A Copper-Mediated Disulfiram-Loaded pH-Triggered PEG-Shedding

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Copper-mediated disulfiram-loaded pH-triggered PEG sheddable TAT peptide-modified lipid nanocapsules for use in tumor therapy Ling Zhang, Bin Tian, Yi Li, Tian Lei, Jia Meng, Liu Yang, Yan Zhang, Fen Chen, Haotian Zhang, Hui Xu, Yu Zhang, and Xing Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06488 • Publication Date (Web): 26 Oct 2015 Downloaded from http://pubs.acs.org on October 31, 2015

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 42

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

Copper-mediated disulfiram-loaded pH-triggered PEG sheddable TAT peptide-modified lipid nanocapsules for use in tumor therapy Ling Zhang†, Bin Tian†, Yi Li‡, Tian Lei†, Jia Meng†, Liu Yang†, Yan Zhang§, Fen Chen †, Haotian Zhang ‡, Hui Xu†, Yu Zhang**,†and Xing Tang*,†

†

Department of Pharmaceutics, College of Pharmacy, Shenyang Pharmaceutical

University, Shenyang, Liaoning, PR China ‡

Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang,

Liaoning, PR China §

Normal College, Shenyang University, Shenyang, China

ABSTRACT Disulfiram, which exhibits marked tumor inhibition mediated by copper, was encapsulated in lipid nanocapsules modified with TAT peptide (TATp) and pH-triggered sheddable PEG to target cancer cells based on tumor environmental specificity. PEG sheddable lipid nanocapsules (S-LNCs) were fabricated from LNCs by decorating short PEG chains with TATp (HS-PEG1k-TATp) to form TATp-LNCs, and then covered by pH-sensitive graft copolymers of long PEG chains (PGA-g-PEG2k). The DSF-S-LNCs had sizes in the range 60-90 nm and were stable in the presence of 50% plasma. DSF-S-LNCs exhibited higher intracellular uptake and anti-tumor activity at pH 6.5 than pH 7.4. The pre-incubation of Cu showed that the DSF cytotoxicity was based on the accumulation of Cu in Hep G2 cells. Pharmacokinetic studies showed the markedly improved pharmacokinetic profiles of DSF-S-LNCs (AUC= 3921.391 mg/L·h, t1/2z=1.294 h) compared with free DSF (AUC=907.724 mg/L·h, t1/2z=0.252 h). The in vivo distribution of S-LNCs was

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

investigated using Cy5.5 as a fluorescent probe. In tumor-bearing mice, the delivery efficiency of S-LNCs was found to be 496.5% higher than free Cy5.5 and 74.5% higher than LNCs in tumors. In conclusion, DSF-S-LNCs increased both the stability and tumor internalization, and further increased the cytotoxicity because of the higher copper content.

KEYWORDS Disulfiram; copper; lipid nanocapsules; pH sensitive; sheddable; TAT peptide


Page 2 of 42

Page 3 of 42

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


1. INTRODUCTION Disulfiram (DSF), approved by the FDA for treating alcoholism because of its ability to irreversibly inhibit aldehyde dehydrogenase (ALDH), has the potential to be used as a treatment for neoplastic diseases.1, 2, 3 DSF has the ability to induce apoptosis, 4 inhibit cell proliferation and reduce angiogenesis,5 thereby suppressing

tumor

growth,6 invasion and metastasis.7,8 Moreover, used in conjunction with copper (Cu), the action of DSF could be enhanced.9,10 Cu, an essential trace element for humans, plays a key role in tumor cell proliferation and angiogenesis and acts selectively on tumor tissues than normal tissues.11,12 The cytotoxicity of DSF

more mainly

depends on the formation of intracellular metabolite-metal complexes with Cu.13 Cu participates in neovascularization, protein aggregation and oxidative stress involving tumor growth, invasion and metastasis.12 As a divalent metal ion chelator, DSF is degraded to diethyldithiocarbamate acid, which strongly chelates with Cu to form a copper

(II)

diethyldithiocarbamate

complex

(Cu(DETC)2),12

proteasome activity,14,15 acts on ALDH and the NF-κB pathway,

which 16,17

inhibits

suppresses

superoxide dismutase (SOD) and triggers the production of reactive oxygen species (ROS) ,18,19 activates the JNK/c-jun pathway,13 and inactivates enzymes associated with tumorigenesis.20,21 The intracellular action of Cu(DETC)2 is responsible for the anti-tumor function of DSF. Therefore, Cu may be used as a tumor-specific target for DSF to improve anti-tumor efficacy.9, 14 However, under the action of gastric acid or glutathione (GSH) reductase in blood, free DSF is known to be readily reduced to diethyldithiocarbamic acid, which is unstable and undergoes further metabolism.22, 23, 24 Although DSF has great potential for use in cancer therapy, free DSF cannot accumulate in tumor regions due to its instability in blood. Lipid nanocapsules (LNCs) consist of oily cores and shells that are non-ionic hydrophilic and lipophilic surfactants.25 LNCs have been shown to accept a high loading of hydrophobic cargo as well as being able to reverse multidrug resistance.26,



