Synthesis and Characterization of Nitric Oxide-Releasing Platinum(IV

Subscriber access provided by UNIV OF TASMANIA

Article

Synthesis and Characterization of Nitric Oxide-Releasing Platinum (IV) Prodrug and Polymeric Micelle Triggered by Light Swapan Pramanick, Jihoon Kim, Jinhwan Kim, Gurusamy Saravanakumar, Dongsik Park, and Won Jong Kim Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00749 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on January 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a 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.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Synthesis and Characterization of Nitric Oxide-Releasing Platinum (IV) Prodrug and Polymeric Micelle Triggered by Light Swapan Pramanick, †# Jihoon Kim, †# Jinhwan Kim, †‡ Gurusamy Saravanakumar,† Dongsik Park‡ and Won Jong Kim *†‡

†Center for Self-assembly and Complexity, Institute for Basic Science, 77 Cheongam-ro, Nam-gu, Pohang 37673, Republic of Korea. ‡ Department of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 37673, Republic of Korea. # These authors contributed equally to this work. *

Corresponding author. Tel.: +82-54-279-2104; fax: +82-54-279-3399

E-mail address: [email protected]

ACS Paragon Plus Environment

Bioconjugate Chemistry 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

Abstract Herein, we report the proof of concept of photo-responsive chemotherapeutics comprising nitric oxidereleasing platinum prodrugs and polymeric micelles. Photo-activatable nitric oxide–releasing donors were integrated into the axial positions of a platinum (IV) prodrug, and the photo-labile hydrophobic groups were grafted in the block copolymers. The hydrophobic interaction between nitric oxide donors and the photo-labile groups allowed for the loading of platinum drugs and nitric oxide–releasing donors in the photo-labile polymeric micelles. After cellular uptake of micelles, light irradiation induced the release of nitric oxide, which sensitized the cancer cells. Simultaneously, photo-labile hydrophobic groups were cleaved from micelles, and the nitric oxide–releasing donor was altered to be more hydrophilic, resulting in the rapid release of platinum (IV) prodrugs. The strategy of using platinum (IV) prodrugs and nitric oxide led to enhanced anticancer effects.


Page 2 of 35

Page 3 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Platinum (Pt)-based drugs represented by cisplatin have been widely used as chemotherapeutics for various cancers over the last three decades. However, their use is limited by several major problems, including drug resistance, biomolecule-associated deactivation, and side effects due to non-specific delivery.1-3 As Pt-based drugs are exploited for the treatment of more than 50% of all cancers,4 research aimed at addressing these issues is urgently required. Compared to active Pt(II) drugs, Pt(IV) prodrugs have two additional coordination sites in their axial positions, allowing for additional functionalities and enhanced stability against nucleophiles.1-9 Pt(IV) prodrugs undergo intracellular bioreduction and afford Pt(II) drugs the opportunity to exert their anticancer effects. Therefore, the use of Pt(IV) prodrugs have been suggested as a practical way to overcome biomolecules-associated deactivation of platinum-based drugs.5-9 One of the most critical impediments for the application of Pt-based drugs is ineffectiveness in drugresistant cells. In an effort to surmount this problem, several molecules, enzymes and nucleic acids have been simultaneously utilized with Pt-based drugs, which can interfere with DNA-repair systems, prevent the approach of glutathione, or down-regulate the protein expression associated with drug resistance.3,10-12 However, these strategies have limitations, such as low efficiency and high cost. Accordingly, in this study, we have focused on a physiological sensitizer, nitric oxide (NO), to enhance the Pt-based drug effect. NO is a gaseous mediator in vivo that participates in the progression or treatment of various diseases, including autoimmune diseases, bacterial infections, cardiovascular diseases, cancer, and wound healing.13-18 Focused on anticancer chemotherapy, NO enhances the efficiency of chemotherapy by reducing the number of active P-glycoproteins and MDR-associated proteins (MRPs),19,20 depleting glutathione that inactivates the anticancer drugs,19,21 inactivating hypoxia-induced factors (HIF) that induces multidrug resistance (MDR) under anaerobic conditions,19,22 and inhibiting nuclear factor kappa B (NF-κB) that regulates the drug resistance.19,23 These outstanding characteristics have recently attracted the attention of researchers to exploit it as a sensitizer for drug-resistant tumors.19,24-28 There are several points to consider seriously when selecting NO-donors for the design of Pt(IV) prodrugs facilitating NO release. First, NO donors should be stable during the loading and cellular uptake



