635002, 21475026 and 81501461), Natural Science Foundation of Fujian Province of China (No. 2015H6011, 2017J05131, 2017J01199), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), the Independent Research Project of State Key Laboratory of Photocatalysis on Energy and Environment (No. 2014B02). REFERENCES (1)

Allen, T. M.; Cullis, P. R., Drug Delivery Systems: Entering the Mainstream. Science

2004, 303, 1818-1822. (2)

Xie, J.; Lee, S.; Chen, X., Nanoparticle-Based Theranostic Agents. Adv. Drug Delivery

Rev. 2010, 62, 1064-1079. (3)

Song, X. R.; Wang, X.; Yu, S. X.; Cao, J.; Li, S. H.; Li, J.; Liu, G.; Yang, H. H.; Chen,

X., Co9Se8 Nanoplates as A New Theranostic Platform for Photoacoustic/Magnetic Resonance Dual-Modal-Imaging-Guided Chemo-Photothermal Combination Therapy. Adv. Mater. 2015, 27, 3285-3291.


19


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

(4)

Page 20 of 24

Wang, R.; Hu, H.; Cai, Q.; Zhao, N.; Zhu, Y.; Xu, F., Versatile Functionalization of

Amylopectin for Effective Biomedical Applications. Sci. China Chem. 2015, 58, 1461-1470. (5)

Petros, R. A.; DeSimone, J. M., Strategies in the Design of Nanoparticles for Therapeutic

Applications. Nat. Rev. Drug Discovery 2010, 9, 615-627. (6)

Song, X. R.; Yu, S. X.; Jin, G. X.; Wang, X.; Chen, J.; Li, J.; Liu, G.; Yang, H. H., Plant

Polyphenol-Assisted Green Synthesis of Hollow CoPt Alloy Nanoparticles for Dual-Modality Imaging Guided Photothermal Therapy. Small 2016, 12, 1506-1513. (7)

Guo, T.; Lin, Y.; Li, Z.; Chen, S.; Huang, G.; Lin, H.; Wang, J.; Liu, G.; Yang, H. H.,

Gadolinium Oxysulfide-coated Gold Nanorods with Improved Stability and Dual-Modal Magnetic Resonance/Photoacoustic Imaging Contrast Enhancement for Cancer Theranostics. Nanoscale 2017, 9, 56-61. (8)

Blanco, E.; Shen, H.; Ferrari, M., Principles of Nanoparticle Design for Overcoming

Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941-951. (9)

Walkey, C. D.; Chan, W. C., Understanding and Controlling the Interaction of

Nanomaterials with Proteins in a Physiological Environment. Chem. Soc. Rev. 2012, 41, 27802799. (10)

Schottler, S.; Landfester, K.; Mailander, V., Controlling the Stealth Effect of

Nanocarriers through Understanding the Protein Corona. Angew. Chem. Int. Ed. 2016, 55, 88068815. (11)

Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M.; Somasundaran, P.; Klaessig,

F.; Castranova, V.; Thompson, M., Understanding Biophysicochemical Interactions at the Nanobio Interface. Nat. Mater. 2009, 8, 543-557.


20

Page 21 of 24

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


(12)

Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A., Biomolecular Coronas Provide

the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779-786. (13)

Hu, C. M.; Fang, R. H.; Luk, B. T.; Zhang, L., Polymeric Nanotherapeutics: Clinical

Development and Advances in Stealth Functionalization Strategies. Nanoscale 2014, 6, 65-75. (14)

Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S., Poly(ethylene glycol) in Drug

Delivery: Pros and Cons as Well as Potential Alternatives. Angew. Chem. Int. Ed. 2010, 49, 6288-6308. (15)

Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M., PEGylation as A Strategy for

Improving Nanoparticle-based Drug and Gene Delivery. Adv. Drug Delivery Rev. 2016, 99, 2851. (16)

Pombo Garcia, K.; Zarschler, K.; Barbaro, L.; Barreto, J. A.; O'Malley, W.; Spiccia, L.;