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

27

In addition, PEG coating (hydrosterate-PEG660) gives LNCs stealth properties, 28, 29,

30

allowing them to avoid capture by the reticuloendothelial system (RES) and remain

long enough in the blood circulation to passively accumulate in tumor tissues through the enhanced permeability and retention (EPR) effect. However, PEG coating can sterically hinder direct interaction between LNCs and cells.31, 32 Cell penetrating peptides (CPPs) have been intensively investigated by researchers involved in cancer therapy，and they have been successfully used to improve cellular uptake of a large variety of cargos.35 TAT peptide (TATp, YGRKKRRQRRR) is one kind of CPPs, containing two positively charged amino acids, arginine and lysine. TATp is reported to form hydrogen bonds between the guanidine head group of arginine and negative charged compounds (phosphates and sulfates) on the cell surface membrane.35 However, TATp-modified carriers also have limitations: (1) non-specificity in terms of cell diversity, which may result in toxic effects in normal tissues, and (2) being cleaved by the large number of proteolytic enzymes in the circulation.36, 37 Environmental-responsible nanocarrier delivery systems can prolong the circulation of TATp-modified carriers in blood and allow TATp to act at tumor sites. They protect TATp by stimulus-sensitive bonding with polymers during their delivery, and the bond is broken by using the characteristics of the tumor site (pH, temperature, enzymes) and the “hidden” TATp is exposed to increase the intake.32, 38-41

In this paper, we will report the design of DSF-loaded PEG sheddable lipid nanocapsules (DSF-S-LNCs) that respond to the extracellular low pH, exposing TAT peptide and conjugating with intracellular Cu abundant in tumors, resulting in increased tumor targetability, internalization and toxicity. The structure and treatment strategy of DSF-S-LNCs are illustrated in Scheme 1. The functions of DSF-S-LNCs include (1) passive tumor targeting by the EPR effect due to prolonged circulation by long PEG chains; (2) overcoming the nonselective intracellular uptake and instability on the way to tumor sites through shielding TATp by means of long PEG chains; (3)


Page 4 of 42

Page 5 of 42

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


exposure of TATp resulting from the shedding of the long PEG chains at tumor sites, due to pH sensitive poly glutamate acid (PGA); (4) increased cellular internalization by TATp-mediated endocytosis; (5) release of DSF and formation of Cu(DETC)2 in Cu-rich tumor cells to act on protease mediated apoptosis, resulting in increased tumor toxicity.

Scheme 1. Structure and treatment strategy of DSF-S-LNCs

2. EXPERIMENTAL SECTION 2.1 Materials 2.2 Synthesis of HS-PEG1k-TATpand PGA-g-PEG HS-PEG1k-TATp was synthesized according to the method reported in the literature



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 42

with some modifications.36, 41 Excess NHS-PEG1000-maleimide was initially linked to 12-hydroxystearic acid (HS) with the assistance of triethylamine (TEA) by stirring in anhydrous chloroform at room temperature for 2 h. Then, TATp was added to the mixture, followed by stirring overnight. Then the solvent was evaporated and the excess TATp and salts were removed by dialysis (MWCO 2000 Da). Finally, the product was obtained by freeze-drying. Poly (glutamic acid)-graft-methoxy poly (ethylene glycol) (PGA-g-PEG) was synthesized as described previously.42,

43, 44

Briefly, poly (L-glutamic acid) (PGA,

10000 Da), NHS and mPEG-NH2 (2000 Da) were dissolved in borate saline buffer (0.05 M, pH 8.5). Then, EDC was added to the solution and the mixture was stirred overnight at room temperature. Upon the completion of the reaction, the mixture was purified by dialysis (MWCO 14000 Da) against deionized water for 24 h. The final product was obtained by freeze-drying overnight and stored at -20 ◦C. In order to confirm the structure of the polymers, 1H NMR (400 MHz, Bruker AVANCE III HD) and MS analyses (Xevo TQ, Waters) were carried out. The grafting ratio of PGA-g-PEG was based on the proton resonance absorptions. 42

2.3 Preparation of DSF-LNCs, DSF-TATp-LNCs and DSF-S-LNCs DSF-LNCs were prepared by the phase-inversion method.45,

46

Briefly, a mixture

consisting of DSF, MCT, Kolliphor HS15, Lipoid S75, NaCl and water was heated to allow complete solubilization and then subjected to heating cycles between 60 and 85 ◦

