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Enhancing Photochemical Internalization of DOX through a Porphyrin-based Amphiphilic Block Copolymer Jia Tian, Lei Xu, Yudong Xue, Xiaoze Jiang, and Weian Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01037 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Biomacromolecules

Enhancing Photochemical Internalization of DOX through a Porphyrin-based Amphiphilic Block Copolymer

Jia Tian2, Lei Xu2, Yudong Xue2, Xiaoze Jiang1, Weian Zhang1,2*

1

State Key Laboratory for Modification of Chemical Fibers and Polymer

Materials, Donghua University, Shanghai 201620, China 2

Shanghai Key Laboratory of Functional Materials Chemistry, East China University

of Science and Technology, Shanghai 200237, China

*Corresponding author. E-mail address: [email protected] (W. Zhang)

1

ACS Paragon Plus Environment

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Abstract Drug resistance is a primary obstacle that seriously reduces therapy efficiency of most chemotherapeutic agents. To address this issue, the photochemical internalization (PCI) was employed to help the anticancer drug escape from lysosome and improve their translocation to the nucleus. Herein, a pH-sensitive porphyrin-based amphiphilic block copolymer (PEG113-b-PCL54-a-porphyrin) was synthesized, which was acted not only as a carrier for the delivery of DOX, but also as a photosensitizer for PCI. PEG113-b-PCL54-a-porphyrin as a drug carrier exhibited a higher drug loading capacity, entrapment efficiency and DOX release content. The PCI effect of PEG113-b-PCL54-a-porphyrin was studied by confocal laser scanning microscopy, and the results showed that most of DOX could be translocated into the nucleus for DOX-loaded

PEG113-b-PCL54-a-porphyrin

micelles.

Moreover,

the

IC50

of

pH-sensitive DOX-loaded PEG113-b-PCL54-a-porphyrin micelles, was much lower than

that

of

its

counterpart

without

pH

responsiveness,

DOX-loaded

PEG113-b-PCL54-porphyrin micelles. Therefore, this drug delivery system based on pH-sensitive porphyrin-containing block copolymer would act as a potential vehicle for overcoming drug resistance in chemotherapy.

2


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Introduction In cancer chemotherapy, most conventional anticancer drugs have many inherent problems to transverse in and out of blood vessels freely,1–3 for example, the poor solubility of anticancer drugs always leads them to aggregate together in blood vessels; furthermore, the non-selectivity of drugs, which means the anticancer drugs not only transfer to tumor sites but also the normal tissues, leads severe systemic side effects to patients; additionally, the enhanced efflux of drugs by transporter results in multiple drug resistance.4–8 Under these circumstances, drug delivery system (DDS) based on polymeric micelles for anticancer drugs has attracted great interests,9–14 since DDS could significantly prolong the circulation time of drugs, and improve tumor-selective targeting and therapeutic efficacy.15–20 Moreover, the controlled size of these polymeric micelles could help them penetrate and retain into tumor sites for an extended period of time via EPR effect (Enhanced Permeability and Retention effect).21–23 Many research works even clinical trials have reported the advantages of DDS in cancer treatment.24–28 For example, Zhang and coworkers synthesized novel PEG-b-PCL copolymers bearing different amounts of acid-labile β-carboxylic amides functional groups, which could encapsulate a high loading content of Doxorubicin （DOX） by the electrostatic interaction.29 Stimulus-responsive DDSs were also developed since they are expected to be both site-specific and time-release controlled to meet the request of the practical use.30–33 Wu and coworkers demonstrated pH- and redox dual responsive micelles, which displayed specific tumor targeting ability and higher drug-release controllability under the tumor cells microenviroment.34 3


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Although DDS has demonstrated to greatly improve the efficiency of drug delivery, multiple drug resistance is another great challenge in chemotherapy, which can reduce the cell-proliferation inhibition degree of anti-cancer drugs.35,36 DOX, as a mostly clinic used anti-cancer drug, which belongs to the anthracycline family, can permeate the membrane in its neutral situation but will be relatively impermeable when it is protonated.37–39 The weak alkaline DOX can distribute throughout the cytoplasm and nucleus. While being taken up in the acidic compartment (like lysosome), it can predict that DOX will be protonated and jailed in these compartments. This will lead the protonated DOX to accumulate in acidic compartment, followed by being pumped outside of cells and dispersed into the external medium.40–43 Thus, it is necessary for developing a novel DDS to help these protonated DOX escape from acidic compartment and translocate into nucleus.44,45 Photochemical internalization (PCI),46–50 a new strategy based on photodynamic therapy