Stephan, H.; Graham, B., Zwitterionic-Coated "Stealth" Nanoparticles for Biomedical Applications: Recent Advances in Countering Biomolecular Corona Formation and Uptake by the Mononuclear Phagocyte System. Small 2014, 10, 2516-2529. (17)

Ishida, T.; Kiwada, H., Accelerated Blood Clearance (ABC) Phenomenon upon Repeated

Injection of PEGylated Liposomes. Int. J. Pharm. 2008, 354, 56-62. (18)

Abu Lila, A. S.; Kiwada, H.; Ishida, T., The Accelerated Blood Clearance (ABC)

Phenomenon: Clinical Challenge and Approaches to Manage. J. Controlled Release 2013, 172, 38-47. (19)

Underhill, D. M.; Goodridge, H. S., Information Processing During Phagocytosis. Nat.

Rev. Immunol. 2012, 12, 492-502.


21


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

(20)

Page 22 of 24

Liu, X.; Pu, Y.; Cron, K.; Deng, L.; Kline, J.; Frazier, W. A.; Xu, H.; Peng, H.; Fu, Y. X.;

Xu, M. M., CD47 Blockade Triggers T cell-mediated Destruction of Immunogenic Tumors. Nat. Med. 2015, 21, 1209-1215. (21)

Brown, E., Integrin-Associated Protein (CD47) and Its Ligands. Trends Cell Biol. 2001,

11, 130-135. (22)

Jaiswal, S.; Jamieson, C. H.; Pang, W. W.; Park, C. Y.; Chao, M. P.; Majeti, R.; Traver,

D.; van Rooijen, N.; Weissman, I. L., CD47 is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis. Cell 2009, 138, 271-285. (23)

Barclay, A. N.; Van den Berg, T. K., The Interaction Between Signal Regulatory Protein

alpha (SIRPalpha) and CD47: Structure, Function, and Therapeutic Target. Annu. Rev. Immunol. 2014, 32, 25-50. (24)

Chao, M. P.; Weissman, I. L.; Majeti, R., The CD47-SIRPalpha Pathway in Cancer

Immune Evasion and Potential Therapeutic Implications. Curr. Opin. Immunol. 2012, 24, 225232. (25)

Oldenborg, P. A., Role of CD47 as A Marker of Self on Red Blood Cells. Science 2000,

288, 2051-2054. (26)

Hu, C.-M. J.; Fang, R. H.; Zhang, L., Erythrocyte-Inspired Delivery Systems. Adv.

Healthcare Mater. 2012, 1, 537-547. (27)

Rodriguez, P. L.; Harada, T.; Christian, D. A.; Pantano, D. A.; Tsai, R. K.; Discher, D.

E., Minimal "Self" Peptides that Inhibit Phagocytic Clearance and Enhance Delivery of Nanoparticles. Science 2013, 339, 971-975.


22

Page 23 of 24

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)

Ai, H.; Flask, C.; Weinberg, B.; Shuai, X. T.; Pagel, M. D.; Farrell, D.; Duerk, J.; Gao, J.,

Magnetite-Loaded Polymeric Micelles as Ultrasensitive Magnetic-Resonance Probes. Adv. Mater. 2005, 17, 1949-1952. (29)

Huang, G.; Zhu, X.; Li, H.; Wang, L.; Chi, X.; Chen, J.; Wang, X.; Chen, Z.; Gao, J.,

Facile Integration of Multiple Magnetite Nanoparticles for Theranostics Combining Efficient MRI and Thermal Therapy. Nanoscale 2015, 7, 2667-2675. (30)

Zhou, Z.; Tian, R.; Wang, Z.; Yang, Z.; Liu, Y.; Liu, G.; Wang, R.; Gao, J.; Song, J.; Nie,

L.; Chen, X., Artificial Local Magnetic Field Inhomogeneity Enhances T2 Relaxivity. Nat. Commun. 2017, 8, 15468.


23


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 24 of 24

Table Of Contents (TOC) graphic


24

stealth magneto-nanomicelles for theranostics combining efficient MRI

Recommend Documents