C (i.e. 60 ◦C→85 ◦C→60 ◦C). The system was then kept at 78 ◦C and quenched by

adding deionized water (0 ◦C) and, finally transferred to an ice-water bath for another 5-minute agitation, leading to formation of stable LNCs. DSF-TATp-LNCs were prepared by modifying LNCs with post-insertion as described in previous reports.29,

47

To “add” functional TATp to the surface of DSF-LNCs,

DSF-LNCs and TATp were incubated in selected ratios at 60 ◦C. The mixture was vortex-mixed every 15 min and quenched in an ice bath for 1 min. Free HS-PEG1k-TATp was removed by Sphedex G50 column chromatography.


Page 7 of 42

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 DSF-TATp-LNCs were further combined with PGA-g-PEG to obtain DSF-S-LNCs by electrostatic absorption.48, 49 Aqueous solutions of PGA-g-PEG (pH 8.5) in a series of concentrations were prepared. DSF-S-LNCs were obtained by adding DSF-TATp-LNCs to the PGA-g-PEG solution drop by drop at predetermined volume ratios (2:1.5, v/v) followed by stirring for over 4 h. Free PGA-g-PEG was removed by dialysis. All LNCs were passed through a 0.22 µM filter membrane in order to sterilize and remove the large particles generated during the preparation processes.

2.4 Characterization

of

DSF-LNCs,

DSF-TATp-LNCs

and

DSF-S-LNCs 2.4.1 Measurements of particle size and zeta potential The particle size and zeta potential values were measured by dynamic light scattering (DLS) using a NicompTM 380 Zeta Potential/Particle Sizer (Nicomp Particle Sizing Systems, Santa Barbara, CA, USA). 2.4.2 Morphological observations Transmission electron microscopy (TEM) was used to observe the morphology of the preparations. Samples were deposited on a carbon film copper grid and negatively stained with sodium phosphotungstate solution (1%, w/w). The analyses were performed using a JEOLJEM-2000EX transmission electron microscope equipped with an Energy Dispersive Spectroscopy (EDS) Si (Li) detector. 2.4.3 Drug loading The drug loading (DL) of the DSF preparations was determined by a UV-VIS spectrometer at 254 nm. The DSF preparations were linear over the concentration range 1-20 µg/mL (y=0.0441x+0.0541, R=0.9992). The DSF preparations were



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

dissolved in isopropanol and diluted 200-fold in methanol. After filtration through a 0.22 µM filter membrane, the absorbance of each solution was measured. The external standard method was used to calculate the concentration of DSF. The DL (%) was calculated using the following formula.50

(%) =

× 100 +

Where WDSF was the amount of DSF measured in LNCs; Wexcipients was the amount of all excipients used in formulation. 2.4.4 Stability The stability of the structure of DSF-TATp-LNCs and DSF-S-LNCs in the circulation was examined by measuring the particle size. The DSF-TATp-LNCs and DSF-S-LNCs were put into 50% rat plasma at 37 ◦C and then the changes in particle sizes of different formulations were monitored at predetermined interval by DLS. 2.4.5 pH Sensitivity To test the shedding potential of the PEG shell of DSF-S-LNCs in response to pH, S-LNCs were incubated in PBS at pH 7.4 and pH 6.5 at 37 ◦C.51 The particle sizes and zeta potentials were measured by DLS. The morphology of the DSF-S-LNCs was examined visually by TEM.

2.5 In vitro release The release profiles of DSF from different formulations were studied using a dialysis method. Briefly, dialysis bags (MWCO 14000 Da) containing 500 µL DSF-loaded formulations and 500 µL dissolution medium were incubated in flasks containing 20 mL pH 7.4 and pH 6.5 PBS as well as 2% Tween 80 at 37 ◦C at a shaking rate of 100 rpm. At predetermined time intervals, the dissolution medium in each flask was withdrawn for testing and replaced with 20 mL fresh dissolution medium. The drug concentration was determined as described above.


Page 8 of 42

Page 9 of 42

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


2.6 Cell culture Hep G2 cells were grown in DMEM medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum.52 Cu-enriched Hep G2 cells were generated by continuously culturing the cells in 30 µM copper gluconate for a minimum period of 3 days. Cultures were maintained at 37 ◦C in a humidified 5% CO2 incubator. In all studies, the cells were subcultured every 2-3 days and used for experiments at passages 3-7.