(PDT),51–60

is

that

reactive

oxygen

induces

the

breakdown

of

endosome/lysosome membrane under appropriate light exposure.61–66 By effectively improving the therapeutic effect of anticancer drugs, this strategy has been successfully employed in drug and gene-release system such as DOX,67–69 camptothecin,70 bleomycin,71 nucleic acid72,73 and peptides.74 PCI strategy was first reported by Berg et al. as a method for light-enhanced cytosolic release of membrane-impermeable molecular therapeutics entrapped in endocytic vesicles.75 Then, Kataoka et al. constructed a new functional polyion complex vesicle which contained a photosensitizer, Al (III) phthalocyanine chloride disulfonic acid, for 4


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efficient photo-induced release of photoactive agent in intracellular drug delivery.76 Lai et al. have developed DOX-loaded 4-armed porphyrin-polylactide nanoparticle (PPLA-NPs) drug delivery system which could effectively increase the translocation of DOX from cytoplasm into nucleus after irradiation.77 Additionally, Na et al. have demonstrated single-oxygen producible polymeric micelles conjugated with chlorine e6 and pluronic F127 (Ce6-PF127) for overcoming DOX resistance in cancer under relatively weak laser irradiation condition78. Thus, it is interesting to combine the PCI effect and pH sensitive photosensitizer in DOX releasing system to overcome multiple drug resistance in cancer treatment. In this contribution, a well-defined pH-sensitive porphyrin photosensitizer was designed, which was conjugated an amphiphilic biodegradable block copolymer with a pH-sensible acetal bond, PEG113-b-PCL54-a-porphyrin (Scheme 1). The merits of PEG113-b-PCL54-a-porphyrin as the carrier for DOX were: (1) the amphiphilic PEG-b-PCL block copolymer is biodegradable and biocompatible; (2) The acetal bond is pH sensitive, which could be cleaved under lower pH, especially under the microenvironment of cancer cells; (3) The hydrophobic porphyrin was covalently bonded to amphiphilic block copolymer, which could effectively improve DOX loading efficiency; (4) A cell proliferation inhibition capability was enhanced under the

PCI

effect.

To

confirm

above

advantageous

properties,

PEG113-b-PCL54-a-porphyrin was self-assembled in water, and the assembled morphologies were characterized by TEM and DLS. The singlet oxygen quantum yields were determined by UV-Visible spectrophotometer and fluorescence 5


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spectrophotometer. DOX release at the low pH environment were evaluated in vitro. The toxicity of these micelles against A549 cells were evaluated by MTT assay. Confocal laser scanning microscopy (CLSM) was employed to evaluate the cellular uptake and intracellular distribution of DOX and photosensitizers.

Scheme

1.

Illustrations

for

the

preparation

of

DOX-loaded

PEG113-b-PCL54-a-porphyrin micelles (a), and PCI effect for the DOX escape from lysosome (b).

6


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Experimental Materials Methoxy poly(ethylene glycol) (PEG, Mn = 5 000), ε-caprolactone (ε-CL) and Tin(II) 2-ethylhexanoate (Sn(Oct)2) were all purchased from Sigma-Aldrich. Pyridinium p-toluenesulfonate

(PPTS),

2-chloroethyl

1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide

vinyl

ether

hydrochloride

(CEVE),

(EDC),

and

4-dimethylaminopyridine (DMAP) were all purchased from Aladdin Reagents of China. Hochest 33342 and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime. CellLight Lysosomes-GFP was purchased from Lifetecnologies. Toluene, dichloromethane (DCM) and N, N-dimethylformamide (DMF) were dried over calcium hydride and distilled before use. All other chemicals were of analytical grade and used as received. TPP-OH was synthesized according to our previous literature.79 PEG-b-PCL-porphyrin was synthesized as shown in Scheme S1. The 1H NMR spectra of PEG-b-PCL-a-Cl block copolymer, TPPC6-COOH, PEG-b-PCL-Porphyrin block copolymer were displayed in Figure S1-S3, respectively. The process, 1H NMR，GPC and DLS data of cleavage of acetal group were shown in Figure S4-S7.

Synthesis

of

monomethoxy-poly(ethylene

glycol)-b-poly(ε-caprolactone)

(PEG-b-PCL) PEG (1 g, 0.2 mmol), Sn(Oct)2 (0.81 g, 0.002 mmol) and anhydrous toluene (50 mL) were placed in a dry glass tube equipped with a magnetic stirring bar and distilled 3 h 7


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to remove residual water. Subsequently, ε-CL (1.24 g, 10.9 mmol) was added into the tube and vacuumed for another 30 min. After the tube was flame-sealed under vacuum, the polymerization was conducted in an oil bath at 120 °C for 24 h. Then the reaction mixture was dissolved with DCM, and precipitated in cold diethyl ether to obtain a white product. Yield: 2.15 g (96%), Mn = 12 700 g/mol, Mw/Mn = 1.18.