process because unstable NO-donors have limited potential in the current combined drug and NO delivery system in anticancer therapy.25-28 Second, stimuli-responsive NO-releasing donors are strongly preferred, as the physiological functions of NO are highly dependent on its reacting site and concentration.13-18 Third, a NO-donor has to improve the loading efficiency of Pt-based drugs. Among a variety of NO-donors with distinguished characteristics based on stability, stimuli, and water dispersibility,29-32 the nitrobenzene was selected because it exhibits high stability in physiological conditions and rapid NO release under light irradiation.31-35 In addition, the lipophilic nitrobenzene was believed to increase the hydrophobicity of the Pt(IV) prodrug, which could contribute to prodrug loading into the various nanoparticles, including polymeric micelles and liposomes.3-9 To deliver the NO-releasing Pt(IV) prodrugs with minimum side effects and high delivery efficiency, we exploited photo-labile polymeric micelles since simple light irradiation facilitates the spatio-temporal trigger of drug release and subsequent targeted therapy.36-40 In addition, we expected that photo-initiated micellar degradation with the same light source would be beneficial for one-pot drug and NO release. Accordingly, 4,5-dimethoxy-2-nitrobenzyl (DMNB) was chosen as a functional moiety for photo-labile micelles because its hydrophobic group could be cleaved from the polymeric backbone upon exposure of light with the same wavelength as nitrobenzene,41-45 causing the disassembly of micelles. Furthermore, because 2-(trifluoro)phenol, the byproduct of light-exposed nitrobenzene, is more water soluble than untreated nitrobenzene due to the hydrogen bonds with water molecules, this photo-responsive phase transition of nitrobenzene was also expected to contribute to the enhanced photo-responsive drug release. To summarize, we report a novel strategy for the synthesis of photo-triggered NO-releasing Pt(IV) prodrugs with photo-responsive nitrobenzene and polymeric micelles with photo-labile DMNB (Figure 1A). After intracellular uptake of the micelles, a light irradiation facilitates the NO release from nitrobenzene, which alleviates drug-resistance of the cancer cells. Simultaneously, the DMNB moiety is released from block copolymers, and hydrophobic nitrobenzene is altered to be more hydrophilic. These processes accelerate the disassembly of micelles and reduce the hydrophobic interactions between of Pt(IV) prodrugs and micelles, resulting in the rapid release of Pt(IV) prodrugs (Figure 1B).


Page 4 of 35

Page 5 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic illustration of (A) the reaction mechanism of the photo-activable NO-releasing Pt (IV) prodrug and the photo-labile polymeric micelle and (B) its therapeutic mechanism inside cell.

Results and discussion



Synthesis of NO-releasing Pt (IV) prodrug with nitrobenzene and photo-labile block copolymers For the synthesis of photo-triggered NO-releasing Pt(IV) prodrug, amine functionalized nitrobenzene was prepared prior to the conjugation to Pt(IV) prodrugs. The long alkyl chain was introduced between the primary amine and nitrobenzene for the efficient reaction with diamminebis(3-carboxypropanoato)dichloroplatinum(IV) (Pt(IV)-diCOOH) and the enhanced hydrophobicity of the Pt(IV) prodrug. The presence of the long alkyl chain induced the red-shift in the absorbance spectrum of nitrobenzene, in accordance with previous reports (Figure S1).33,34 The Pt(IV) prodrug with nitrobenzene (Pt(IV)-NO, compound 8) was synthesized by conjugating carboxylic groups of Pt(IV)-diCOOH (Figure 2A, compound 3) to amine functionalized nitrobenzene via EDC/HOBt chemistry (Figure 2B). Successful synthesis of Pt(IV)-NO was corroborated by 1H NMR , 13C NMR and electrospray ionization mass (ESIMS). In 1H NMR using DMSO-d6, the disappearance of two D2O-exchangable primary amine protons (NH2) of nitrobenzene at δ 3.39 ppm and the appearance of a D2O-exchangable proton triplet at δ 7.79 ppm demonstrated the formation of a new amide bond (-CO-NH-) between nitrobenzene and the carboxylic groups of Pt(IV) prodrugs. ESI-MS results were in accordance with the expected structure of Pt(IV)-NO ([M + H]+ = 1109 m/z and [M + Na]+ = 1131 m/z), and the absorption spectrum of Pt(IV)-NO exhibited similar spectra with long alkyl chain-bearing nitrobenzene. These results demonstrated the successful synthesis of Pt(IV)-NO. The Pt(IV) prodrug with benzene groups (Pt(IV)-C, compound 13) was prepared as a control drug incapable of releasing NO by conjugating carboxylic groups of Pt(IV)-diCOOH to amine functionalized benzene prepared by Gabriel type synthesis (Figure 2C).