2.7 Cytotoxicity assays The cytotoxicity of Cu, DSF and DSF-loaded preparations was assessed by MTT assay as described previously.21 The cytotoxicity of Cu and/or DSF was tested using Hep G2 cells cultured in Cu-free medium. Experiments involving cells cultured in Cu-free medium were carried out by culturing 5×103 cells/well in 96-well plates with Cu-free medium. On the following day, the cells were cultured in Cu-free medium containing different concentrations of DSF or copper gluconate for 48 h and 72 h, respectively. Experiments involving cells cultured in Cu-free medium that were treated with DSF for 24 h followed by copper gluconate treatment were carried out by culturing 5×103 cells/well in 96-well plates with Cu-free medium. On the following day, the cells were washed with pre-warmed PBS and cultured with different concentrations of copper gluconate ((DSF) Cu). Experiments involving cells cultured in Cu-containing medium were carried out by culturing 5×103 cells/well in 96-well plates with medium containing different concentrations of copper gluconate. On the following day, the cells were washed with pre-warmed PBS and cultured in Cu-free medium containing DSF ((Cu) DSF). After a 48 h or 72 h incubation of the cell cultures with appropriate test drugs, 10 µL MTT solution (5 mg/mL) was added to each well. Cells were incubated for another 4 h and then the medium was removed and the MTT-formazan generated by living cells was dissolved in 100 µL dimethyl sulfoxide (DMSO). The absorbance of each well at a wavelength of 570 nm was measured using a microplate reader. The relative cell



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

viability (%) was calculated using the following equation.

(%) =

!" × 100 #$#"

The cytotoxicity of DSF-loaded preparations was investigated using Cu-enriched Hep G2 cells. Cu-enriched Hep G2 cells were seeded at a density of 5×103 cells/well in 96-well plates for 24 h. On the following day, the cells were washed with pre-warmed PBS and exposed to Cu-free medium containing different concentrations of DSF, DSF-LNCs, DSF-TATp-LNCs and DSF-S-LNCs (pre-treated at pH 7.4 or 6.5 prior to their addition to cells) for 48 h. After that, the cell viability was measured by the MTT method. To determine the cytotoxicity of blank formulations, Cu-enriched Hep G2 cells seeded in 96-well plates were exposed to a series of concentrations of LNCs, TATp-LNCs and S-LNCs for 48 h. Then, the cell viability was again measured by the MTT method.

2.8 Apoptosis analysis Cu-enriched Hep G2 cells were seeded in 6-well plates (5×104 cells/well) and incubated for 24 h. Then, the cells were reacted with DSF-S-LNCs (DSF 3 µM) pre-treated at pH 6.5 or pH 7.4. After 24 h, the cells were harvested and washed twice with ice-cold PBS, stained with Annexin V-FITC (FITC=fluorescein isothiocyanate) and propidium iodide (PI) following the manufacturer’s instructions for the Annexin V-FITC/PI KIT. Finally, the samples were analyzed using a FACS Cabilibr flow cytometer within 1 h.

2.9 Intracellular uptake studies To investigate the cellular uptake of LNCs, Cy5.5, instead of DSF, was loaded into the LNCs. Different LNCs carried Cy5.5 at a concentration of 20 ng/mL. Adherent Cu-enriched Hep G2 cells were incubated on glass cover slips placed in six-well culture plates at a density of 5×104 cells/well for 24 h. The medium was then replaced by fresh medium containing function-modified Cy5.5-loaded LNCs for another 1 h or 4 h at 37 ◦C, 5% CO2. Then, the cells were washed with cold PBS (3 times, 5 min/wash) and fixed with 4% paraformaldehyde solution. The cell nuclei were stained


Page 10 of 42

Page 11 of 42

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 4’, 6’-diamidino-2-phenylindole (DAPI) for 20 min, followed by washing with PBS (3 times, 5 min/wash). Finally, the coverslips were placed on microscope slides and visualized by a confocal laser scanning microscopy (CLSM, LSM710, ZEISS, Germany).

2.10

Pharmacokinetic studies

Animal experiments were approved by the Ethics Committee of Shenyang Pharmaceutical University and carried out under the Guide for Care and Use of Laboratory Animals. Male Sprague-Dawley (SD) rats (n=4, body weight range 200-220 g) were used for the pharmacokinetic studies. Free DSF, DSF-LNCs and DSF-S-LNCs (20 mg/kg, respectively) were injected (1 mL/100 g) through the tail veins and blood samples (200 µL) were collected from the ophthalmic venous plexus at intervals (0.08, 0.17, 0.25, 0.33, 0.50, 0.67, 0.83, 1, 2, 4, 6 h). The blood samples were pre-stabilized with stabilizing agent and subjected to solid phase extraction, then analyzed by the UPLC-ESI-MS/MS method described earlier.24 Pharmacokinetic parameters were calculated using DAS 2.0 software.