Synthesis of monomethoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-acetal-ethylenechloride (PEG113-b-PCL54-a-Cl) PEG113-b-PCL54 (1.27 g, 0.1 mmol) and PPTS (2.5 mg, 0.01 mmol) were refluxed in anhydrous toluene to remove the trace amount of water and then dissolved in 25 mL of anhydrous DCM in a dry flask. CEVE (0.1 ml, 1 mmol) in DCM (5 mL) solution was added dropwise into the above mixture at 0 °C under a dry argon atmosphere over 30 min. After further stirring for 30 min at room temperature, the reaction was quenched by Na2CO3 aqueous solution (5 wt%). The mixture was diluted with 30 mL DCM and washed with brine for 3 times. The organic layer was collected and dried with anhydrous MgSO4, concentrated and further precipitated into cold diethyl ether. Yield: 1.27 g (94%).

Synthesis of monomethoxy-poly(ethyleneglycol)-b-poly(ε-caprolactone)-a-porphyrin (PEG113-b-PCL54-a-porphyrin) PEG113-b-PCL54-a-porphyrin was synthesized via etherification reaction between 8


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PEG113-b-PCL54-a-Cl and TPP-OH. PEG113-b-PCL54-a-Cl (0.64 g, 0.05 mmol) and TPP-OH (0.063 g, 0.1 mmol) were dissolved in 20 mL of anhydrous DMF. The reaction was carried out at 60 °C for 10 h under argon. 10 mL of ultrapure water was added into the flask to quench the reaction, and the product was extracted with DCM. Finally, the PEG113-b-PCL54-a-porphyrin was precipitated in cold diethyl ether. Yield: 0.61 g (91 %), Mn = 12 900 g/mol, Mw/Mn = 1.24.

Preparation of Blank and Doxorubicin-loaded Micelles Blank micelles and DOX-loaded micelles were prepared via a membrane dialysis method, respectively. Briefly, for blank micelles, copolymers (50 mg) were dissolved in 5 mL of DMSO. This solution was added drop-wise into deionized water (5 mL) under magnetic stirring. After stirring for 2 h at room temperature, the solution was dialyzed against pure water for 1 day to remove DMSO. For DOX-loaded micelles, the preparation method was similar to that of blank micelles. Predetermined amount of DOX•HCl and 3 equivalent of triethylamine were dissolved in 5 mL of DMSO, followed with adding 50 mg copolymers and stirred for 2 h at 25 °C. The solution was filtered through a 0.45 µm filter and dialyzed against pure water for 1 day to remove DMSO. The DOX loaded in the micelles was extracted using DMSO, and the amount of DOX was determined by UV-Vis spectrophotometer at 500 nm according to calibration curve. Drug loading content (DLC) and encapsulation efficiency (EE) were calculated according to the following formula, respectively. 9


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DLC(%) =

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weight of drug in micelles

× 100%

(1)

weight of drug loaded micelles EE(%) =

weight of drug in micelles weight of drug in feed

× 100%

(2)

Measurement of Critical Micelle Concentration (CMC) The CMC was determined using pyrene as a fluorescence probe according to the previous literature.80 A predetermined amount of pyrene solution in acetone was added into a series of volumetric flasks to ensure that the pyrene concentration in the final solution was 6 × 10-7 mol/L and acetone was allowed to evaporate. The micelle solutions with various concentrations were added to each of the flasks and the mixture was kept at room temperature overnight. Emission wavelength was carried out at 335 nm, and excitation spectra were recorded ranging from 350 to 450 nm. The excitation emission slits were set at 10 nm and 2.5 nm, respectively. The fluorescence absorbance values of I372 and I383 were collected for the calculation of critical micelle concentration.

Fluorescence Quantum Yields The quantum yields were checked according to the previous literature.81,82 Fluorescence emission spectra were performed at Thermo Scientific Lumina fluorescence spectrophotometer. The excitation and emission slits were both set at 10 nm. The absorption spectra were acquired at Thermo Scientific Evolution 220 UV spectrophotometer. H2TPP was used as a secondary standard. Predetermined amount of H2TPP was dissolved in THF, and block copolymer micelles at pH = 7.4 and 5.0 10


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were prepared, respectively. The quantum yields were calculated as follows:

Yu = Ys

Au I s λs nu2

(3)

As I u λu ns2

Where, Y is the fluorescence quantum yield; I represents the absorbance; A represents integrated fluorescence intensity; λ is excitation wavelength and n is refractive index of solution. The subscript “s” and “u” represent the standard and the sample, respectively. Ys is the quantum yield of H2TPP (Ys = 0.11)

Singlet Oxygen Quantum Yields Singlet oxygen quantum yield (Φ) determinations were carried out using the experimental described in previous literature.83 Φ value was determined in air using the relative method with H2TPP as the reference. DPBF was used as chemical quencher for singlet oxygen. Briefly, 2 mL solution of the mixture of micelles and 1, 3-diphenylisobenzofuran (DPBF) was introduced to cell and illuminated with light in the presence of air. Eq. (4) was employed for the calculations: Φu = Φ s

