Targeted Ultrasound-Triggered Phase Transition ... - ACS Publications

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Targeted Ultrasound-Triggered Phase Transition Nanodroplets for Her2-overexpressing Breast Cancer Diagnosis and Gene Transfection Di Gao, Jinbiao Gao, Ming Xu, Zhong Cao, Luyao Zhou, Yingqin Li, Xiaoyan Xie, Qing Jiang, Wei Wang, and Jie Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00761 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 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.

Molecular Pharmaceutics 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 56

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

Molecular Pharmaceutics

Targeted Ultrasound-Triggered Phase Transition Nanodroplets for Her2-overexpressing Breast Cancer Diagnosis and Gene Transfection

Di Gaoa,†, Jinbiao Gao a,†, Ming Xub, Zhong Caoa, Luyao Zhoub, Yingqin Lia, Xiaoyan Xieb, Qing Jianga, Wei Wangb,*, Jie Liua,*

a

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen

University, Guangzhou, Guangdong 510006, China b

Department of Medical Ultrasonics, Institute of Diagnostic and Interventional

Ultrasound, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong

†

510080, China

These authors contribute equally to this work.

* E-mail: [email protected] [email protected]

1

ACS Paragon Plus Environment


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 56

Abstract For successful gene therapy, it is imperative to accumulate therapeutic gene in tumor tissues followed by efficiently delivering gene into targeted cells. Ultrasound irradiation, as a noninvasive and cost-effective external stimulus, has been proved to be one of the most potential external-stimulating gene delivery strategies recently in further improving gene transfection. In this study, we developed a tumor-targeting ultrasound triggered phase-transition nanodroplets AHNP-PFP-TNDs comprised of a perfluorinated poly (amino acid) C11F17-PAsp (DET) as a core for simultaneously loading perfluoropentane (PFP) and nucleic acids, and a polyanionic polymer PGA-g-PEG-AHNP as the shell for not only modifying the surface of nanodroplets but also introducing an anti-Her2/neu peptide (AHNP) aiming to targeted treatment of Her2-overexpressing breast cancer. The results showed the average diameter of AHNP-PFP-TNDs was below 400 nm, nearly spherical in shape. And the modification of PGA-g-PEG-AHNP not only increased the serum stability of the nanodroplets

but

also

improved

the

affinity

between

nanodroplets

and

Her2-overexpressing breast cells. Both intratumor and intravenous injection of AHNP-PFP-TNDs into nude mice bearing HGC-27 xenografts showed that the gene transfection efficiency and the ultrasound contrast effect were significantly enhanced after exposed to the ultrasound irradiation with optimized ultrasound parameters. Therefore, this targeting nanodroplets system could be served as a potential theranostic vector for tumor targeting ultrasound diagnosis and gene therapy.

Keywords gene delivery, Her2 targeting, perfluoropentane, ultrasound irradiation, cavitation

2


Page 3 of 56

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


Introduction Along with the development and progress of molecular biology technology, gene therapy has become a new widely investigated therapeutic approach for cancer treatment. Therein to safety and effective gene-delivery systems are essential on account of their advantages on protection of gene from degradation and release the gene at a given site. Although numerous gene delivery systems have been developed 1-2

including virus vectors

and non-virus vectors

3-5

, inadequate suppression of

proliferation and metastasis of cancer cell still remains an urgent issue to be solved, since that virus vectors are accompanied with severe immunological inflammation or even carcinomatosis

6

transfection efficiency

and non-virus vectors are usually involved in low gene 7-8

. Therefore, more desirable gene-delivery strategies should

be taken into consideration to cover the existing shortages in gene therapy. Recently, the gene delivery strategy of combining ultrasound (external stimulus) and cationic micro-sized bubbles (gene carriers) has been the subjects of many investigations 9. It is generally known that ultrasound irradiation is more secure, noninvasive, cost-effective and site-specific when compared with the other physical approaches

10-11

. However, a main drawback of the micro-sized bubbles is their poor

ability to accumulate in tumor tissues via the enhanced permeability and retention effect (EPR effect). Moreover, the gas-phase perfluorocarbon (PFC), usually encapsulated in the microbubbles, is easily leaked from the shell materials of microbubbles under physics distortion and sonication resulting in a short lifetime during blood circulation

12

. For overcoming these defects, liquid-phase PFC

nanodroplets that using liposomes 13 or polymers 14 to load a low-boiling-point liquid PFC have been studied to replace the cationic microbubbles. At physiological temperature (37 °C), these nanodroplets could stay in the liquid state but to realize the effective acoustic droplet vaporization (ADV) to obtain the microbubbles under ultrasound irradiation with adequate high acoustic pressure. Therefore, as a potential ultrasound contrast agent (UCA), the generated microbubbles could dramatically enhance the acoustic impedance for ultrasonography. Furthermore, the nanodroplets have been proved useful in high-intensity focused ultrasound (HIFU) sensitization

15

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 56

and drug delivery therapeutically 16. However, there have been no studies on using the nanodroplets as gene carrier to co-loading liquid PFC and DNA for gene therapy so far. In our previous study 17, we have synthesized a new amphiphilic poly (amino acid) C11F17-PAsp(DET), which could effectively load the perfluoropentane (PFP) based on the high compatibility between these two fluorinated structure and condense DNA using hydrophilic cationic segments. This new material allowed us to prepare a PFP-loaded US-triggered phase-transition cationic nanodroplets, which combined the function of the ultrasound contrast agent and gene delivery vector within a single delivery nanocarrier. The PFP-TNDs nanodroplets could realize the phase transition to generate the microbubbles at the tumor sites for ultrasonography through acoustic droplet vaporization (ADV) after the ultrasound irradiation, meanwhile the gene transfection efficiency of PFP-TNDs could be enhanced dramatically due to the cavitation effect and sonoporation after increasing the ultrasonic pressure. Breast cancer is one of the most malignant diseases for women severely impacting the lives of female patients. Compared to the mammography, ultrasound imaging showed high accuracy in distinguishing benign and malignant masses for diagnosing breast cancer. Meanwhile, ultrasound examination is more sensitive, convenient and safer than mammography, which could also effective avoid false positives in mammography usually causing unnecessary biopsies

18

. Therefore,

combining the advantages of established gene delivery system PFP-TNDs mentioned above and the ultrasonography in diagnosing breast cancer, here we chose to construct a potential targeted gene carrier for diagnosis and therapy of breast cancer. Human epidermal growth factor receptor-2 (Her2/neu) has been known as an excellent therapeutic target for the treatments of breast cancer, which is overexpressed in nearly 30% of breast cancers 19. Therefore, a series of active drug delivery systems based on Herceptin, an anti-Her2 antibody, has been designed for the treatment of breast cancers

20-22

. However, this targeting strategy still remains defects of resistance and

side effects of Herceptin itself

23-24

. To tackle this problem, a 1.5 kDa cyclic peptide

mimic of Herceptin named Anti-Her2/neu peptide (AHNP) has been used to substitute the Herceptin with the results demonstrated that this target ligand has high affinity for 4


Page 5 of 56

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


the Her2/neu receptor and activity against tumor growth in vivo 25. Based on the advantages of AHNP peptide, to obtain ideal gene delivery efficiency in vivo, here we further developed a tumor-targeting cationic nanodroplet AHNP-PFP-TNDs comprised of an anti-Her2/neu peptide (AHNP) for the active targeted treatment of Her2 receptors overexpressing breast cancer (Scheme 1). Besides, further improvement in the performance of ultrasound on enhancing US contrast effect and gene transfection in vivo was another important purpose of this research. In this study, the physicochemical properties of nanodroplets were evaluated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Moreover, the DNA delivery ability, US responsiveness, cellular uptake, and gene expression against Her2 positive cells with or without US irradiation were also analyzed through in vitro studies. Finally, in vivo ultrasound imaging and gene transfection experiment using subcutaneous tumor xenograft model in nude mice were conducted to evaluate the effect of ultrasound on enhancing US contrast effect and gene delivery ability of the AHNP-PFP-TNDs.