2.11

In vivo biodistribution in tumor-bearing mice

The in vivo biodistribution of different LNCs were assessed in tumor-bearing BALB/cA nude mice using the FX Pro in vivo imaging system (Carestream Health). Cy5.5 was used to substitute DSF for near-infrared (NIR) fluorescence imaging. About 5×106 tumor cells were subcutaneously injected into the right flank of the 7- to 8-week–old male BALB/cA nude mice (Beijing Huafukang Biology Inc) and tumors were allowed to grow to an average size of 100-200 mm3 before the experiment. Free Cy5.5, Cy5.5-LNCs and Cy5.5-S-LNCs (0.1 mg/mL, respectively) were injected (100 µL/10 g) via the tail vein. Whole body optical imaging was performed at 6, 12 and 24 h post-injection. Then, the mice were sacrificed, and the imaging signal of Cy5.5 in the main organs and tumors was monitored after 24 h.

3. RESULTS AND DISCUSSION 3.1 Synthesis

and

characterization

of

HS-PEG1k-TATp


and


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

PGA-g-PEG The chemical structures of HS-PEG1k-TATp and PGA-g-PEG were confirmed by 1

H-NMR (solvent: D2O)(Figure 1). In Figure 1A, the symmetric double peaks at 7.18

- 6.63 ppm belonged to the aromatic protons of tyrosine in TATp. The multiple peaks at 3.59 were due to the -CH2-CH2-O- in PEG. The peaks at 2.47-3.22 and multiple peaks at 1.25-1.79 belonged to protons of –CH2-NH-NH-NH2 in arginine and protons of -CH2-CH2- in 12-hydroxystearic acid. As shown in Figure 1B, the intense peak at 3.59 ppm and the proton peak at 3.27 were assigned to the PEG chain (-CH2-CH2-O-) and the methyl protons (-CH3), respectively. The peaks at 2.18 and 4.22 were ascribed to the methylene protons (-CH-CH2-CO-) and methylidyne proton (-NH-CH-CO-) in glutamic acid. The grafting percentage (X) was calculated as shown below. After calculation, the grafting percentage of PGA-g-PEG was 8.3%.

%.'( 182 × . = ).*+ 136 Where A3.59 is the characteristic proton peak area of PEG at δ=3.59 and A2.18 is the characteristic proton peak area of PGA at δ=2.18. In addition, the molecular weight of HS-PEG1k-TATp was confirmed by MS analysis. As shown in Figure 2, the results showed multiple hydrogenation peaks for TATp carrying more charges. Calculation showed that the molecular weight of HS-PEG1k-TATp was 2901.8 in agreement with theory (2902), suggesting the successful synthesis of HS-PEG1k-TATp.


Page 12 of 42

Page 13 of 42

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. 1H-NMR of HS-PEG1k-TATp (A) and PGA-g-PEG (B) conjugates



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 2. The mass spectrum of HS-PEG1k-TATp

3.2 Assessment of DSF-LNCs, DSF-TATp-LNCs and DSF-S-LNCs As a previous report, LNCs with a 49.7 nm particle size and a narrow size distribution (PI=0.059) were obtained.26 LNCs are composed of lipid cores and mixed monolayers containing Kolliphor HS-15 and lipoid. Hence, LNCs showed a zeta potential about -15.46 mV, identical to previous data and close to those of other PEG covered particles such as PEGylated liposomes.26, 29, 36 PGA-g-PEG aqueous solution with different concentrations at pH 8.5 was mixed with the TATp-LNCs. The amount of PGA-g-PEG was chosen to be between 2% and 15% (PGA-g-PEG% in Mol of lipid surfactant) in order to investigate the effect on particle size and zeta potential. After LNCs had been added to HS-PEG1k-TATp, the zeta potential of TATp-LNCs increased sharply from ~-15.46 mV (LNCs) to ~5 mV (Table 1). This result can be attributed to the positively charged arginine and lysine of TATp after insertion of HS-PEG1k-TATp on the surface of LNCs. As shown in Table 1, the particle size and zeta potential of DSF-S-LNCs both changed after adding PGA-g-PEG compared with LNCs and TATp-LNCs. The particle sizes remained almost unchanged when the PGA-g-PEG percentage was no more than 5%, but the zeta potential became negative, and it failed to reach that of