Ru I s Rs I u

(4)

Where Φs is the singlet oxygen quantum yield for the standard H2TPP (Φs = 0.62); R is the DPBF photobleaching rate in the presence of samples and the standard; I is the rate of light absorption by the samples and the standard, respectively. To avoid chain reactions induced by quenchers (DPBF) in the presence of singlet oxygen, the concentration of quencher (DPBF) was lowered than 3 × 10−5 M. The subscript “s” and “u” represent the standard and the sample, respectively 11


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In Vitro DOX Release Study DOX-loaded micelle solution was diluted to 1.5 mg/mL in phosphate buffered saline (PBS, 0.01 M, pH = 7.4), and transferred into a dialysis membrane tubing (the molecular mass cutoff is 12 kDa). The dialysis bags were incubated in 20 mL of PBS at 37 °C with gentle shaking (100 rpm). At desired time intervals, 1 mL of release media was withdrawn and replenished with an equal volume of fresh media. The amount of DOX was determined by UV-Vis spectrophotometer at 500 nm according to the calibration curve.

Cell Culture A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2.

In vitro cytotoxicity A549 cell suspension with a density of 5 000 cells/well (200 µL) was seeded in 96-well plates overnight. For dark cytotoxicity, the cells were treated with different doses of DOX, PEG113-b-PCL54, PEG113-b-PCL54+DOX, PEG113-b-PCL54-porphyrin, PEG113-b-PCL54-porphyrin+DOX,


and

PEG113-b-PCL54-a-porphyrin+DOX micelles, respectively. The plates were cultured at 37 °C for another 24 h in dark and evaluated by means of the standard MTT assay. 12


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The results were measured at 560 nm by using a SpectraMax spectrometer. The cell viability was calculated as follows: cell viability (%) = (ODtest – ODblank)/(ODcontrol ODblank) × 100, where ODtest is the absorbance in the presence of sample solutions and ODblank is the absorbance without treatment. For phototoxicity, the cells were separately treated with various concentrations of PEG113-b-PCL54-porphyrin, PEG113-b-PCL54-porphyrin+DOX,


and

PEG113-b-PCL54-a-porphyrin+DOX micelle solutions and incubated for 18 h. Then the plates were irradiated with a light emitting diode (LED) lamp (420 nm, 100 mW cm-2) for 0.5 min. After culturing for 24 h, the cell viability was also evaluated using the MTT assay.

Confocal laser scanning microscopy (CLSM) measurements CLSM was used to evaluate the localization of DOX and the PCI effects of PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles. A549 cells with a density of 1.5 × 105 cells per dish were seeded onto the glass bottom dish and cultured with DMEM supplemented with 10% FBS for 24 h. Then the culture medium was replaced by PEG113-b-PCL54-porphyrin, PEG113-b-PCL54-porphyrin+DOX, PEG113-b-PCL54-a-porphyrin

micelles

and

PEG113-b-PCL54-a-porphyrin+DOX

micelles (porphyrin concentration: 5 µg/mL) containing culture medium (mixed with predetermined amount of CellLight Lysosomes-GFP) for 16 h. CellLight Lysosomes-GFP (excitation/emission maxima: 485 nm/520 nm) was used here to stain the lysosomal and incubated with cells more than 16 h. After being thoroughly 13


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washed with PBS solution, the cells were irradiated with a light emitting diode (LED) lamp (420 nm, 100 mW cm-2) for 0.5 min and incubated with fresh full medium for another 2 h. Cells were fixed with 4% paraformaldehyde in PBS solution for 25 min, then stained with hochest 33342 to mark the nucleus. The CLSM was performed by using a fluorescence microscope (Nikon AIR).

Result and Discussion Synthesis

and

Characterization

of


Block

Copolymer.

Scheme 2. Synthetic route of PEG113-b-PCL54-a-porphyrin copolymer.

The PEG113-b-PCL54-a-porphyrin copolymer was synthesized by the combination of ring-opening

polymerization

(ROP)

and

coupling

reaction

(Scheme

2).

PEG113-b-PCL54 copolymer was first synthesized using the terminal hydroxyl group of PEG chain to initiate the ROP of ε-CL in bulk under the catalysis of Sn(Oct)2 at 120 °C for 24 h. PEG113-b-PCL54-a-Cl was further synthesized by coupling reaction 14


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between CEVE and PEG113-b-PCL54 at 0 °C in the presence of the catalyst, PPTS. Then PEG113-b-PCL54-a-porphyrin was finally obtained by the etherification reaction between

PEG113-b-PCL54-a-Cl

and

TPP-OH.