Scheme 1. Schematic diagram of the AHNP-PFP-TNDs for the targeting ultrasound-assisted gene transfection.

Materials and methods 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 56

Materials For the chemical synthesis, β-Benzyl-L-aspartate acid (BLA) was obtained from GL Biochem Ltd. (China). 1H,1H,2H,2H,3H,3H-Perfluoroundecylamine (C11F17-NH2) and triphosgene

were

bought

from

Sigma-Aldrich Chemical Co..

AHNP

(FCDGFYACYKDV) was obtained from China Peptides Co. Ltd. (China). HOOC-poly (ethylene glycol)-NH2 (HOOC-PEG-NH2, 5000 Da) was purchased from Jenkem Technology Co. Ltd. (China).

N-methyl-2-pyrrolidone (NMP) and

Diethylenetriamine (DET) purchased from Aladdin Industrial Co. (China) were used after distillation. Poly (γ-glutamic acid) (γ-PGA, 5 kDa) was bought from Nanjing Sai Taisi Biotechnology Co. Ltd. (China). The reagents for nuclear magnetic resonance (chloroform-d (CDC l3), deuterium oxide (D2O) and dimethyl sulfoxide-d6 (DMSO-d6)) were bought from Sigma-Aldrich Chemical Co.. For the preparation and characterization of nanodroplets, perfluoro-n-pentane (PFP)

was

purchased

from

Strem

Chemicals.

Hoechst

33342,

3-(4,

5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) and Branched polyethylenimine (PEI) (25 kDa) were bought from Sigma. Gel red was purchased from Biotium Inc.. pLucDNA (pGL4.13 encoding the firefly luciferase), Reporter Lysis 5× Buffer and Luciferase Assay reagent were purchased from Promega Co.. Micro-BCA protein assay was bought from Thermo Fisher Scientific Inc. Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit was purchased from Mirus (Madison, WI). For the cell lines, SK-BR-3 cells and HGC-27 cells were obtained from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in DMEM (Hyclone) with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin–streptomycin at 37 °C under a 5% CO2 humidified atmosphere.

Synthesis

and

characterization

of

C11F17-poly

{N-[N’-(2-aminoethyl)]

aspartamide} (C11F17-PAsp (DET)) Based on our previous work β-Benzyl-L-aspartate

acid

17

(BLA)

, C11F17-PAsp (DET) was synthesized from in

three

chemical

reactions.

Briefly, 6


Page 7 of 56

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


β-benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) was firstly synthesized by the method of phosgenation of BLA using triphosgene through the Fuchs-Farthing method 26-27

. Then, C11F17-poly (β-benzyl -L-aspartate) (C11F17-PBLA) was prepared by the

ring-opening

polymerization

of

BLA-NCA

initiated

by

1H,1H,2H,2H,3H,3H-Perfluoroundecylamine (C11F17-NH2). For the synthesis of the end-product C11F17-PAsp (DET), the diethylenetriamine (DET) was used to the aminolysis reaction of C11F17-PBLA. To confirm the chemical structure of products in each step, proton nuclear magnetic resonance spectroscopy (1H NMR, 400MHz Bruker AvanceⅢ spectrometer) was selected with the products dissolved in the corresponding solvent. Specifically, BLA-NCA was detected in chloroform-d (CDCl3). C11F17-PBLA and C11F17-PAsp (DET) was dissolved in dimethyl sulfoxide-d6 (DMSO-d6) and deuterium oxide (D2O) respectively for the measurement. In addition, by integrating the area of characteristic peak on 1H NMR spectra, the molecular weight of prepared polymers was calculated.

Synthesis and characterization of PEG-AHNP AHNP (30 mg) and NH2-PEG-COOH (89.5 mg, about 1.2 equiv) were firstly dissolved in 4 mL of anhydrous DMSO. Secondly, NHS (2.45 mg, about 1.2 equiv) pre-dissolved in DMSO was quickly added into above solution with well mix. After dropping 4.21 mg EDC (about 1.2 equiv) and 20 µL triethylamine (TEA) into the solution then, the mixture was reacted at 35 °C for 24 h under nitrogen atmosphere. The resultant PEG-AHNP solution was dialyzed with a dialysis membrane (MWCO 3,500 Da) against DMSO for 2 days and deionized water for another 2 days followed by lyophilization to obtain the end-product PEG-AHNP which was stored at -20 °C. The chemical structure of PEG-AHNP was confirmed through the 1H NMR measurement by dissolving the product in dimethyl sulfoxide-d6 (DMSO-d6). In addition, the conjugation yield (AHNP content) in PEG-AHNP conjugates was detected by measuring the UV absorption of peptide AHNP at wavelength of 280 nm.

Synthesis and characterization of PGA-g-PEG-AHNP 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 56

For the synthesis of PGA-g-PEG-AHNP, 9.4 mg γ-glutamic acid (γ-PGA, 5000 Da) was firstly dissolved in 3 mL borate saline buffer (0.05 M, pH = 8.5) and 42.2 mg PEG-AHNP (about 1.2 equiv) was dissolved in 1 mL anhydrous DMSO respectively, followed by well mixture to obtain a transparent solution. Then, NHS (0.754 mg, about 1.2 equiv) pre-dissolved in borate saline buffer was quickly added into above solution. After dropping 1.258 mg EDC (about 1.2 equiv) into the solution, the mixture was reacted under nitrogen atmosphere at 25 °C for 24 h. To obtain the end-product PGA-g-PEG-AHNP, the solution was lyophilized after two-day dialysis against deionized water. The synthesized PGA-g-PEG-AHNP was stored at -20 °C. The 1H NMR spectrum was used to confirm the chemical structure of the conjugation. Besides, PEG-AHNP grafting percentage of PGA-g-PEG-AHNP was determined based on the integration of proton resonance absorptions in 1H NMR spectrum and calculated as the percentage of PGA carboxylate groups.