Page 6 of 35

Page 7 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Synthetic scheme and chemical structures of (A) Pt(IV)-diCOOH (compound 3), (B) NOreleasing prodrug (Pt(IV)-NO, compound 8), (C) control prodrug (Pt(IV)-C, compound 13), and (D) photolabile polymer (E-polymer, compound 19) and control polymer (C-polymer, compound 16).

The block copolymer with photo-labile DMNB (E-polymer) was synthesized via three steps. First, poly(ethylene oxide)-b-poly(benzyl-L-glutamate) (PEO114-b-PBLG20) was prepared by using macroinitiator PEO-NH2 and monomer γ-Benzyl L-glutamate N-carboxyanhydride (γ-BLG NCA) since the ring-opening polymerization (ROP) of N-carboxyanhydride (NCA) has been widely utilized for the synthesis of biocompatible and biodegradable polypeptides.46,47 The successful polymerization was confirmed with 1H NMR by observing the disappearance of a proton of NCA at δ 4.37 ppm and all peaks corresponding to PEO and PBLG. The composition of PEO and PBLG was estimated by comparing the integral ratios of the



Page 8 of 35

peaks at δ 3.64 ppm from ethylene protons (-CH2CH2O-) of PEO and the peaks at δ 7.26 ppm from aromatic protons of PBLG. Gel permeable chromatography (GPC) analysis confirmed a single peak (Mw/Mn = 1.14) which was shifted toward short elution time, indicating the increase in molecular weight compared to the PEO and successful polymerization (Table 1 and Figure S2). Due to its structural similarities with E-polymer, PEO114-b-PBLG20 was chosen as a control polymer (C-polymer) without photo-responsiveness (Figure 2D, compound 16). Second, the benzyl groups were removed from PEO114b-PBLG20 via palladium catalyzed hydrogenation to afford the poly(ethylene oxide)-b-poly(L-glutamic acid) (PEO114-b-PLGA20) with free carboxylic groups. The disappearance of aromatic protons peaks at δ 7.26 ppm and methyl protons adjacent to aromatic rings (Ar-CH2) at δ 5.10 ppm confirmed the complete debenzylation of PEO114-b-PBLG20. Third, poly(ethylene oxide)-b-poly(4,5-dimethoxy-2-nitrobenzyl Lglutamate) (PEO114-b-PDMNBLG20) (E-polymer) was synthesized as a photo-responsive polymer (Figure 2D, compound 19) by conjugating DMNB to the carboxylic groups of PEO114-b-PLGA20 via Steglich esterification. The chemical structure of the block copolymer was confirmed by 1H NMR.

Table 1. Characterization of E-polymer and C-polymer Sample

[M]0/[I]0a

Mnb (NMR)

Mnc (GPC)

Mw/Mnc

PEG-NH2

-

5000

6134

1.05

E-polymer

20

10505

10844

1.34

C-polymer

25

9380

8373

1.14

a

molar ratio of monomer over initiator in the polymerization

b

determined by 1H-NMR using CDCl3 as solvent.

c

determined by GPC using polystyrene as standard and THF as eluent at a flow rate of 1 mg/mL.

