Subscriber access provided by MT ROYAL COLLEGE
Communication
Peptide-Functionalized Phase-transformation Nanoparticles for LIFU-Assisted Tumor Imaging and Therapy LeiLei Zhu, HongYun Zhao, ZhiYi Zhou, YongHong Xia, Zhigang Wang, Haitao Ran, Pan Li, and JianLi Ren Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b05087 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 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.
Nano Letters 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 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Nano Letters
Peptide-Functionalized
Phase-transformation
Nanoparticles for LIFU-Assisted Tumor Imaging and Therapy LeiLei Zhu,† HongYun Zhao,† ZhiYi Zhou,† YongHong Xia, † ZhiGang Wang, †,‡ HaiTao Ran, †,‡ Pan Li, †,‡
and
JianLi Ren*,†,‡ †
Chongqing Key Laboratory of Ultrasound Molecular Imaging,
Chongqing 400010, China; ‡
Ultrasound Department of the Second Affiliated Hospital of Chongqing
Medical University, Chongqing 400010, China.
ABSTRACT: In this study, we successfully developed a novel tumor homing-penetrating
peptide
-
phase-transformation
nanoparticles
functionalized
drug-loaded
(tLyP-1-10-HCPT-PFP
NPs)
combining LIFU for tumor ultrasound molecular imaging and precise therapy. With the nanoscale particle size, tLyP-1-10-HCPT-PFP NPs could pass through the tumor vascular endothelial cell gap. Induced by tLyP-1
peptide
with
targeting
and
penetrating
efficiency,
tLyP-1-10-HCPT-PFP NPs could increase tumor accumulation and penetrate deeply into the extravascular tumor tissue, which penetrating through Combined
extracellular with
matrix,
LIFU
which
cellular
membrane
was
low-intensity
ACS Paragon Plus Environment
to and
cytoplasm. focused,
Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 34
tLyP-1-10-HCPT-PFP NPs could phase-transformate into microbubbles and enhance tumor ultrasound molecular imaging for tumor diagnosis. What’s more, after further irradiation by LIFU, intracellular ‘explosion effect’ caused by ADV, UTMD and release of 10-HCPT could realize physico-chemical synergistic antitumor therapy.
KEYWORDS: Tumor homing-penetrating peptide, phase-transformation, low-intensity focused ultrasound, ultrasound molecular imaging, precise therapy
Ultrasound molecular imaging has made remarkable progress over the last decade, and recent studies have shown that ultrasound contrast agents not only enhance the imaging for diagnosis, but also serve as vehicles to carry drugs or gene for therapy.1-5 Although the traditional organic microbubbles can significantly enhance the echo signals of the ultrasonography due to the fact that they can function as the reflectors for ultrasound by the resonance at the diagnostic frequency,6,7 their extremely large particle sizes (typically several micrometers) restrict them only to image in the blood pool because only the particle sizes of less than 700 nm can infiltrate the large pores which are present in the leaky vasculature of tumors.8,9
Comparatively, nanosized
bubbles only have the very limited contributions to improve the contrast-enhanced ultrasound imaging due to the substantial decrease of the nonlinear backscattering with the reduction of their diameters.10-12 Fortunately, the nanoparticles (NPs) or microspheres with perfluorocarbon could go through phase-transformation to microbubbles via acoustic droplet vaporization (ADV) technology which was first proposed by kripfgans et al.12 Furthermore, the technique of ultrasound targeted microbubble destruction (UTMD) for drug or gene delivery has shown an attractive
ACS Paragon Plus Environment
Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Nano Letters
application in disease treatment, especially in cancer therapy.13-16
UTMD increases
the permeability of cell membrane through sonoporation, effectively facilitating intracellular transfer of macromolecules.3,17
But UTMD was passive targeting and
microbubbles in the sound field were all destructed by UTMD, which did not achieve the precise location in the true sense. Therefore, to enhance ultrasound imaging and achieve precise targeted microbubble destruction, our lab invented low intensity focused ultrasound (LIFU).