The

chemical

structure

of

PEG113-b-PCL54-a-porphyrin block copolymer was characterized by 1H NMR (Figure 1). Besides the characteristic peaks at 4.09, 2.34, 1.67, 1.40 ppm are ascribed to the ethylene protons of PCL block, and 3.66 and 3.40 ppm are attributed to the protons of PEG block, 8.88, 8.23, 8.21 7.76, 7.30 and -2.77 ppm are ascribed to the protons of porphyrin moiety. Moreover, the integral ratio of a : g : o was nearly 3 : 1 : 2, which further confirmed the PEG113-b-PCL54-a-porphyrin block copolymer was successfully synthesized.

Figure 1. 1H NMR spectrum of PEG113-b-PCL54-a-porphyrin block copolymer.

15


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In addition, the unimodal peaks in the GPC traces for PEG113, PEG113-b-PCL54 PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin block copolymers also revealed that the copolymerization and coupling reaction were successfully performed (Figure 2). The molecular weight of two copolymers determined by 1H NMR and GPC was listed in Table S1. The molecular weight distributions of PEG113-b-PCL54, PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin are 1.18, 1.30 and 1.24, respectively. Additionally, the molecular weights measured with GPC analysis were slightly higher than those calculated by 1H NMR, which could be resulted from the difference in hydrodynamic volumes between block copolymers and polystyrene standards.

Figure 2. GPC traces of PEG113 (a), PEG113-b-PCL54 (b), PEG113-b-PCL54-porphyrin (c), and PEG113-b-PCL54-a-porphyrin (d).

16


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Preparation and Characterization of Copolymer Micelles. The amphiphilic block copolymer can self-assemble into micelles in aqueous solution using the dialysis method. The formation of micelles was first evaluated by measuring CMC with the fluorescence technique using pyrene as a probe, since it can transfer from the aqueous environment to the non-polar region of the micellar interior core, which results in significant spectroscopic changes. The ratio of pyrene fluorescence intensities excited at 383 and 372 nm (I383/I372) was plotted as a function of the logarithm

of

copolymer

concentration

for

PEG113-b-PCL54,

PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin. As illustrated in Figure 3, the intensity ratio remained almost unchanged at low copolymer concentrations and increased sharply when the copolymer concentrations reached a certain value, indicating the pyrene was trapped into the micelle core with more hydrophobic environment, which indicated the formation of micelles. The CMC values for PEG113-b-PCL54, PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin were determined as 5.18 × 10-3, 1.04 × 10-3 and 1.89 × 10-3 mg/mL as shown in Table 1. Lower CMC value for PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin indicated they have a good thermodynamic stability. This is because of the presence of hydrophobic porphyrin at the end of PEG113-b-PCL54 chain, which could promote hydrophobic interaction within the core of micelles and benefit for the formation of self-assembled micelles.

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Figure 3. Plots of the I383/I372 ratio against log C of polymeric micelles.

To further evaluate the properties of block copolymer micelles, both the mean size and the morphology of these PEG113-b-PCL54 and PEG113-b-PCL54-a-porphyrin micelles were studied by DLS (Figure 4) and TEM (Figure 5), respectively. DLS results

showed

the

size

of

PEG113-b-PCL54-porphyrin

and

PEG113-b-PCL54-a-porphyrin micelles was about 119.2 and 113.5 nm, respectively, which was much smaller than that of PEG113-b-PCL54 micelles (215.4 nm). TEM images also revealed the formation of spherical aggregates from these block copolymers in water (Figure 5). The big difference between micellar sizes obtained from three block copolymers may be because of porphyrin moiety attached at the end of polymer chain. The π-electronic stacking between aromatic porphyrin macrocycles and the hydrogen bonding formed between -NH group and -CO- group may contribute greatly to the lower micellar diameters from porphyrin-containing copolymers. 18


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Figure 4. Size distribution of DOX-free and DOX-loaded micelles determined by DLS.

Table 1. Characteristics of DOX-free and DOX-loaded micelles. Polymers

DOX-free micelles Diameter

PDIa

(nm)a

DOX-loaded micelles

CMC

Diameter

(mg/mL)b

(nm)a

PDIa

DLC

EE

(%)c

(%)c

PEG113-b-PCL54

215

0.208

5.18 × 10-3

123.8

0.118

6.19

30.9


119

0.072

1.04 × 10-3

70.1

0.227

11.12

62.6


114

0.108

1.89 × 10-3

68.8

0.232

11.04

62.1

a

Determined by DLS.

b

c

CMC was determined by fluorescence spectra with pyrene as the fluorescence probe.