Preparation of targeting nanodroplets AHNP-PFP-TNDs Targeting nanodroplets AHNP-PFP-TNDs was formulated through a three-step process. Firstly, the PFP-NDs (PFP/C11F17-PAsp(DET) nanodroplets) were prepared via the general oil/water (o/w) emulsion process: 10 mg C11F17-PAsp(DET) was dissolved in deionized water (2 mL) in the first step followed by cooling to 4 °C. Subsequently, 60 µL perfluoro-n-pentane (PFP, 3%, v/v) was carefully added into the C11F17-PAsp(DET) aqueous solution. After undergoing a probe sonication process with the amplitude of 30% (1 s power on, 1 s power off, 90 s) in an ice bath with a probe-type sonicator (Misonix Sonicator S-4000), the PFP-NDs was acquired and stored at 4 °C prior to use. Secondly, The PFP-BNDs (PFP/C11F17-PAsp(DET)/ LucDNA binary nanodroplets) was formulated through gently mixing plasmid DNA and resultant PFP-NDs at various N/P ratios (molar ratio of primary amine groups in the N-substituted polyaspartamides to the phosphate groups in DNA) followed by 30 min incubation at 4 °C. For the last step on preparing the targeting nanodroplets AHNP-PFP-TNDs with different C/N ratios (molar ratio of primary amine groups in the N-substituted polyaspartamides to the free carboxyl groups in PGA-g-PEG-AHNP), 8


Page 9 of 56

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


a certain amount of PGA-g-PEG-AHNP dissolved in 10% glucose solution was mixed with PFP-BNDs aqueous solution of an equal volume with the purpose of obtaining an isosmotic solution with glucose concentration of 5%. After gently pipetting and another 30 min incubation (4 °C), AHNP-PFP-TNDs were prepared. Note that the PFP-TNDs without AHNP ligand was also prepared as a control in our work with the similar method above, where the PGA-g-PEG-AHNP was replaced by PGA-g-PEG with the same PEG grafting percentage.

Characterization of targeting nanodroplets AHNP-PFP-TNDs In order to get a better understanding of the characterization of prepared nanodroplets including their size distribution, surface charges and morphology, dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90) and Transmission Electron Microscope (TEM, JEM 1400) were selected for the measurement. For the DLS detection, the nanodroplets were measured in deionized water of certain concentration at 25 °C. All the detections were performed using a 633 nm wavelength beam at 90° scattering angle. For the TEM imaging, the nanodroplets were carefully dropped onto a copper grid and negatively stained to display the morphology by aqueous solution of uranyl acetate.

Gel red exclusion assay A successful gene carrier should definitely have the great potential to condense DNA, which could be confirmed by performing gel red exclusion assay 28. Briefly, an aliquot of 100×gel red solution was added to the nanodroplets solution followed by a 10 min incubation at 25 °C in the dark. Certainly, a naked DNA solution with equivalent DNA was used as control. With the assistance of microplate reader (BioTek Synergy 4), the fluorescence intensity of the mixture was measured (excitation wavelength: 510 nm, emission wavelength: 590 nm). According to the following equation, the relative fluorescence intensity of nanodroplets (PFP-BNDs, PFP-TNDs and AHNP-PFP-TNDs) was calculated to present the DNA condensation ability:

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 56

Where FSample, FDNA and FGel Red represent the fluorescence intensity values of nanodroplets, naked DNA and gel red, respectively.

Serum stability To observe the serum stability of resultant nanodroplets PFP-BNDs and AHNP-PFP-TNDs, we measured the size change of nanodroplets under mimic physiological conditions by a solution study. During the experiment, nanodroplets were firstly incubated in phosphate buffer saline solution (PBS, 0.01 M, pH = 7.4) containing 10% FBS at 37 °C with gentle stir. Then, 100 µL samples were withdrawn from the nanodroplets solutions to dilute to measure the particle size using DLS at predetermined time intervals. All measurements were performed in triplicate.

In vitro US imaging The method for evaluating the ultrasound imaging could refer to our previous work

17

. Briefly, the AHNP-PFP-TNDs solution with degassed water as dilution

medium was added into a Plastic Pasteur pipettes firstly followed by heating to 37 °C for 20s. Then, quickly move the pipettes into a pure water tank to perform contrast-enhanced ultrasonography (CEUS) imaging in the tank. For the details about ultrasonic device used during the experiment, a diagnostic Aplio500 (Toshiba Medical Systems, Tokyo, Japan) equipped with a 3 cm* 1 cm transducer head (375BT) was applied to generate the focused pulsed (pulse length 250 ms, mean frequency 4 KHz) at different frequencies and mechanical index (MI). After analyzing the obtained digital files including images and films, the ultrasound contrast effect was investigated. Note that the gray-scale intensity was measured from the obtained US images by ImageJ software.

In vitro gene transfection studies Optimization of AHNP-PFP-TNDs To screen out the optimal C/N ratios for 10


Page 11 of 56

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


AHNP-PFP-TNDs, the gene transfection efficiency of nanodroplets were evaluated at various C/N ratios (2/5 to 10/5). Besides, SK-BR-3 cells and HGC-27 cells, both Her2 positive cell lines, were chosen to test the targeting efficacy of peptide AHNP. Here, we took the SK-BR-3 cells as an example to describe the methods in detail. About 1 ×105 SK-BR-3 cells were seeded in a 48-well plate and incubated in DMEM medium (500 µL per well) with 10% FBS overnight at 37 °C under a 5% CO2 atmosphere. Then, the nanodroplets containing 0.5 µg of LucDNA were added to each well followed by further incubation for 6 h. It is worth mentioning that PEI/LucDNA polyplex was selected as a positive control during the experiment. Subsequently, the culture medium was replaced by fresh DMEM containing 10% FBS to incubate for another 42 h before analyzing the gene transfection efficiency. For luciferase assay, the cells were rinsed with PBS twice and lysed in 100 µL of l ×reporter lysis buffer (Promega). The cell lysate (20 µL) was collected from the supernatant after 10 min centrifugation with rotating speed of 12000 rpm and mixed with luciferase assay buffer (100 µL) to detect the fluorescence intensity using a luminometer (Promega) recorded as relative light units (RLU). In addition, the BCA protein assay (Pierce, USA) was selected to evaluate the protein content in the supernatant. Note that the gene transfection efficiency was calculated as RLU per mg protein in our experiment. All the transfection experiments were performed in triplicate. Competitive inhibition experiment For the further investigation of the targeting efficacy of AHNP, competitive inhibition experiments were conducted on SK-BR-3 cells and HGC-27 cells then. Typically, certain concentrations of free AHNP dissolved in serum-free culture medium were pre-incubated with SK-BR-3 cells and HGC-27 cells for 1 h. After the incubation, the serum-free media was changed to the media with 10% FBS containing the same amount of free AHNP. Then, the nanodroplets containing 0.5 µg of LucDNA were added to each well. After another 48 h incubation, the luciferase gene transfection efficiency of the samples was measured in accordance with the procedures described above. Transfection of AHNP-PFP-TNDs with US irradiation In order to observe the effect of ultrasound irradiation on enhancing gene transfection in vitro, we conducted 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 56

the gene transfection experiment with various ultrasound parameters. Briefly, the SK-BR-3 cells and HGC-27 cells were seeded at a density of 2 ×105 cells/well in 6-well

flat

bottomed

plates

and

allowed

to

adherence

overnight.

The

AHNP-PFP-TNDs containing 2 µg LucDNA were added to the well. Then, to achieve the effective ultrasound irradiation, the 375BT ultrasonic probe, which was 3.0 cm in length and 1.0 cm in width, was directly immersed into the culture medium while the ultrasound parameters (frequency and MI values) were adjusted in our experiment. At the time point of 6 h post ultrasound irradiation, the medium was changed to fresh medium

for

another

42

h

incubation.

Note

that

ultrasound

untreated

AHNP-PFP-TNDs group was used as control. The gene transfection efficiency of the nanodroplets s was measured based on procedures mentioned above.