Page 14 of 42

Page 15 of 42

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


LNCs. However, after the PGA-g-PEG percentage was 10% or higher, the particle size of DSF-S-LNCs increased significantly and the zeta potential decreased to values lower than LNCs. This phenomenon could be explained as shown in Figure 3. At the beginning of the addition of PGA-g-PEG, the negative charges on which were far less than the positive charges (NnegativeNpositive)(Figure 3C). If this continued, less glutamate units of each molecule of PGA-g-PEG would be attached to the particles but more stretch into in the water, which would result in both an increase in particle size and polydispersity, and a decrease in the zeta potential of DSF-S-LNCs. Stefanick et al found that long PEG molecules folded into globular mushroom-like structures rather than a linear conformation, burying the conjugated peptide into the PEG coating and sterically hindering their association with the cell surface, while shorter PEG molecules acting as a peptide linker produced increased cellular uptake.34 Thus, nanocarriers consisting of a short PEG (PEG1000) linker and a coating of long PEG (PEG2000) are necessary. The PEG1000 used as linkers could adopt a linear conformation, extend peptides beyond the PEG660 coating of LNCs and facilitate the interaction with the cell surface. In contrast, PEG2000 could play a role in shielding the peptides to reduce opsonization by blood proteins, then being abandoned after the nanocarriers arrived at the target location to expose the peptides.



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 42

Figure 3. Structure of S-LNCs with different amounts of PGA-g-PEG.

(A) NnegativeNpositive. Table1. Average particle size, zeta potential and DL of DSF preparations.

DSF-LNCs DSF-TATp-LNCs 2% PGA-g-PEG 5% PGA-g-PEG DSF-S-LNCs 10% PGA-g-PEG 15% PGA-g-PEG

Particle size (nm)

PI

Zeta potential (mV)

DL (%)

49.7

0.059

-15.46 ± 0.75

3.72 ± 0.17

51.2

0.045

5.05 ± 1.69

3.45 ± 0.52

48.2

0.057

-2.17 ± 2.92

3.62 ± 0.25

50.5

0.086

-9.63 ± 1.90

3.76 ±0.83

70.0

0.179

-35.56 ± 1.84

3.71 ± 0.14

93.7

0.312

-40.13 ± 2.83

3.59 ± 0.36

3.3 Stability Although TAT peptide on the surface of LNCs promotes cellular uptake, the numerous surface cations have an adverse effect on the circulating time of formulations in blood.37 In order to prove that shielding PEG can reduce the interaction between TATp and plasma components, we incubated DSF-LNCs, DSF-TATp-LNCs and DSF-S-LNCs with 50% rat plasma at 37 ◦C and monitored the particle sizes of the formulations at various incubation times. The particle size of DSF-LNCs and DSF-S-LNCs remained almost unchanged over a


Page 17 of 42

4 h incubation, which indicated that DSF-S-LNCs were as stable as DSF-LNCs when in contact with plasma. However, DSF-TATp-LNCs exhibited a significant increase in particle size from about 50 nm to over 1000 nm in 60 min and finally became precipitated (As shown in Figure 4). This observation showed the stability of DSF-S-LNCs in the presence of PEG protection, which plays a role in minimizing the interaction with components in plasma. Thus, shielding TATp is essential to increase the stability of lipid nanocapsules. 1500 Diameter of Particles (nm)

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


1000

DSF-LNCs DSF-TATp-LNCs DSF-S-LNCs

500

0 0

50

100 150 Time (min)

200

250

Figure 4. Change in particle sizes of DSF-LNCs, DSF-TATp-LNCs and DSF-S-LNCs in 50% rat plasma.

3.4 pH Sensitivity DSF-S-LNCs were constructed by shielding DSF-TATp-LNCs with a pH-sensitive anionic block copolymer consisting of PGA and PEG. In an alkaline to near neutral environment, the side carboxyl groups of the PGA block are deprotonated (Figure 5A) and PGA-g-PEG adopts a random coil conformation. PGA-g-PEG was absorbed to TATp-LNCs through the electrostatic interaction with protonated segments in TATp. As a result, sheddable LNCs were formed. Once these sheddable LNCs were exposed to an acidic environment, the binding force between polymers and TATp-LNCs would be weaken due to the reduction in the electrostatic attraction. PGA-g-PEG lost its charges and dissociated from the surface of the LNCs leading to exposure of the positively charged segments. When PGA-g-PEG was in the uncharged state in acidic



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

condition it adopted an α-helix conformation which resulted in a reduction in solubility and formation of flocculate copolymer.51 The effect of pH on DSF-S-LNCs with regard to the incubation time was first studied using DLS. As shown in Figure 5B, the particle size of DSF-S-LNCs did not change markedly over 4 h at pH 7.4 and remained around 70 nm. By contrast, the particles of DSF-S-LNCs at pH 6.5 separated into two clusters: one was about 50 nm, approximately the same size as DSF-TATp-LNCs, while the other one was over 200 nm, which suggested the aggregates of shedding polymers. After filtration to remove the additional aggregates, the surface charge of DSF-S-LNCs at pH6.5 changed from a negative value to a positive one, approaching that of DSF-TATp-LNCs (Figure 5C). This result suggested almost complete removal of the shielding PEG shell and the exposure of DSF-TATp-LNCs. TEM micrographs of DSF-S-LNCs under different conditions at 4 h were examined and the results are displayed in Figure 5D and 5E. At pH 7.4, spherical particles coated with hydration film were observed in Figure 5D, the particle size remained uniform and no large aggregates were formed, which indicated that the morphological structure of DSF-S-LNCs was stable at pH 7.4. However, at pH 6.5, after filtration and removal of the aggregates, the outer hydration films disappeared while the lipid nanocapsules retained a spherical structure, which indicated that the morphology remained unaffected after polymer shedding and DSF-TATp-LNCs were stable under low pH conditions (Figure 5E).