Integrating diagnosis, treatment, monitoring and effect
evaluation, LIFU realized imaging and target drug release at the same time. LIFU could
precisely
focus
on
tumor
tissue,
which
reduced
the
nonspecific
phase-transformation and destruction in normal tissues. Compared with high intensity focused ultrasound (HIFU), acoustic intensity in a focal spot of LIFU was only 0.4– 3.2 W/cm2, which further prevented damage to the surrounding normal tissues. Additionally, tumor-targeting delivery systems have been developed for precise tumor targeting, increased tumor accumulation and enhanced cancer therapy.18-20
To
further enhance the targeting effect, ligands characterized by specific targeting effect were modified onto the surface of vehicle, which could be recognized by specific receptors, carriers, etc.21-23
But nanoparticles mediated by the ligands with poor
penetration effect only enter tumor tissue by the EPR effect and remain near blood vessels. Although angiogenesis is one of the main features of tumors, there are regions that lack of microvessels in the tumors which restrict the ability of targeting delivery systems. Therefore, the penetration of targeting delivery systems is extremely significant for the effective treatment of solid tumors. To overcome this problem, phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels and penetrate deep into the extravascular tumor tissue,24,25 which is called tumor homing-penetrating peptide. tLyp-1 (CGNKRTR), a truncated form of LyP-1 (CGNKRTRGC) with 7 amino acid, was reported as a ligand targeted to the NRP receptor with high affinity and specificity.26
Studies showed that the neuropilin-1 (NRP-1) was frequently over
expressed in several human tumor types, including carcinomas (e.g., pancreas, prostate, breast, ovarian, colon and kidney), melanoma, glioblastoma, leukemias,
ACS Paragon Plus Environment
Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
lymphomasandothers.27-34
Page 4 of 34
tLyp-1 peptide was a kind of targeting peptide with the
function of penetrating tumor vessels and tumor stroma. Herein, the aim of this study was to develop a novel tumor homing-penetrating peptide - functionalized drug-loaded phase-transformation nanoparticles combining LIFU for tumor ultrasound molecular imaging and precise therapy.
As shown in
schematic diagram (Figure 1), our nanoparticles consist of four parts: 1) The liquid core is composed of pefluoropentane (PFP), a hydrophobic fluorocarbon compound with adequate boiling point (about 29
),35 which has been extensively used clinically;
2) The lipid shell consist of biocompatible DSPE-PEG3400-tLyP-1, DPPG, DPPC and cholesterol; 3) 10-Hydroxycamptothecin (10-HCPT) is loaded in the phospholipid bilayer of lipid shell which is one of the natural camptothecin analogs with a powerful anti-tumor activity against a wide range of tumors in clinical practice. The required dose of 10-HCPT for cancer therapy is 4~6 mg per injection which is over twenty times lower than that of paclitaxel or doxorubicin;36-38
4) The
tumor
homing-penetrating peptide tLyP-1 which connected to DSPE-PEG3400-mal via a maleimide-thiol coupling reaction (Supporting Information, Figure S1) is present on the outer shell of nanoparticles.
After reaction, the analysis of HPLC verified that
tLyP-1 peptide was connected to DSPE-PEG3400-mal successfully (Supporting Information, Figure S2). Our study had shown that FITC-tLyP-1 could target to MDA-MB-231cells and penetrate into the deep of the MDA-MB-231 spheroids model39,40 (Supporting Information, Figure S3, S4).
The nanoparticles mediated by
tLyP-1 peptide could penetrate through tumor blood vessels and tumor stroma, and into the deep tumor tissue,26,41-43 which might realize targeted diagnosis and precise therapy of every tumor cell. This strategy was shown in Figure 1, our novel ultrasound contrast agent with smaller size could arrive tumor tissues through the tumor vascular endothelial gap (380~780 nm) by EPR effect. The nanoparticles mediated by tLyP-1 peptide accumulated and penetrated deep into the extravascular tumor tissue. At target site, the nanoparticles were irradiated by LIFU. Then nanoparticles were turned into microbubbles by ADV for enhancing tumor ultrasound imaging. After that, the 10-HCPT were released accurately from the microbubbles
ACS Paragon Plus Environment
Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Nano Letters
destructed by LIFU for precise targeting anti-tumor therapy due to the focusing of LIFU , targeting and penetrating of tLyp-1 peptide. To substantiate our strategy for enhancing ultrasound molecular imaging and precise therapy, we prepared the 10-HCPT-loaded phase-transformation nanoparticles mediated by tLyP-1 peptide (tLyP-1-10-HCPT-PFP NPs) using filming-rehydration method and acoustic vibration method (Details see the methods in the supporting information).