Determined by UV-Vis spectrophotometer. 19


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Preparing of DOX-loaded micelle and in Vitro Drug Release Behavior. DOX was encapsulated into the micelles by a dialysis method using these three block copolymers. The self-assembled morphologies of these DOX-free or loaded micelles were examined by TEM (Figure 5). As shown in Table 1, the sizes of DOX-loaded PEG113-b-PCL54-porphyrin (70.1 nm) and DOX-loaded PEG113-b-PCL54-a-porphyrin (68.8 nm) were smaller than that of DOX-loaded PEG113-b-PCL54 (123.8 nm). This is probably due to the enhanced interaction force between aromatic guest DOX molecules and macrocycle molecular porphyrin moiety. Moreover, DLS results (Table 1) also showed the mean sizes of DOX-loaded PEG113-b-PCL54-porphyrin and DOX-loaded PEG113-b-PCL54-a-porphyrin micelles were smaller than those of their DOX-free micelles. The DLC and EE of these block copolymers were further evaluated and the results were listed in Table 1. When the feed ratio of DOX to block copolymer was 1:5, the DLC

of

PEG113-b-PCL54,


and

PEG113-b-PCL54-a-porphyrin micelles was 6.19%, 11.12% and 11.04%, respectively, corresponding to the EE of 32.99%, 62.6% and 62.1%. It could be seen that the DLC and EE of PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles were much higher than that of PEG113-b-PCL54 micelles which could be attributed to noncovalent interaction such as π-π stacking and hydrogen bond interaction formed by aromatic ring, carbonyl groups, hydroxyl groups and secondary amine.

20


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Figure 5. TEM images of (a): PEG113-b-PCL54; (b): PEG113-b-PCL54 + DOX; (c): PEG 11 3 -b-PCL 54 -porphyrin; (d): PEG 11 3 -b-PCL 5 4 -porphyrin + DOX; (e): PEG 113 -b-PCL 54 -a-porphyrin; (f): PEG 113 -b-PCL 54 -a-porphyrin + DOX

Figure

6.

The

release

behavior

of

DOX

from

PEG113-b-PCL54,

PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles, a: pH=7.4 / PEG113-b-PCL54+DOX / DOX; b: pH=5.0 / PEG113-b-PCL54+DOX / DOX; c: pH=7.4 21


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/ PEG113-b-PCL54-porphyrin + DOX / DOX; d: pH=5.0 / PEG113-b-PCL54-porphyrin + DOX / DOX; e: pH=7.4 / PEG113-b-PCL54-a-porphyrin + DOX / DOX; f: pH=5.0 / PEG113-b-PCL54-a-porphyrin + DOX / DOX.

In vitro release behaviors of porphyrin and DOX were carried out at pH = 7.4 and pH = 5.0, respectively. For the release of porphyrin (Figure S8), at pH = 7.4, there was

almost

no

porphyrin

released

from


and

PEG113-b-PCL54-a-porphyrin micelles; However, at pH = 5.0, it could be detected about 42% of porphyrin released from pH sensitive PEG113-b-PCL54-a-porphyrin micelles, while nearly no porphyrin was released from PEG113-b-PCL54-porphyrin micelles. This is attributed to the breakage of the pH-sensitive acetel bond of PEG113-b-PCL54-a-porphyrin copolymer under acidic environment, resulting in the release of porphyrin moieties. The release of DOX was further estimated from DOX-loaded micelles (Figure 6). The relative quantity of DOX released from DOX-loaded

PEG113-b-PCL54,


and

PEG113-b-PCL54-a-porphyrin micelles at pH = 7.4 were all less than 34.4% (Figure 6a, 6c and 6e). At pH = 5.0, the percentage of DOX released from DOX-loaded PEG113-b-PCL54,


and


micelles were 99.2%, 56.6% and 91.7%, respectively (Figure 6b, 6d and 6f). We could find that the percent of DOX released from PEG113-b-PCL54-a-porphyrin micelles was higher than that from PEG113-b-PCL54-porphyrin micelles at lower pH. Thus, this also confirmed that pH-sensitive PEG113-b-PCL54-a-porphyrin micelles may 22


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be better than PEG113-b-PCL54-porphyrin micelles in the delivery and release of DOX. Besides, upon cellular uptake the DOX loaded PEG113-b-PCL54-porphyrin micelles will encounter the intracellular pH gradients, for example, pH 5.9-6 in early endosomes, pH 5.0-5.5 in lysosomes. The pH degradable acetal groups will increase cleaving from the micelles with the pH gradients, which results in the accessorial release of DOX in the process of intracellular uptake and the therapeutic efficacy.