Page 18 of 42

Page 19 of 42

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. (A) The general structure and change in properties with respect to the pH of PGAs. (B and C) Changes in particle size (B) and zeta potential (C) of DSF-S-LNCs at 4 h. (D and E) TEM images of DSF-S-LNCs at pH 7.4 (D) and pH 6.5 (E) at 4 h.

3.5 In vitro release 3.5.1 The effect of Tween 80 Since DSF is insoluble in water, Tween 80 was used as a solubilizer to obtain sink conditions. It has been postulated that micelles formed by Tween 80 may act as a reservoir and continuously capture DSF released from LNCs due to the high affinity between DSF and Tween 80. In order to investigate the stability of DSF-S-LNCs in Tween 80, the particle size changes in different concentrations of Tween 80 were monitored during the in vitro release study and TEM micrographs of DSF-S-LNCs in 2% Tween 80 solution were obtained. In both 2% and 10% Tween 80 solution, the particle size of DSF-S-LNCs remained unchanged over 168 h, and only PI values became higher when in 10% Tween 80 (Figure 6A). Then, the TEM of DSF-S-LNCs in 2% Tween 80 solution at 4 h showed that hydration-film-coated spherical particles were surrounded by a number of micelles (Figure 6C). Spherical DSF-S-LNCs were



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

still observed at 72 h and the particle size remained unchanged and homogeneous (Figure 6D), which indicated that the morphology of DSF-S-LNCs was stable and not affected by 2% Tween 80. As a result, 2% Tween 80 aqueous solution was selected as the dissolution medium. In addition, it was inferred that the diffusion of DSF from the surface and inner oily core rather than degradation of lipid nanocapsules was the main release mechanism. 3.5.2 In vitro release in Tween 80 The release of DSF from LNCs and S-LNCs was investigated at 37 ◦C at different pH values in 2% Tween 80. The profiles of the in vitro cumulative release of DSF from DSF-LNCs and DSF-S-LNCs compared with free DSF solution are shown in Figure 6B. DSF solution was prepared by dissolving DSF in 2% SDS solution as control. In the control, DSF solution released more than 70% DSF during 12 h in PBS (0.05 M, pH 7.4). However, the percentage of DSF released from DSF-LNCs and DSF-S-LNCs at 72 h was less than 50%. So, LNCs exhibited sustained-release of DSF in this in vitro environment. This release characteristic of DSF obtained in our study essentially agreed with the results of earlier studies.26,

53

This could be attributed to the

distribution of DSF being mainly in the internal oily core and only partly on the surfactant surface. Under the same pH conditions, DSF-LNCs and DSF-S-LNCs exhibited similar cumulative DSF release curves. This implied that the modification of DSF-LNCs had little effect on the release of DSF from DSF-S-LNCs. However, the release of DSF from DSF-LNCs and DSF-S-LNCs was only slightly affected by pH. The mean 72-hour cumulative release of DSF from DSF-LNCs and DSF-S-LNCs at pH 7.4 was 33.94% and 36.20%, while it was 53.02% and 50.48% at pH 6.5. The cumulative release of DSF at pH 6.5 was higher than that at pH 7.4. In our previous study, DSF showed a higher solubility at pH 6.5 than pH 7.4 in Tween 80 solution. This difference in release may be due to the faster dissolution of DSF on the surface at pH 6.5. The subsequent dissolution mainly depended on the diffusion of DSF from the


Page 20 of 42

Page 21 of 42

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


inner oily core to the surface, which was hardly affected by pH. These results implied that the release of DSF-S-LNCs was not significantly different from that of DSF-LNCs, which means that the shedding of PEG would not lead to the leakage of DSF. DSF-S-LNCs were stable under normal physiological conditions and exhibited less release under extracellular conditions. In other words, most of the DSF remained encapsulated in lipid nanocapsules before entering tumor cells.

Figure 6. (A) Change in particle size of DSF-S-LNCs in 2% and 10% Tween 80 solutions. (B) In vitro release profiles of DSF from SDS solution (Free DSF), DSF-LNCs and DSF-S-LNCs in PBS (0.05M, pH 7.4 and pH 6.5 in 2% Tween solution). Data represent mean ± standard deviation (n=3). (C and D) TEM images of DSF-S-LNCs at 2% Tween 80 solution at 4 h (C) and 72 h (D).