Formation and photo-degradability of polymeric micelles


Page 9 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To investigate the photo-degradability of E-polymer, the structural change with light irradiation was observed by 1H NMR. The benzylic methylene protons of DMNB group of PEO114-b-PLGA20 have been disappeared with light irradiation (Figure S3). These results indicate that the DMNB can be released from E-polymer by light irradiation, converting the block of the polymer from hydrophobic to hydrophilic. The amphiphilicity of E-polymer and C-polymer was expected to form self-assembled micelles under aqueous conditions. The critical micellar concentration (CMC) of E-polymer and C-polymer was determined by the pyrene fluorescence method, which demonstrated their ability to load hydrophobic drug and possible form of self-assembled structures in aqueous conditions (Figure S4). Accordingly, Pt(IV)NO was loaded into the E-polymer via the dialysis method to afford the Pt(IV)-NO loaded photo-labile micelles (Pt(IV)-NO/E-micelles). Transmission electron microscopy (TEM) depicted spherical morphologies of Pt(IV)-NO/E-micelles 25 nm in diameter before ultraviolet (UV) irradiation (365nm, 300 µW/cm2) (Figure 3A). Pt(IV)-NO loaded control micelles (Pt(IV)-NO/C-micelles), control drug-loaded photo-labile micelles (Pt(IV)-C/E-micelles) and control drug-loaded control micelles (Pt(IV)-C/C-micelles) were prepared as control groups (Table 2). The drug loading contents were determined by inductively coupled plasma-mass spectrometry (ICP-MS) and the results are summarized in Table 2. Drug loading content of Pt(IV)-NO/E-micelles was higher (27.6%) than those of other formulations, which might be due to the strong hydrophobic interactions between nitrobenzene and DMNB.

Table 2. Physicochemical characterization of micelles Drug loading content

Pt Loading content

Sizea (nm)

Sizea (nm)

(wt.%)

(wt. %)

(dark)

(light)

27.51±0.128

4.86±0.023

75.5±3.1

36.7±6.0

18.18±0.098

3.22±0.017

23.4±1.5

24.6±0.8

20.08±0.102

3.52±0.018

63.1±0.9

30.9±5.7

Sample

Pt(IV)-NO/Emicelle Pt(IV)-NO/Cmicelle Pt(IV)-C/Emicelle



Pt(IV)-C/C-

14.44±0.125

2.53±0.022

Page 10 of 35

24.4±2.1

23.2±2.5

micelle a

determined by particle size analyzer.

The photo-responsive disassembly of micelles was examined by TEM and dynamic light scattering (DLS) (Figure 3). There was no change in the morphology or size of light-exposed C-micelles (Figure 3D-F). Although E-micelles are stable without light irradiation (Figure S5), a majority of E-micelles decreased in size or was disrupted after light irradiation for 20 min (Figure 3A-C).

Figure 3. Physicochemical characterization of photo-responsive Pt(IV)-NO/E-micelles and Pt(IV)-NO/Cmicelles with or without light irradiation. Representative TEM image of Pt(IV)-NO/E-micelles (A) before and (B) after light irradiation. (C) Change of hydrodynamic volume of Pt(IV)-NO/E-micelles before (black) and after (red) light irradiation. TEM image of Pt(IV)-NO/C-micelles (D) before and (E) after light irradiation. (F) Hydrodynamic volume of Pt(IV)-NO/C-micelles before (black) and after (red) light


Page 11 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


irradiation. Scale bar is 50 nm.