It showed that the size and zeta potential of the tLyP-1-10-HCPT-PFP
NPs were (366.30±11.50) nm and (5.70±3.70) mV determined by Malvern Zeta Sizer (Figure 2a, 2b), respectively. While, the size and zeta potential of the 10-HCPT-PFP NPs were (247.50±9.80) nm and (-42.70±5.50) mV (Supporting Information, Figure S5).
Interestingly,
compared
with
10-HCPT-PFP
NPs,
the
zeta
of
tLyP-1-10-HCPT-PFP NPs was raised to positive, which owed to tLyP-1 peptide with positive charge. The nanoparticles displayed spherical shape with moderate uniform size and particle size measured from TEM images was in good agreement with that measured by inverted fluorescence microscope (Figure 2c, 2d). With the help of the particle size advantage, tLyP-1-10-HCPT-PFP NPs could go through the tumor vascular endothelial gap (380~780 nm) to the extravascular tumor tissue. It overcame the first barrier of drug delivery system for effective tumor treatment. Furthermore, the envelopment rate and 10-HCPT-loading rate of our nanoparticles were (86.04±4.27)% and (7.82±0.38)%, respectively. The targeting and penetrating efficiency of tLyP-1-10-HCPT-PFP NPs was evaluated in vitro.
After co-incubation with monolayer MDA-MB-231 cells at 37
for 1h, the confocal laser scanning microscopy (CLSM) images showed that DiI-labeled tLyP-1-10-HCPT-PFP NPs were accumulated obviously around the cytomembrane of MDA-MB-231 cells that NRP-1 was overexpressed.
Meanwhile,
a part of tLyP-1-10-HCPT-PFP NPs penetrated into the cytoplasm. We also found that the amount of tLyP-1-10-HCPT-PFP NPs around the cytomembrane and into the cytoplasm of MDA-MB-231 cells was higher than that of unmodified 10-HCPT-PFP NPs. However, there was no significant accumulation of the two NPs around human umbilical vein endothelial cells (HUVEC) (Figure 3a). Therefore, it showed that
ACS Paragon Plus Environment
Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 34
targeting efficiency of tLyP-1-10-HCPT-PFP NPs to MDA-MB-231 cell was high. The results of quantitative analysis by flow cytometry were in accordance with the results above (Figure 3b-d). Some studies had found that in vitro three-dimensional tumor spheroid was sufficient model to mimic the microenvironment of solid tumor because of several similarities, among which are poor drug penetration, drug resistance, altered protein expression and enzyme activity, and a viable rim with gradients of oxygen tension, nutrients, catabolites and cell proliferation.
In this
study, we had established the three-dimensional spheroid of MDA-MB-231 cell successfully and after co-incubation with DiI-labeled tLyP-1-10-HCPT-PFP NPs or 10-HCPT-PFP NPs at 37
for 24 h, it was found that the red fluorescence of
tLyP-1-10-HCPT-PFP NPs were distributed into tumor spheroids more evidently than that of 10-HCPT-PFP NPs (Figure 4a). The tLyP-1-10-HCPT-PFP NPs could reach more than 80 µm away from the bottom of the tumor spheroids and were distributed throughout the whole spheroids. However, 10-HCPT-PFP NPs were distributed only in the surface layer of the tumor spheroids, which indicated that tLyP-1 peptide could facilitate the transportation of NPs into the tumor spheroids (Figure 4b, 4c). To further validate the active targeting efficiency of tLyP-1-10-HCPT-PFP NPs in vivo, fluorescence imaging was performed in MDA-MB-231 xenograft-bearing nude mice using DiR (a near-infrared dye) to track the NPs. As shown in Figure 5a, red fluorescence was seen in tumor area 1 h~12 h after the injection of DiR-labeled tLyP-1-10-HCPT-PFP NPs, and the fluorescence intensity and range peaked at 1 h. However, there was no obvious red fluorescence in tumor area 1 h~12 h after the injection of DiR-labeled 10-HCPT-PFP NPs. The fluorescence intensity had no obvious difference in other organs between tLyP-1-10-HCPT-PFP NPs and 10-HCPT-PFP NPs. Correspondingly, the conclusion was further determined by ex vivo imaging of the tumor and other organs (Figure 5b).