Cellular uptake The cellular uptake experiment was conducted by flow cytometer. In the first step, a fluorescein-labeled plasmid was prepared according to the protocol of Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit (Mirus, Madison, WI) which actually conjugating the fluorescein to the luciferase plasmid. Then SK-BR-3 cells were seeded in a 6-well plate (2 ×105 cells per well) and incubated overnight. PFP-BNDs, PFP-TNDs and AHNP-PFP-TNDs bearing 2µg of the fluorescein-labeled plasmid were added to the cells subsequently. Through using the similar method in the section of gene transfection, the cells were irradiated by US with optimal ultrasonic parameters followed by another 4 h incubation at 37 °C. Thereafter, the cells were rinsed three times with PBS solution (0.01 M, pH=7.4) and collected using trypsin followed by centrifuge at 1500 rpm for 5 min. After resuspending the cells in 0.5 mL of cold PBS, the fluorescein contents were analyzed using FACS-Callibur (Becton Dickinson, San Jose) at an excitation wavelength of 488 nm (10000 counts per sample).

Cytotoxicity of PFP-loaded nanodroplets The cytotoxicity of prepared nanodroplets plays important role in determining 12


Page 13 of 56

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


the further application of these nanodroplets in vivo. Therefore, SK-BR-3 cells and HGC-27 cells were employed to evaluate the cytotoxicity of PFP-BNDs, PFP-TNDs and AHNP-PFP-TNDs by MTT assay. Take SK-BR-3 cells as an example, firstly the cells were cultured in 10% FBS included DMEM media and seeded in a 96 well plate with 7×103 cells (100 µL media per well) overnight. Then, the nanodroplets of different concentrations were added to the well followed by 48 h incubation. At the end of the

incubation, the cells were washed three times with PBS solution. Following that rinse, 100 µL of fresh medium and 20 µL of PBS containing 5 mg/mL of MTT were added to each well and the cells were incubated at 37 °C for an additional 4 h. For the last step, the medium was removed and 150 µL DMSO was added to dissolve the resultant formazan salt crystals for the quantitative measurement at an absorption wavelength of 570 nm using a microplate reader (BioTek Synergy 4).

In vivo US imaging and gene transfection For the study of in vivo US imaging and luciferase expression evaluation, HGC-27 cells were induced into female Balb/c nude mice (4-5 weeks old, Guangdong Medical Laboratory animal center, China) by means of the subcutaneous injection of 1×107 cells presuspended in 100 µL cold PBS into the flank of both sides and the mice were maintained in the Center for Experiment Animals (AAALAC accredited experimental animal facility). When the tumors reached 200 mm3 in size, the mice were divided into several groups (five mice per group) for in vivo experiments. Firstly, intratumor injection was employed to detect the ultrasound imaging and gene transfection efficacy of AHNP-PFP-TNDs at tumor sites. Meanwhile, the naked DNA

was

chosen

as

control

during

the

experiment.

Briefly,

150

µL

AHNP-PFP-TNDs solution were injected into the tumor of the mice at a dosage of 20 µg DNA per tumor. Then, one of tumors in each mouse was irradiated by ultrasound with low MI value for ultrasound imaging for 10 min to observe the contrast-enhanced ultrasonography (CEUS) imaging followed by high MI US irradiation for 60 s. Through another 48 h gene transfection in vivo, the mice were euthanized and their tumors were collected and rinsed twice with normal saline. Then 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 56

an IKA T25 digital Ultra-Turrax homogenizer (Germany) was used to homogenize the tumor tissues which was immersed into 50% (w/v) lysis buffer (Promega) followed by the process of freezing and thawing. Subsequently, 100 µL supernatants of homogenates which obtained by a centrifugation at 12 000 rpm for 10 min were mixed with luciferase substrate reagent (100 µL) to measure luciferase activity which was reported as RLU per mg of tissue. In addition, we also evaluated the ultrasound imaging and gene transfection of AHNP-PFP-TNDs via tail-vein injection. For this experiment, a total of 25 mice were divided into five experimental groups in the first step: (1) PFP-BNDs groups; (2) PFP-TNDs groups; (3) AHNP-PFP-TNDs groups; (4) PEI/LucDNA polyplex groups; (5) The naked LucDNA groups. Different formulations (150 µL) were administered to the mice at a dosage of 20 µg DNA per mouse. In this experiment, one of tumors in each mice was irradiated by ultrasound with low MI value for ultrasound imaging for 10 min firstly and irradiated with high MI value performing twice at 30 min and 4 h after the injection of formulations respectively. The detection of luciferase gene expression was conducted 48 h after the injection with the same method mentioned above.

Statistical analysis The data were statistically analyzed using two-sided Student's T-test. All data are expressed as the mean ± the standard deviation from at least three repeated experiments. A P value of 0.05 or less is considered to be statistically significant.

Results and discussion Synthesis and characterization of C11F17-PAsp (DET) The synthetic strategy of the fluorinated block copolymers illuminated by the previous work indicated that the high liquid perfluorocarbon compatibility could be obtained through fluorinating polymers

29-30

. With this inspiration, we synthesized

C11F17-PAsp (DET) for efficiently loading Perfluoro-n-pentane (PFP) and condensing DNA, the schematic illustration for the materials synthesis are depicted in Figure S1A. 14


Page 15 of 56

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


As shown in Figure S1A, the reaction of ring-open polymerization (ROP) of BLA-NCA

was

firstly

conducted

by

using

1H,1H,2H,2H,3H,3H-Perfluoroundecylamine (C11F17-NH2) as the initiator with the purpose of introducing the fluorinated groups to the product C11F17-PBLA. Figure S1B showed the 1H NMR spectroscopy of C11F17-PBLA in dimethyl sulfoxide-d6 (DMSO-d6). In the spectroscopy of C11F17-PBLA, the characteristic peak of BLA-NCA at δ 6.16 ppm (-NH- in five-membered ring) disappeared while the resonance at δ 1.63 ppm (-CH2CH2CH2-) appeared, which indicated the successful ring-open polymerization of BLA-NCA meanwhile confirmed the function of initiator C11F17-NH2 during the ROP. In addition, the degree of polymerization was calculated to be 28.2 from the resonance absorption peaks at δ 1.63 ppm mentioned above and δ 7.25 ppm attributed to C6H5CH2- in PBLA segment. It is also worth noting that the corresponding molecular weight equals to 6,500 Da as we expected. The further aminolysis reaction in the scheme (Figure S1A) was conducted by selecting diethylenetriamine (DET) as the aminolysis reagent not only to alter the hydrophilic and hydrophobic property of the polymer but also provide this macromolecule a positive charged segment for DNA binding. The

1

H NMR spectroscopy of

end-product C11F17-PAsp (DET) was exhibited in Figure S1C, where the resonance absorptions for -CO-(NHCH2CH2)2-NH2 at δ 2.86-3.50 ppm appeared and the peaks at δ 4.67-7.33 ppm attribute to the benzyl ester disappeared. The results confirmed the successful aminolysis of C11F17-PBLA by DET. Additionally, the degree of polymerization and molecular weight of obtained C11F17-PAsp (DET) were also calculated from spectroscopy through the absorptions in initiator at δ 1.63 ppm and peaks at δ 2.86-3.50 ppm (-CO-(NHCH2CH2)2-NH2) in the side chains of C11F17-PAsp (DET) in order to evaluate the possible breaking of main-chain amide bonds during the aminolysis reaction. With careful calculation, we confirmed that there was no obvious bond breaking during the aminolysis reaction.