NO-releasing profile from prodrug-loaded micelles It is impossible to measure experimentally the NO release from free Pt(IV)-NO in physiological conditions due to its water-insoluble characteristics. However, the encapsulation of Pt(IV)-NO with the micelle was expected to increase the solubility of Pt(IV)-NO, allowing for the measurement of NO release. Accordingly, the photo-responsive NO releasing behaviour of Pt(IV)-NO/E-micelles was evaluated by using a Sievers NOA chemiluminescence analyzer which detects chemiluminescence generated by the quantitative reaction between NO and ozone in real time. The ESI/MS and 1H NMR demonstrated that axial ligands are not cleaved after light irradiation and the released NO does not form any adducts with the Pt(IV) prodrugs (data not shown). In particular, X-ray photoelectron spectroscopy (Figure S6) clearly showed that the binding energy Pt4f of Pt(IV)-NO was retained before (75.9 and 79.1 eV of 4f(7/2) and 4f(5/2) levels) and after light irradiation (75.3 and 78.2 eV of 4f(7/2) and 4f(5/2) levels) (Figure S6B,C). These are similar to those of K2PtCl6 (75.7 and 79.0 eV of the 4f(7/2) and 4f(5/2) levels)48, but different with those of cisplatin (73.2 and 76.6 eV of the 4f(7/2) and 4f(5/2) levels) (Figure S6A). These results demonstrate that the species of prodrug is retained after light irradiation, implying the photo-stability of the prodrugs. As shown in Figure 4A, the pulsatile NO release profile was observed depending on the repeated exposure of light and dark, clearly demonstrating the light-dependent NO release behaviour of Pt(IV)-NO. These results indicate that the hydrophobic interactions between block copolymer and nitrobenzene stabilize the nitrobenzene to release NO efficiently under light-exposed aqueous conditions. We presumed that the photo-responsive disassembly of E-micelles would show a more rapid release of Pt(IV) prodrugs than C-micelles. In addition, the accelerated drug release was expected during NO release owing to the photo-induced transition from nitrobenzene to the relatively more water soluble 2(trifluoromethyl)phenol. To ascertain our hypothesis, the photo-responsive release of Pt(IV) prodrugs was monitored (Figure 4B). The release of drug from Pt(IV)-C/C-micelles was independent of light irradiation. However, as postulated, Pt(IV)-C/E-micelles and Pt(IV)-NO/C-micelles showed higher drug release in the



light condition than in the dark condition due to the disassembly of micelles by cleavage of hydrophobic DMNB or transition of nitrobenzene into hydrophilic 2-(trifluoromethyl)phenol. Accordingly, Pt(IV)NO/E-micelles exhibited the most photo-responsive behaviors in drug release. These results suggest that the utilization of nitrobenzene not only facilitates the loading of Pt(IV) prodrugs in the micelles, but also improves the photo-responsive drug release behaviors cooperatively with DMNB. After Pt(IV) prodrug is released from the micelles, it is expected to be transformed into its active Pt(II) form under intracellular reductive conditions for the efficient anticancer effects. In XPS results (Figure S7), the binding energy Pt4f of Pt(IV)-NO under intracellular reduction conditions (1 mM ascorbic acid, 24 h)48-50 was same with those of cisplatin (73.2 and 76.6 eV of the 4f(7/2) and 4f(5/2) levels) (Figure S6A), which is totally different from those of Pt(IV)-NO without incubation under reductive conditions (75.9 and 79.1 eV of 4f(7/2) and 4f(5/2) levels). These results imply that the prodrug can exert the Pt(II)-based anticancer effects after being released from micelle and NO release. Prior to investigating the anticancer effects, the potential of Pt(IV)-NO for intracellular NO delivery was evaluated by confocal laser scanning microscopy (CLSM) with DAF-2DA, a typical NO-detecting fluorescence dye (Figure 4C). Cells treated with Pt(IV)-C/E-micelles showed negligible NO release similar to non-treated cells, irrespective of light and dark conditions. However, the cells treated with Pt(IV)-NO/E-micelles exhibited increased NO release only when exposed to the light, demonstrating that Pt(IV)-NO/E-micelle has the ability to release NO on demand at the intracellular level.


Page 12 of 35

Page 13 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (A) Real-time NO release profiles of Pt(IV)-NO/E-micelles under repeated light irradiation and dark conditions. Red arrow and black arrow represent the light on and off, respectively. (B) Cumulative release of drug from Pt(IV)-NO/E-micelles, Pt(IV)-NO/C-micelles, Pt(IV)-C/E-micelles, and Pt(IV)-C/Cmicelles with or without light irradiation. (C) Intracellular release of NO from Pt(IV)-NO/E-micelle observed by DAF-2 DA-assisted CLSM using MCF-7 cells without and with light irradiation. Green color depicts fluorescence of DAF-2 DA after reaction with released NO. Scale bar is 50 µm.