3.6 Sensitivity of copper Tumor cells and tissues have the tendency to take up more Cu than normal ones.12 This feature can be used as a targeting method for anti-cancer therapy.9 Initially, to investigate whether Cu is a requirement for DSF cytotoxicity, we tested the effect of DSF alone on Hep G2 cells in the absence of Cu. Hep G2 cells were cultured in medium containing different concentrations of DSF without copper gluconate. As



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

shown in Figure 7C, DSF alone without Cu was not very toxic to cells at DSF concentrations up to 10 µM. In a follow-up experiment, Hep G2 cells were divided into 3 groups: (1) those cultured in different concentrations of copper gluconate (Cu alone), (2) those pre-cultured in medium containing DSF for 24 h and then cultured in different concentrations of copper gluconate ((DSF) Cu), and (3) those pre-cultured in medium containing different concentrations of copper gluconate for 24 h, then cultured in medium containing DSF ((Cu) DSF). Cu is an essential element for humans. As Figure 7A shows, copper gluconate concentrations up to 100 µM were not cytotoxic to Hep G2 cells. However, Cu caused the death of 80% of cells when a concentration of 300 µM reached. As shown in Figure 7A and 7B, the combination of DSF and Cu ((DSF) Cu and (Cu) DSF) was more toxic than Cu or DSF alone. Our study confirmed that Cu is essential for DSF cytotoxicity as suggested by previous results.13 Interestingly, when comparing cells pre-cultured with DSF or Cu first, we found that pre-culturing Cu with cells first ((Cu) DSF) was always more toxic. This reason for this may be that the key to the effect of DSF lies in Cu(DETC)2, an intracellular Cu-chelate of DSF; while pre-uptake of DSF could result in formation of chelates with proteins containing thiols, resulting in a reduction in the amount of DSF available to form Cu(DETC)2. In order to measure the Cu content in cells, 1×107 cells were collected with/without Cu enrichment culturing. Cells were washed with PBS three times to remove any Cu in the medium. The total Cu in each sample was determined by atomic absorption chromatography (nova AA® 400, Analytik Jena AG, Germany). The transport of Cu into cells takes place in a time-dependent and saturable manner.12 To simulate the in vivo Cu status of cancer cells, Hep G2 cells were cultured in medium containing 30 µM Cu.52 Subsequently, the Cu-enriched cells were washed with PBS three times to remove any residual Cu in the medium and then used for cell viability and apoptosis assays. Blank cells (-Cu) were washed with 30 µM Cu once and then three times with PBS, as a control. As shown in Figure 7D, the content of copper in cells cultured in


Page 22 of 42

Page 23 of 42

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


Cu medium was significantly higher than in regular media. The results showed that intracellular Cu accumulation was achieved by pre-cultured Cu, and cells cultured in DMEM containing Cu were used for subsequent experiments.

Figure 7. (A and B) Viability of Hep G2 cells after treatment with Cu alone (blue), DSF (3 µM) 24 h incubation followed by Cu ((DSF) Cu) (red) and Cu 24 h incubation followed by DSF (3µM) ((Cu) DSF)(green) for 48 h (A) and 72 h (B). (C) Viability of Hep G2 cells treated with DSF alone for 48 h (blue) and 72 h (red). (D) The content of copper in cells after cell culture without Cu (-Cu) and with Cu (+Cu) at a concentration of 30 µM for 3 days.

3.7 Cell viability The in vitro cytotoxicity of lipid nanocapsules was evaluated by MTT assay. LNCs are a biodegradable drug delivery system composed of natural compounds, except HS-PEG. Thus, LNCs and its “upgrades” are expected to have a low toxicity.25 As shown in Figure 8A, the cell viability of Cu-enriched Hep G2 cells treated with blank LNCs, TATp-LNCs and S-LNCs was almost more than 90% at concentrations ranging from 1 to 1000 µM, confirming non-toxicity and excellent biocompatibility. The in vitro antitumor activity of DSF- LNCs, DSF-TATp-LNCs and DSF-S-LNCs (pre-incubated at pH7.4 and pH 6.5, respectively) was studied in Cu-enriched Hep G2



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

cells and compared with free DSF (Figure 8B). The IC50 values (the concentration inhibiting cell growth by 50%) of each preparation are shown in Table 2 (calculated using Graphpad Prism software). The results showed that the cytotoxicity of DSF formulations was dose-dependent. There were significant differences (p

A Copper-Mediated Disulfiram-Loaded pH-Triggered PEG-Shedding

Recommend Documents