Synthesis and characterization of PGA-g-PEG-AHNP To formulate the targeting nanodroplets AHNP-PFP-TNDs, anionic polymer 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 56

PGA-g-PEG-AHNP was necessary to be prepared through a two-step chemical reaction. For the first step, the AHNP (chemical structure shown in Figure 1A) was conjugated to NH2-PEG-COOH through dehydration condensation in organic solvent in consideration of the relative hydrophobic of AHNP molecule. The successful synthesis of PEG-AHNP was demonstrated by 1H NMR spectroscopy (Figure 1C), which exhibited the characteristic absorption peak of PEG at 3.62 ppm (–CH2CH2–O–) and AHNP at 6.93-7.24 ppm (C6H5– and C6H4–). In addition, we also confirmed the successful synthesis of PEG-AHNP through UV-Vis spectra that the absorption peak at 281nm attributed to AHNP could be found for the prepared PEG-AHNP while not appeared for the naked PEG (Figure 1B). To measure the conjugation rate of PEG-AHNP, we constructed a calibration curve by plotting the intensity of UV-Vis absorption of free AHNP versus the known concentrations. Through the calculation, the conjugation rate was confirmed to be 98.3%.

Figure 1. (A) Chemical structure of AHNP peptide. (B) UV-Vis spectra of AHNP at 16


Page 17 of 56

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


concentration ranging from 1 mg/mL to 0.0625 mg/mL. The PEG and PEG-AHNP at concentration of 3 mg/mL were used during the measurement. (C) 1H NMR spectra of PEG-AHNP in DMSO-d6. (D) 1H NMR spectra of PGA-g-PEG-AHNP in D2O. (temperature, 25 °C; polymer concentration, 5 mg/mL).

Secondly, the prepared PEG-AHNP was grafted to the side chain of γ-PGA with the ideal grafting percentage of 10% based on our previous work 17. Considering the hydrophilic of γ-PGA and the relative hydrophobic of PEG-AHNP, the chemical reaction was conducted in two-phase reaction system. Figure 1D shows the 1H NMR spectroscopy of PGA-g-PEG-AHNP in deuterium oxide (D2O) where the resonance at 3.51 ppm attributed to PEG (–CH2CH2–O–) and 2.36 ppm attributed to γ-PGA (-CH2CH2COO-). The simultaneous appearance of two absorption peaks in the spectrum indicated the successful preparation of PGA-g-PEG-AHNP. It is worth mentioning that the grafting percentage of PEG-AHNP to γ-PGA was 9.2% that approximated to our expectation of 10%.

Preparation and Optimization of PFP-loaded nanodroplets The PFP-loaded nanodroplets PFP-NDs (PFP/C11F17-PAsp(DET) nanodroplets) were primarily prepared via an o/w emulsion process that the amphiphilic polymer C11F17-PAsp(DET) as a cationic surfactant was dissolved in deionized water to well emulsify the amphiphobic PFP. After the probe sonication process, it is obvious that the solution turned from clear to milky and this state could remain for more than 24 h. As a comparison, in the absence of polymer C11F17-PAsp(DET) in the solution, the same sonication procedure could only formulate the emulsion with faintish opalescence, and the dispersed PFP would easily aggregate to the bottom of the containers in a short period of time. Hence, it is indicated that C11F17-PAsp(DET) played a significant role for the emulsification. The size and distribution of nanodroplets PFP-NDs were then evaluated by dynamic light scattering (DLS) with the results shown that the diameter of PFP-NDs was around 340 nm with a narrow distribution (PDI=0.051). Besides, the surface charges of PFP-NDs was further 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 56

measured using Malvern Zetasizer Nano ZS90 with the result of +68.2 mV (Table 1), not only confirming the existence of cationic segment in the polymer C11F17-PAsp(DET) but also demonstrating that the fabricated positive nanodroplets were perfect candidates to condense negative DNA through electrostatic interaction. Subsequently, the PFP-BNDs (PFP/C11F17-PAsp (DET)/LucDNA binary nanodroplets) were formulated and the N/P ratio of 20 was concluded as optimal which was used for the subsequent experiments.

Table 1. Characterization of PFP-NDs and the optimized PFP-BNDs and AHNP-PFP-TNDs. Data are given as mean ± SD (n = 3). Sample a

Size (nm)

PDI

Zeta Potential (mV)

PFP-NDs

340± 22

0.051

68.2 ± 2.7

PFP-BNDs

444± 20

0.227

54.5± 3.5

PFP-TNDs

302± 11

0.115

9.7± 2.9

AHNP-PFP-TNDs

356± 27

0.122

14.1 ±3.1

a: The PFP-NDs, PFP-BNDs, PFP-TNDs and AHNP-PFP-TNDs represent PFP/ C11F17-PAsp (DET) nanodroplets, PFP/C11F17-PAsp (DET)/ LucDNA binary nanodroplets, PFP/C11F17-PAsp (DET)/ LucDNA/ PGA-g-PEG ternary nanodroplets and

PFP/C11F17-PAsp

(DET)/

LucDNA/

PGA-g-PEG-AHNP

nanodroplets,

respectively.

To develop the ideal AHNP modified multiple nanodroplets for targeted gene delivery, anionic polymer PGA-g-PEG-AHNP was employed to surface-modify the positive PFP-BNDs. Actually, there are three reasons why we choose this anion polymer for the surface modification: (1) the modification by PGA-g-PEG-AHNP could decline the strong positive surface charge of PFP-BNDs, to a great extent, through adjusting the C/N ratio (C/N ratio stands for the molar ratio of primary amine groups in the N-substituted polyaspartamides to the carboxyl groups in PGA-g-PEG-AHNP), since that nanoparticles with strong positive charge have been 18


Page 19 of 56

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


proved to be not stable in physiological environment, causing a short lifetime during blood circulation as well as a high cytotoxicity in vivo

31

. (2) Through introducing

PEG onto the surface of prepared PFP-BNDs, the circulation half-life of nanodroplets could be extended due to the reduced opsonization according to the previous researches 32-33. (3) The peptide AHNP was selected as a ligand for active targeting to Her2/neu-positive cancer cells to enhance the cellular uptake meanwhile relatively compensate the adverse effects from PEGylation that confining the interaction between nanoparticles and cells 31. Firstly, to ensure the success of such surface modification, the size and zeta potential

of

AHNP-PFP-TNDs

(PFP/C11F17-PAsp

(DET)/LucDNA/

PGA-g-PEG-AHNP ternary nanodroplets) were characterized. As shown in Figure 2, raising the amount of PGA-g-PEG-AHNP declined the zeta potential of the AHNP-PFP-TNDs from +47.8 (C/N=0/5) to −32.4 mV (C/N=10/5). These results confirmed that PGA-g-PEG-AHNP was coated onto the surface of PFP-BNDs. Moreover, the mean size of the AHNP-PFP-TNDs dropped from 450 to 350 nm after being modified with PGA-g-PEG-AHNP since that the anion polymer was capable of packing the shell of the nanodroplets through electrostatic interaction.

Figure 2. Particle size and zeta potential of AHNP-PFP-TNDs at different C/N ratios (N/P=20).