Therapeutic evaluation of light-responsive prodrug-loaded micelle The therapeutic potential of Pt(IV)-NO/E-micelle was evaluated in HCT -116 and MCF-7 cells (Figure 5, Figure S8, and Table S1). Bare E-micelles and bare C-micelles showed negligible cytotoxicity,



indicating good biocompatibility of the polymers. The increased cytotoxicity of free Pt(IV)-NO was observed under light irradiation, whereas the therapeutic effects of free Pt(IV)-C were independent of light irradiation, demonstrating that NO enhanced the sensitivity of Pt(IV) drug. As reported previously,19 the enhanced anticancer effects might be due to the role of NO in reducing drug resistance via depletion of glutathione21 and inhibition of P-glycoprotein,19,20 MRPs,19 HIF,22 and NF-κB.23 The utilization of micelles improved the cytotoxicity of Pt(IV) prodrugs compared to the free Pt(IV)-NO and Pt(IV)-C. These results may be attributed to the amphiphilic characteristics of Pt(IV)-NO and Pt(IV)-C because amphiphilic Ptbased drugs show poor intracellular uptake due to the entrapment in lipid bilayers of the cellular membrane.7 That is, these data imply the requirement of drug delivery systems for efficient intracellular delivery of amphiphilic Pt(IV) prodrugs. Light irradiation has negligible effects on the cytotoxicity of Pt(IV)-C/C-micelles. However, Pt(IV)-NO/C-micelles and Pt(IV)-C/E-micelles slightly enhanced anticancer effects under light irradiation due to the drug-sensitizing effects or enhanced drug release. As expected, the Pt(IV)-NO/E-micelles exerted significantly improved anticancer effects upon light irradiation, which was likely due to the combined effects of NO and rapid drug release. Among various mechanism of NO in chemosensitization, we postulated that the light-responsive NO release in the cells increases the cellular uptake of platinum drugs by inhibiting drug efflux associated with P-glycoproteins and MRPs, which enhances the chemotherapeutic effects.19,20 Therefore, we measured Pt contents in the cells after treatment of each micelle with or without light irradiation (Figure 5C). There is no difference in Pt uptake profile among non-illuminated micelles, implying that the process of influx and efflux of Pt drug reached to equilibrium states within 18 h incubation of micelles. Accordingly, it is noteworthy that the improved uptake of Pt in light-irradiated micelles happened only during 20 min light irradiation and 30 min further incubation. It is also of importance to note that the possible effect of NO in enhanced drug influx can be excluded because micelles were washed out from the media before light irradiation. The NO-releasing drug micelles (Pt(IV)-NO/E-micelles and Pt(IV)-NO/C-micelles) showed higher Pt uptake than NO-nonreleasing drug micelles (Pt(IV)-C/E-micelles and Pt(IV)-C/C-micelles), respectively, indicating that NO might make an effect to the efflux system of Pt drug. In addition, the light-responsive micelles (Pt(IV)NO/E-micelles and Pt(IV)-C/E-micelles) showed higher Pt uptake than light-non-responsive micelles (Pt(IV)-NO/C-micelles and Pt(IV)-C/C-micelles), respectively, implying that light irradiation accelerated


Page 14 of 35

Page 15 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the drug release into the cells by disruption of drug-loaded micelles. These results imply that the improved anticancer effects of Pt(IV)-NO/E-micelles are ascribed not only to the light-responsive burst drug release, but also to the role of NO in inhibiting drug efflux. The mode of cell death was investigated by Annexin Vfluorescein isothiocyanate (FITC)/propidium iodide (PI) assay-assisted flow cytometry (Figure S9). We found that the anticancer effects of Pt(IV)-NO/E-micelles were through necrosis and late apoptosis (Figure 5D). In contrast, control groups induced an insignificant level of necrosis and apoptosis, which were correlated with the results of methylthiazole tetrazolium (MTT) assay.



Figure 5. In vitro anticancer effects of Pt(IV)-NO/E-micelle with and without light irradiation in (A) HCT116 and (B) MCF-7 cell lines. Statistical analysis was performed as compared with the light-exposed Pt(IV)-NO/E-micelle (*P

Synthesis and Characterization of Nitric Oxide-Releasing Platinum(IV

Recommend Documents