Fluorescence and Singlet Oxygen Quantum Yields Herein,

we

separately

measured

the

fluorescence

quantum

yields

of

PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles at pH = 7.4 and 5.0, and the results were listed in Table 2. The fluorescence quantum yields of PEG113-b-PCL54-porphyrin micelles at pH = 7.4 and 5.0 were 0.09 and 0.13, while PEG113-b-PCL54-a-porphyrin micelles at pH = 7.4 and 5.0 were 0.08 and 0.17, respectively. The fluorescence quantum yield of PEG113-b-PCL54-a-porphyrin micelles was slightly higher than that of PEG113-b-PCL54-porphyrin micelles at pH = 5.0. In the PDT or PCI process, the singlet oxygen quantum yield of photosensitizers is a key parameter to determine the PDT/PCI efficiency. The Φ value was estimated using a chemical method with DPBF as a quencher, where the amount of DPBF was monitored using UV-Vis spectrophotometer. As shown in Table 2, the singlet oxygen quantum yields of PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles were almost same (Φ = 0.46) at pH = 7.4. However, at pH = 5.0, the singlet oxygen

quantum

yields

of


23


and

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PEG113-b-PCL54-a-porphyrin micelles were 0.53 and 0.79, respectively. The results showed that PEG113-b-PCL54-a-porphyrin micelles not only had a higher fluorescence quantum yield but also a singlet oxygen quantum yield at lower pH environment by the introduction of the pH-sensitive acetal bond. It could be because that the acid environment led to cleaving the acetal bond and improving the release of porphyrin moieties, thus increasing fluorescence and singlet oxygen quantum yields of the micelles.

Table 2. Fluorescence (Y) and Singlet Oxygen (Ф) quantum yields of PEG113-b-PCL54-porphyrin

and


micelles at pH = 7.4 and 5.0, respectively. Y

Samples

Ф

pH = 7.4

pH = 5.0

pH = 7.4

pH = 5.0


0.09

0.13

0.46

0.53


0.08

0.17

0.46

0.79

PCI Effects on the Cytotoxicity of A549 Cells The dark cytotoxicity and phototoxicity of free DOX, PEG113-b-PCL54 + DOX, PEG113-b-PCL54-porphyrin + DOX and PEG113-b-PCL54-a-porphyrin + DOX micelles were separately studied against A549 cells by using MTT assay. Firstly, the cytotoxicity of block copolymers was evaluated, and the results displayed that all the block copolymers nearly had no cytotoxicity (Figure S9a). For dark cytotoxicities of 24


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all samples as shown in Figure S9b, the cell-proliferation inhibition degree was gradually higher as the increasing of DOX concentration. The inhibition degree of PEG113-b-PCL54-a-porphyrin + DOX micelles was slightly higher than that of PEG113-b-PCL54-porphyrin + DOX micelles. This can be ascribed to the breakage of pH-sensitive acetal bond in PEG113-b-PCL54-a-porphyrin micelles which promoted the DOX release under lower pH. PCI effects of these samples were studied by using a LED lamp (420 nm, 100 mW cm-2). Herein, we fixed the irradiation time for 0.5 min and varied the sample concentrations to screen out the appropriate drug concentration for the PCI effect. As shown in Figure 7, no significant phototoxicities were observed at low porphyrin concentration (< 5 µg/mL) for either DOX-free PEG113-b-PCL54-porphyrin or PEG113-b-PCL54-a-porphyrin micelles, indicating that there was no PDT effect at this porphyrin concentration. However, for DOX-loaded PEG113-b-PCL54-porphyrin and PEG113-b-PCL54-a-porphyrin micelles under the irradiation of light at the low porphyrin concentration (< 5 µg/mL), they all showed clearly enhanced cell proliferation inhibition in the comparison with their dark cytotoxicity, which could be ascribed to PCI effect. For the high porphyrin concentration (> 5 µg/mL), the extra cytotoxicity of these DOX-loaded micelles could be attributed to the main contribution

of

PDT

effect.

Additionally,

the

IC50

of

DOX-loaded

PEG113-b-PCL54-a-porphyrin micelles was 9.08 µg/mL (calculated as DOX concentration),

which

was

much

lower

than

that

of

DOX-loaded

PEG113-b-PCL54-porphyrin micelles (18.43 µg/mL). These results indicated that 25


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Page 26 of 39

porphyrin containing micelles may induce singlet oxygen mediated lysosome disruption, help DOX escape from the lysosome and increase the therapy efficiency.

Figure 7. Cell viability determined by using MTT assay: (a) phototoxicity of PEG113-b-PCL54-porphyrin, PEG113-b-PCL54-porphyrin+DOX and dark toxicity of PEG113-b-PCL54-porphyrin+DOX (b) phototoxicity of PEG113-b-PCL54-a-porphyrin, PEG113-b-PCL54-a-porphyrin+DOX

and

dark

toxicity

of

PEG113-b-PCL54-a-porphyrin+DOX.

Intracellular Distribution of DOX-Loaded Micelles without or with PCI Effects To puzzle out the possible mechanism behind the PCI effect and DOX-loaded micelles in the synergism cell-proliferation inhibition degree, confocal microscopy was

further

performed.