It suggested that
modification with tLyP-1 peptide improved the active tumor-targeting efficiency of NPs in vivo.
Meanwhile，12 h after the injection, tissue frozen section were
prepared and the images were captured using CLSM.
The fluorescent intensity of
DiI-labeled tLyP-1-10-HCPT-PFP NPs in tumor was considerably higher than that of
ACS Paragon Plus Environment
Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Nano Letters
DiI-labeled 10-HCPT-PFP NPs. In most of normal tissues, the fluorescence intensity had also no obvious difference between tLyP-1-10-HCPT-PFP NPs and 10-HCPT-PFP NPs (Figure 5c).
It indicated that tLyP-1-10-HCPT-PFP NPs could only increase the
targeting effect to tumors rather than normal tissues. These results above should attribute to tLyP-1 peptide which could specifically combine with NRP-1 overexpressed on MDA-MB-231 cell and contained a cryptic CendR motif ((R/K)XX(R/K)) which was responsible for cell internalization and tissue penetration.44-46 To test the phase-transformation effect of tLyP-1-10-HCPT-PFP NPs by heating, NPs were dealt with different temperature. From the optical microscopy observation, it was found that tLyP-1-10-HCPT-PFP NPs realized phase-transformation when the temperature reached about 45
(Figure 6a), while there were no obvious change in
size and shape when the temperature were about 37
and 40
.
It proved that PFP
was loaded into the core of the NPs successfully. As far as we know, the boiling pint of PFP was only 29
. While in this study, we found that the phase-transformation
temperature of tLyP-1-10-HCPT-PFP NPs was about 45
. It demonstrated that the
phase-transformation temperature of tLyP-1-10-HCPT-PFP NPs increased with the cover of lipid shell. Furthermore, tLyP-1-10-HCPT-PFP NPs could not turn into microbubbles at physiological temperature (37
), which avoided the embolism.
To confirm that tLyP-1-10-HCPT-PFP NPs could enhance ultrasound imaging by ADV in vitro and in vivo, NPs were irradiated by LIFU. As shown in Figure 6b, tLyP-1-10-HCPT-PFP NPs also realized phase-transformation by LIFU in vitro. With the increase of acoustic intensity and time, the B-mode and contrast-enhanced ultrasound (CEUS) mode imaging of tLyP-1-10-HCPT-PFP NPs enhanced after being irradiated by LIFU. Interestingly, this phenomenon described above was only observed within the scope of 0.4~2 W/cm2, while the B-mode and CEUS mode imaging of tLyP-1-10-HCPT-PFP NPs receded at 2.8 W/cm2.
The light microscopy
images of tLyP-1-10-HCPT-PFP NPs after irradiation by LIFU showed that the size of nanoparticles increased when acoustic intensity was within the scope of 0.4~2 W/cm2, while the phase-transformation nanoparticles decreased at 2.8 W/cm2 (Supporting
ACS Paragon Plus Environment
Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Information, Figure S6).
Page 8 of 34
It demonstrated that some of the phase-transformation
nanoparticles were exploded at 2.8 W/cm2, which provide a basis for drug release triggered by LIFU.
Drug release experiment in vitro showed that drug release rate of
LIFU group was about four times of control group at 12 h, the drug release rates of LIFU group were also higher than that of control group at 24 h, 48 h, 60 h, 72 h (Supporting Information, Figure S8).
As for ultrasound imaging, quantitative
analysis of grayscale values was calculated using an Ultrasound Image Analyzer (DFY-
type, Institute of Ultrasound Imaging, Chongqing Medical University, China)
which was an independent research and development product of our lab. The analysis result was in conformity with ultrasound imaging in the B-mode and CEUS mode (Figure 6c, 6d).
It indicated that the grayscale values of the B-mode and CEUS
mode imaging of tLyP-1-10-HCPT-PFP NPs enhanced obviously after being irradiated by LIFU at 2 W/cm2 for 3 min (P