To further evaluate the active targeting efficacy of AHNP-PFP-TNDs, in vitro 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

Page 20 of 56

gene transfection experiment and related competitive inhibition experiments were conducted against two Her2 positive cells, SK-BR-3 cells

34-35

and HGC-27 cells

36

.

Firstly, the in vitro gene transfection experiments were conducted with results shown in Figure 3A and 3B. It is obvious that the gene transfection efficiency of AHNP-PFP-TNDs significantly increased at certain C/N ratios for both cells demonstrating the positive effect of AHNP modification on active tumor targeting. Specifically, as illustrated in Figure 3A, the AHNP-PFP-TNDs nanodroplets significantly enhanced their gene transfection efficacy against SK-BR-3 cells at C/N ratios of 4/5 and 2/5, when compared to the control group of PFP-TNDs. In particular, at C/N ratio of 4/5, the transfection efficiency of the Her2 targeting nanodroplets AHNP-PFP-TNDs was 5.93-fold as high as that of ternary nanodroplets PFP-TNDs without AHNP ligand. However, with the further increase of C/N ratios (e.g., C/N=10/5 and 5/5), the difference of gene transfection efficiency between AHNP-PFP-TNDs and PFP-TNDs disappeared. A possible explanation might be that (1) the PEG shell density of the nanodroplets increased for the sample at relative high C/N ratio, resulting in an obstacle for the interaction between nanodroplets and cell membrane

31

. (2) Based on the previous research, the Her2/neu receptors exist as

"clusters" on cell membranes, which greatly influence the ligand binding due to less binding sites on cellular membrane. For the samples with intermediate number of ligands (C/N=4/5 and 2/5), the nanodroplets would be allowed to bind to the cluster properly resulting in better cellular uptake. By comparison, nanodroplets with the exorbitant ligand density might take up too many Her2/neu receptors for each cluster while deter other nanodroplets to bind to the same cluster

37-39

. Actually, the similar

results could be obtained from the gene transfection experiment of AHNP-PFP-TNDs against another Her2 positive cell line, HGC-27 cells (Figure 3B), that the transfection efficacy increased significantly at C/N ratios of 5/5 and 4/5. It is worth noting that there was no enhancement of gene transfection for the AHNP-PFP-TNDs at lower C/N ratio of 2/5 for this cell line, possibly due to the decreased receptor binding ability for those nanodroplets with low ligand density. On the basis of these results, the AHNP-PFP-TNDs with C/N ratio of 4/5 were selected for subsequent studies. 20


Page 21 of 56

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 3. Gene transfection efficiency of the PFP-TNDs and AHNP-PFP-TNDs at various C/N ratios against SK-BR-3 cells (A) and HGC-27 cells (B). The PEI/LucDNA polyplex are included as positive control; Gene transfection efficiency of the AHNP-PFP-TNDs against SK-BR-3 cells (C) and HGC-27 cells (D) after the cells were pretreated with free-AHNP at different concentrations. The PFP-TNDs nanodroplets were used as a reference sample for comparison.

For competitive inhibition assay, the cells were firstly pre-incubated with free AHNP at the concentration of 10 µM and 20 µM to saturate the Her2 receptors on the cell membrane. Then cells were incubated with nanodroplets in DMEM medium additionally containing free AHNP with the concentration mentioned above. As shown in Figure 3C and 3D, the luciferase expression of ternary nanodroplets PFP-TNDs remained nearly constant but the transfection was largely influenced for the peptide modified nanodroplets AHNP-PFP-TNDs, which experienced a dramatic decline due to the blockage of Her2/neu-mediated pathway. As for a well designed gene delivery system, strong DNA binding ability was 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

Page 22 of 56

essential for their further application. Through gel red exclusion assay, we proved the good DNA condensing ability of prepared AHNP-PFP-TNDs. For the basic principle of this experiment, an alternative non-toxic dye named gel red was used to detect the amount of free DNA. Briefly, the dye molecules could be intercalated into the free DNA bases leading to a remarkable enhancement of fluorescence intensity of solution. Therefore, the high fluorescence intensity means the existence of free DNA, but the weak fluorescence signal reflects DNA was well condensed by the gene carriers due to the prevented effective dye-DNA interactions 40. In Figure 4A, both PFP-TNDs and AHNP-PFP-TNDs with high C/N ratio (e.g., C/N = 10/5) exhibited higher fluorescence intensity compared to PFP-BNDs because the electrostatic interactions between cationic PFP-NDs and DNA was collapsed when introducing excessive anionic PGA-g-PEG-AHNP. However, the relatively low fluorescence intensity for the AHNP-PFP-TNDs at lower C/N ratios (e.g., C/N = 4/5) indicated that the DNA binding was not be easily influenced by moderate surface modification. Additionally, the serum stability of different formulations was also observed in the solution study with the results shown in Figure 4B. After incubation in a mimic physiological condition for 24 h, the particle size of binary nanodroplets PFP-BNDs increased quickly to more than 700 nm in the first 2h. In the same period, the mean count rate value, corresponding to the nanodroplets concentration in aqueous solution, experienced a sharp decrease which possibly resulted from the protein adsorption of nanodroplets in FBS containing solution. However, the nanodroplets modified with either PGA-g-PEG or PGA-g-PEG-AHNP remained fairly stable throughout the incubation period. Actually, there were some similar results obtained from our previous work

41

indicating that the introduction of the PEG layer to the

positive-charged nanoparticles could efficiently reduce opsonization and consequently increase the circulation half-life of nanoparticles

31

. Besides, the PFP of low boiling

point (29 °C) could still remain as liquid status at 37 °C since that the Laplace pressure was applied at the boundary of the nanodroplets, which allowed the nanodroplets to remain the same state at 37 °C for more than 24 h 14, 42.

22


Page 23 of 56

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 4. (A) Results of Gel Red exclusion assay on the AHNP-PFP-TNDs and PFP-TNDs with various C/N ratios; (B) Average particle size vs. incubation time for the PFP loaded nanodroplets with N/P ratio of 20 and C/N ratio of 4/5 incubated in PBS (0.01 M, pH = 7.4) containing 10% FBS at 37 °C; Data are given as mean ± SD (n = 3).

Lastly, transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the morphology and physicochemical characteristics of the nanodroplets with the optimal formulations. As shown in Table 1, the mean size of AHNP-PFP-TNDs is 356±27 nm, which is much smaller than the size of binary nanodroplets PFP-BNDs. Moreover, the surface charges of nanodroplets dropped continuously from 68.2±2.7 mV for PFP-NDs to 54.5±3.5 mV for PFP-BNDs followed by 14.1±3.1 mV for the final AHNP-PFP-TNDs, demonstrating that the LucDNA and anionic PGA-g-PEG-AHNP could efficiently neutralize the positive charges of PFP-NDs (Table 1). Additionally, TEM images in Figure 5A and 5B displayed that PFP-BNDs and AHNP-PFP-TNDs were nearly spherical in shape and the AHNP-PFP-TNDs presented a narrower size distribution and a good dispersibility compared to the PFP-BNDs. As shown in Figure 5C and 5D, the similar results were obtained through DLS analysis for both nanodroplets. In summary, the anion polymer PGA-g-PEG-AHNP played a significant role in decreasing the particle size, reducing the positive surface charges, and increasing the serum stability.