Cells

were

treated

with

DOX-loaded

PEG113-b-PCL54-porphyrin and DOX-loaded PEG113-b-PCL54-a-porphyrin micelles for 2 hours. After that, they were irradiated with a LED lamp and cultured for another 26


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2 hours. As shown in Figure 8, the DOX was incubated both in cytoplasm and nucleus for DOX-loaded PEG113-b-PCL54-porphyrin micelles, while nearly all of the DOX was translocated in the nucleus delivered with PEG113-b-PCL54-a-porphyrin micelles. This confirmed that the DOX encapsulated in PEG113-b-PCL54-a-porphyrin micelles penetrated into the nucleus with a higher efficiency than that loaded in the micelles without acetal groups under light irradiation.

Figure 8. Intracellular distribution of DOX loaded on PEG113-b-PCL54-porphyrin micelles and PEG113-b-PCL54-a-porphyrin micelles. The CLSM was carried out 2 hours after irradiation. Nuclei were stained with Hoechst 33342 (blue). 27


Biomacromolecules

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 9. Cellular uptake and internalization of porphyrin-containing polymer micelles. The cells were prestained by CellLight Lysosomes-GFP (green) and the nucleus were stained with Hoechst 33342 (blue). (a) and (c): cells were treated with PEG113-b-PCL54-porphyrin micelles; (b) and (d): cells were treated with PEG113-b-PCL54-a-porphyrin micelles; (a) and (b) without irradiation; (c) and (d) with irradiation;

We then studied the intracellular distribution of PEG113-b-PCL54-porphyrin micelles and PEG113-b-PCL54-a-porphyrin micelles according to the fluorescence of porphyrin. 28


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Cells were cultured with CellLight Lysosomes-GFP and porphyrin-containing micelles for more than 16 h, irradiated for 0.5 min and cultured for another 2 hours. Cells treated without light irradiation as the control. CellLight Lysosomes-GFP (Ex: 485 nm; Em: 520 nm) was used here to stain lysosome. As shown in Figure 9, most of the red fluorescence was overlay with the green fluorescence which means these porphyrin-containing micelles were co-localized with CellLight Lysosomes-GFP in the compartment of lysosome. But PEG113-b-PCL54-porphyrin micelles presented a weaker red fluorescence compared to the PEG113-b-PCL54-a-porphyrin micelles whenever with or without light irradiation. Interestingly, after light irradiation, both PEG113-b-PCL54-porphyrin micelles and PEG113-b-PCL54-a-porphyrin micelles treated cells all showed fluorescence diffusion phenomenon (PCI effect). Moreover, the cells cultured with PEG113-b-PCL54-a-porphyrin micelles presented the higher degree of fluorescence diffusion than the cells treated with PEG113-b-PCL54-porphyrin micelles. This could be because that the pH-sensitive acetal bond could break inside lysosome (pH = 5), result in the release of porphyrin and the increase of fluorescence intensity and singlet oxygen quantum yields to enhance the PCI effect, which agreed well with the results mentioned above.

Conclusion In summary, we have constructed a well-designed pH-sensitive porphyrin-containing nanocarrier, which could improve the DOX cytotoxicity with the contribution of PCI effect. PEG113-b-PCL54-a-porphyrin micelles presented a higher singlet oxygen 29


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Page 30 of 39

quantum yields and DOX release content at the low pH. The IC50 of DOX-loaded PEG113-b-PCL54-a-porphyrin micelles was 9.08 µg/mL (calculated as DOX concentration),

which

was


much

micelles

lower

than

(18.43

that

µg/mL).

of

The

DOX-loaded photosensitizer

bio-distribution showed that PEG113-b-PCL54-a-porphyrin micelles presented the higher

degree

of

fluorescence

diffusion

than

the

cells

treated

with

PEG113-b-PCL54-porphyrin micelles under the irradiation of appropriate light. Moreover, PEG113-b-PCL54-a-porphyrin micelles could effectively help the DOX escape from lysosome and translocate into nucleus by PCI under light irradiation. Thus, this pH-sensitive porphyrin-containing block copolymer would be designed for advanced drug delivery system to overcome drug resistance in chemotherapy.

Supporting Information: < Synthesis of mPEG113-b-PCL54-Porphyrin copolymer,1H NMR spectra, the cleavage of acetal group in acidic condition, the release behavior of porphyrin, Cell viability of dark toxicity determined by using MTT assay >

Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21574039) and the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University.

30


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For Table of Contents Use Only

Enhancing Photochemical Internalization of DOX through a Porphyrin-based Amphiphilic Block Copolymer

Jia Tian2, Lei Xu2, Yudong Xue2, Xiaoze Jiang1, Weian Zhang1,2*

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Enhancing Photochemical Internalization of DOX ... - ACS Publications

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