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 56

Figure 5. TEM images and particle size distributions of PFP-BNDs (A and C, N/P=20, C/N=0) and AHNP-PFP-TNDs (B and D, N/P=20, C/N=4/5).

In vitro US imaging Based on previous studies

43

, to obtain clear US images at tumor site, there are

two successful elements should be taken into consideration that are efficient US-induced phase transition (ADV) of PFP-loaded nanodroplets and enough stability of the generated bubbles in tumor tissues. Briefly, dramatic deviations of acoustic impedance among different transmitting medium are important reasons why bubbles are essential for the desirable ultrasonography. Actually, the acoustic impedance for water, liquid PFP and bubbles are 1.4 MRayl, approximate 0.3 MRayl and much less than 0.3 MRayl, respectively. Besides, thermal (heating) and acoustic (thermal and/or mechanical) largely influence the phase transition of nanodroplets, which closely related to US parameters, including the temperature, frequency, and MI. To better understand the phase transition of PFP-loaded nanodroplets, it is necessary to investigate these factors comprehensively. 24


Page 25 of 56

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


Actually, we have carried out a series of studies in vitro about US imaging of prepared PFP-loaded nanodroplets. Firstly, the influence of temperature on PFP phase transition was investigated. From the results shown in Figure 6A, for the group of AHNP-PFP-TNDs, the better ultrasound contrast effect could be obtained at body temperature of 37 ° C when compared to the lower temperature such as the normal ambient temperature of 25 ° C, which means the increased temperature holds a crucial status to promote the phase transition of PFP through decreasing the ADV threshold. However, for the control group of water, there is no such obvious difference between two selected temperatures. It is worth mentioning that there is a potential difference between the tumor and normal tissues, that the tumor cells usually have higher temperatures because of their enhanced metabolic activities, which would be better for the PFP-loaded nanodroplets to realize the efficient phase transition 44. Subsequently, we evaluated whether ultrasound frequency significantly affect the US contrast effect meanwhile the optimal frequency was also screened out for the further investigation. As shown in Figure 6B, a brighter US image was observed at a frequency of 3.5 MHz than the other two frequencies (2.5 MHz and 5.0 MHz) under CEUS mode. Moreover, the ImageJ analysis of gray-scale intensity of obtained photographs also confirmed the superiority of 3.5 MHz which displayed the highest gray-scale intensity in Figure 6C. Virtually, for the generated microbubbles with certain size, they usually possess a corresponding resonant frequency to enhance the backscattering signal and prolongate the ultrasonic imaging

45-46

. Therefore, the

frequency of 3.5 MHz was selected for the following research since that this frequency was more proper for the ultrasound imaging of AHNP-PFP-TNDs under CEUS mode. In our previous work related to theranostic agent PFP-TNDs-10%PEG

17

, we

found that ultrasound with various MI values might play different roles during the diagnosis and treatment: at a relatively lower MI value, the phase-transition of PFP-loaded nanodroplets could have be able to effectively triggered to obtain the stable microbubbles for ultrasonic diagnosis. However, in terms of the sonoporation for treatment, the collapse of microbubbles was urgently needed to enhance the 25



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 26 of 56

permeability of cell membrane, since that this collapse could induce a strong microjets accompanied with enhanced inertial cavitation, which could be realized at a higher MI value. To detect whether the AHNP-PFP-TNDs have the similar responsiveness to different MI values, we also investigated the influence of MI on US-triggered phase transition of AHNP-PFP-TNDs, that ultrasound images were obtained with MI value ranging from 0.04 to 1.53. Figure 6E illustrated that the gradually brighter images could be observed at MI values from 0.04 to 0.42, which were further confirmed by ImageJ analysis of gray-scale intensity in Figure 6D. Nevertheless, the darkened images and decreased gray-scale intensity were recorded when the MI values raised to 1.05 and 1.53, which demonstrating that the number of microbubbles declined resulting from the collapse of generated microbubbles at higher MI values. Additionally, the AHNP-PFP-TNDs were stable enough with ultrasound irradiation under lower MI value of 0.08 (widely used in clinical diagnosis) for more than 20 min in vitro (Figure 6F). Based on the results mentioned above, AHNP-PFP-TNDs, as an efficient ultrasound contrast agent (UCA), had great potential for ultrasonic diagnosis at low MI values of 0.08 and ADV effect at high MI values of 1.53.

26


Page 27 of 56

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 6. (A) In vitro US images of AHNP-PFP-TNDs (500 µg/mL) at various temperatures. Degassed water was selected as control. US images and gray-scale intensities of AHNP-PFP-TNDs diluted in degassed water (500 µg/mL) at different frequencies (B and C; MI = 0.08) and MI values (E and D; frequency of 3.5 MHz) using CEUS mode. (F) In vitro US images of AHNP-PFP-TNDs (500 µg/mL) at 27



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 28 of 56

predetermined time intervals (frequency of 3.5 MHz and MI of 0.08).

In vitro Gene transfection of AHNP-PFP-TNDs with US exposure Basically, there is a positive correlation between gene transfection efficiency of AHNP-PFP-TNDs and their inertial cavitation ability. Therefore, through transfecting Her2-overexpressing SK-BR-3 cells and HGC-27 cells with LucDNA-loaded AHNP-PFP-TNDs after US exposure, the cavitation ability of AHNP-PFP-TNDs was evaluated with the results presented in Figure 7. During the experiments, US parameters including ultrasonic frequency and MI value were altered to help us to better understand the behavior of ultrasound-triggered enhancement of gene transfection. It worth mentioning that the ultrasonic exposure time was not studied here because we have found that PFP-TNDs did not show the time-dependent transfection behaviors in our previous research 17. Firstly, three ultrasonic frequencies (2.5 MHz, 3.5MHz and 5MHz) were selected during the experiment to investigate the influence of frequency on gene transfection. As shown in Figure 7A and 7B, the gene transfection efficiency potentiated most obviously for the AHNP-PFP-TNDs at 3.5 MHz against both two cell lines. Comparably, the gene expression of AHNP-PFP-TNDs at other two frequencies displayed only a slight increase, even though the transfection efficacy at 5 MHz was higher than it of 2.5 MHz. Actually, there were visible differences of the US contrast effect according to in vitro US imaging and the corresponding results mean that it is more conducive to generate microbubbles at 3.5 MHz (Figure 6B). Then, the involvement of MI value in the gene transfection of AHNP-PFP-TNDs was studied through altering the ultrasonic MI values from 0.08 to 1.5. Note that MI value of 0.08 is widely used in clinical diagnosis and 1.5 is the maximum of the transducer used during the cell transfection experiment. As shown in Figure 7, the gene transfection efficiency of AHNP-PFP-TNDs was significantly higher when ultrasonic irradiated at higher MI. Specifically, compared to the AHNP-PFP-TNDs without US irradiation, there was a 25-fold increase in gene transfection efficiency against SK-BR-3 cells after an ultrasound treatment at a frequency of 3.5 MHz and a 28


Page 29 of 56

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


MI value of 1.50. Besides, for the HGC-27 cells, the gene transfection efficiency of AHNP-PFP-TNDs with US irradiation at same parameter was about 10-fold that of nanodroplets without US. As a note, the naked DNA was difficult to transfect the cells with or without US assistance, investigated in our previous work

17

. Therefore, we

confirmed that the nanodroplets and ultrasound irradiation play important roles in promoting gene transfection.

Figure 7. Luciferase expression by Her2-overexpressing SK-BR-3cells (A) and HGC-27 cells (B) transfected with LucDNA using AHNP-PFP-TNDs with or without US treatment at ultrasonic frequencies and MI value (exposure time of 60s).

According to the results from in vitro US imaging and gene transfection related study, the enhancement of gene transfection was determined by amount of generated microbubbles and their subsequent acoustic behaviors closely related to the ultrasound parameters. In Figure 6, the brighter US images at 3.5 MHz mean that this frequency could promote the evaporization of nanodroplets and the cavitation of generated microbubbles. Moreover, the bubbles underwent collapsed under US irradiation at higher MI value and the stronger microjet was generated simultaneously to further improve cavitation and sonoporation, resulting in dramatic augment in gene transfection efficacy.

Cellular uptake 29



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 30 of 56

To discuss the factors about influencing the gene transfection of nanodroplets, including targeting ligand AHNP and ultrasound irradiation, cellular uptake of fluorescence-labeling plasmid was evaluated using flow cytometry on SK-BR-3 cell lines. As shown in Figure 8A, the dramatically higher mean fluorescence intensity (MFI) was observed in the cells incubated with AHNP-PFP-TNDs nanodroplets compared to the PFP-TNDs, which could be attribute to the targeting effect of peptide AHNP, strongly interacting with the Her2/neu receptors overexpressing on the cell membranes. Moreover, there was an obvious right-shift of fluorescence for AHNP-PFP-TNDs in Figure 8B, the fluorescence histogram. Therefore, it can be concluded that the AHNP-PFP-TNDs nanodroplets showed the potential for targeting the Her2/neu receptors positive cell lines.

Figure 8. Mean fluorescence intensities (A) and fluorescence histograms (B) of the SK-BR-3

cells

AHNP-PFP-TNDs

incubated (with

for or

4h

with

without

the

PFP-BNDs,

ultrasound

PFP-TNDs

irradiation)

and

bearing

fluorescein-labeled LucDNA. Data are given as mean ±SD (n=3). (MI=1.50, frequency=3.5MHz, exposure time =60s).

Then, we still detected the effect of ultrasound irradiation on the cellular uptake of labeled LucDNA. In Figure 8A, under ultrasound irradiation, the MFI in the cells enhanced significantly for both PFP-TNDs (2-fold compared to PFP-TNDs without 30


Page 31 of 56

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


ultrasound

irradiation)

and

AHNP-PFP-TNDs

(2.8-fold

compared

to

AHNP-PFP-TNDs without ultrasound irradiation), which was consistent with results shown in Figure 8B. Based on above studies, the enhancement of cellular uptake of labeled gene could be explained by the sonoporation after US irradiation, which could generate transient pores on the cell membrane

47-48

. Besides, it should be mentioned

that the generated pores could reseal in a few seconds after the ultrasound irradiation, reported by precious work 49.

In vitro cytotoxicity To determine the cytotoxicity of as-prepared LucDNA loaded nanodroplets PFP-BNDs, PFP-TNDs and AHNP-PFP-TNDs, the MTT assay was conducted on both SK-BR-3 cell lines and HGC-27 cell lines. As displayed in Figure 9, there was no obvious cytotoxicity for the AHNP-PFP-TNDs on both two cell lines even at highest sample concentration (80µg/mL), which means the excellent biocompatibility of this formulation. Note worthily, among three different samples, the cell viability of PFP-TNDs and AHNP-PFP-TNDs slightly enhanced compared to the PFP-BNDs except the samples with highest concentration on SK-BR-3 cells (Figure 9A), demonstrating that the advantage of surface modification by anion polymers through lowering the surface positive charge of nanodroplets.

Besides, the possibility of the decline of the cell viability caused by excessive sonoporation during ultrasound irradiation was also taken into consideration. Through the MTT assay, the influence of selected ultrasound parameters and the established experimental model during our experiment on cell viability was further evaluated. Briefly, the cytotoxicity was firstly detected at various MI values ranging from 0.08 to 1.50 to observe whether severe rupture of the microbubbles at relatively higher MI value could affect the cell viability. The results showed that there was no notably cytotoxicity for the AHNP-PFP-TNDs, even at the highest MI value of 1.50. Meanwhile, even the exposure time increased to 60s, the results also showed no dramatic influence on the cell viability (data not shown). Therefore, the selected 31



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 32 of 56

ultrasound parameters and established US models during our experiments was not impacting the cell proliferation, which is important for AHNP-PFP-TNDs to be further used as an efficient theranostic agent.

Figure 9. Cell viability of the SK-BR-3 cells (A) and HGC-27 cells (B) after incubation with the different nanodroplets samples at various concentrations.

In vivo ultrasound imaging and gene transfection experiment Based on the results obtained from in vitro gene transfection study that the luciferase gene transfection efficiency could be enhanced significantly when the gene was delivered by AHNP-PFP-TNDs with the aid of ultrasound irradiation, the effect of AHNP-PFP-TNDs and ultrasound irradiation on promoting gene transfection in vivo was further evaluated via establishing bilateral subcutaneous HGC-27 xenografted model in nude mice (shown in Figure 11B). During our experiments, the low MI value of 0.08 was primarily chosen to investigate the ultrasonic contrast effect of different samples. Then, the ultrasound with higher MI values of 1.5 was used to irradiate the tumor site followed by evaluating the gene transfection efficiency after another 48h gene expression. Firstly, AHNP-PFP-TNDs were intra-tumor injected into the nude mice and the ultrasound images were illustrated in Figure 10A, where naked DNA was selected as control. As shown in the figure, the contrast effect at tumor site enhanced significantly at MI of 0.08 after intra-tumor injection of AHNP-PFP-TNDs. However, there was no obvious change of the contrast effect for the group of naked 32


Page 33 of 56

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


DNA. Moreover, the effect of high acoustic pressure ultrasound irradiation on gene transfection was investigated through increasing the MI value to 1.5 and US irradiating the tumor site for 60s. The gene transfection efficiency was shown in Figure 10B. In general, it is hard for the naked DNA to realize gene transfection at tumor site due to the electrostatic repulsion between negative DNA and cell membrane, which could block the cellular uptake. Indeed, the gene transfection efficiency of DNA was very low even with ultrasound irradiation. Compared to the naked DNA, the tumors which injected of AHNP-PFP-TNDs showed the definite gene expression after 48 h post-injection. Meanwhile, there was an obvious enhancement of gene transfection level for the tumors with ultrasound irradiation at high MI value (MI=1.5). The overall experimental results above revealed that the nanodroplets AHNP-PFP-TNDs could experience phase transition with ultrasound irradiation, which not only help to enhance the ultrasound imaging but also promote the gene transfection resulted from the increased cellular uptake of the targeting nanodroplets, leading to the higher gene expression.

Figure 10. (A) In vivo ultrasound images of tumors with intra-tumor injection of the 33



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 34 of 56

AHNP-PFP-TNDs and naked LucDNA at frequency of 3.5MHz, MI of 0.08. (B) Luciferase gene expression efficiencies in the tumors of Balb/c nude mice at 48 h after intra-tumor injection of the AHNP-PFP-TNDs and naked LucDNA (with or without ultrasound irradiation). *represents p

Targeted Ultrasound-Triggered Phase Transition ... - ACS Publications

Recommend Documents