pH Dual-Sensitive Nanocarriers for Enhanced

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Controlled Release and Delivery Systems

Near Infrared/pH Dual-Sensitive Nanocarriers for Enhanced Intracellular Delivery of Doxorubicin Kaikai Wen, Mengxue Zhou, Huiru Lu, Ying Bi, Lifo Ruan, Jun Chen, and Yi Hu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01051 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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ACS Biomaterials Science & Engineering

Near Infrared/pH Dual-Sensitive Nanocarriers for Enhanced Intracellular Delivery of Doxorubicin Kaikai Wen,†,‡,§ Mengxue Zhou,†,‡,§ Huiru Lu,† Ying Bi,† Lifo Ruan,†,‡ Jun Chen*,†,‡, and Yi Hu,*,†,‡ †CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Multi-disciplinary Research Division, Institute of High Energy Physics, Chinese Academy of Sciences (CAS), 19B Yuquan Road, Beijing 100049, China. ‡University

of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049, China

* Corresponding author. E-mail: [email protected], [email protected].

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ACS Biomaterials Science & Engineering 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 designed near infrared (NIR)/pH dual-sensitive nanocarriers and evaluated its application to intracellular drug delivery. The nanocarriers were prepared based on amphiphilic poly(β-amino ester) (PBAE)

containing o-nitrobenzyl moieties

in the backbones and upconversion nanoparticles (UCNPs). UCNPs can convert NIR to UV that subsequently removes PEG segments from PBAE copolymers, which could enhance the protonation of PBAE in endo/lysosomes and facilitate the escape of the nanoparticles from lysosomes. In addition, we found the colocalization of the nanoparticles with mitochondria inside the cells, presumably resulting from high hydrophobicity and positive charges of the nanoparticles. The results showed that the nanocarriers with the aid of NIR could enhance the intracellular delivery of DOX, as compared with free DOX and NIR-free control. Furthermore, PBAE@UCNPs-DOX with NIR potently inhibited tumor growth in mice. Therefore, the intelligent micellar nanoparticles might provide a simple yet effective nanoplatform to achieve mitochondrion-targeting drug delivery.

Keywords: near infrared; poly(β-amino ester); upconversion nanoparticles; intracellular drug delivery; mitochondrion


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INTRODUCTION The stimulus-responsive polymers that change upon endogenous stimuli (e.g. reducing substances, the acidity of tumor, etc.)1-4 or external stimuli (e.g. temperature, light, ultrasound, etc.)5-7 can facilitate tumor-targeting drug delivery.8 Among them, light-responsive nanocarriers emerge as a promising tool for spatiotemporally controlled drug delivery to the tumor cells.9-11 For instance, Deng et al.12 have recently integrated UV light-cleavable linkage into the nanocarriers for enhanced DNA delivery. This strategy has achieved remotely controllable degradation of nanocarriers for enhanced DNA delivery. Nevertheless, UV light is not a biocompatible stimulus for drug delivery. To circumvent this problem, upconversion nanoparticles (UCNPs) which can convert near-infrared (NIR) light to UV may be employed as an internal UV light source in the nanocarriers.13 For the intracellular drug delivery, endocytosis is a major way for cells to uptake the nanoparticles.14 However, the degradation of nanoparticles and laden cargos in endosomes (pH ~5.5–6.5) and lysosomes (pH ~4.5–5.5) remains a dominant barrier for intracellular drug delivery. Accordingly, an ideal nanoparticle would be capable of rapidly escaping from endo/lysosomes. Poly(β-amino ester) (PBAE) is a group of amino group-containing cationic polymers, which have been utilized to construct a variety of nanocarriers for delivery of genes, drugs and proteins.15, 16 It can be easily synthesized via mild and simple Michael-type addition polymerization, which can produce a library of structurally versatile materials.15 Their high buffer capacity facilitates the escape of polycation-containing nanoparticles from endo/lysosomes via the “proton sponge” effect. For instance, by introducing tertiary amine with a pKb of about 6.5 in the backbones or side chains, a sharp pH sensitivity and subsequent rapid intracellular drug release can be achieved in the polymeric nanocarriers.17 After drug transportation from endo/lysosome to the cytosol, it is important to deliver the drugs to the subcellular targets, such as cell nucleus and mitochondrion.18 As doxorubicin (DOX) is a DNA-binding chemotherapeutic drug, cell nucleus was widely believed as a primary site of action for DOX.19 However, recent studies have suggested that delivering DOX to mitochondria could potentially overcome DOX



resistance in tumors.20,

21

Therefore, targeted delivery of nanodrugs to subcellular

organelles may offer a novel strategy for enhanced chemotherapeutic efficacy. In this study, we designed and synthesized a novel NIR/pH dual sensitive nanocarrier containing amphiphilic PBAE/PEG with o-nitrobenzyl (ONB) moieties in the backbones and UCNPs. Upon NIR light, UCNPs could convert NIR to UV that subsequently removed PEG from PBAE (Scheme 1). The protonation of PBAE facilitated the escape of the nanoparticles from endo/lysosomes to cytosol. Afterward, released nanoparticles were found to be mainly accumulated in mitochondria, presumably because of electrostatic and hydrophobic interaction.18, 22 Therefore, the smart micellar nanoparticles may provide a simple yet useful tool for mitochondriontargeting drug delivery.

Scheme 1. Illustration of PBAE@UCNPs-DOX micelles for NIR-triggered drug release in tumor cells.

EXPERIMENTAL SECTION Materials and Instruments. All chemicals were purchased from Sigma-Aldrich and used without further purification unless otherwise noted. Methoxy PEG acrylate (5k) was purchased from JenKem Technology USA Inc. MCF-7 cells were purchased from the Bogoo Biotech. Inc. (Shanghai, China) and were cultured in Dulbecco’s Modified


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Eagle Medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS). UVvis spectra were collected on a Varian Cary-50 UV-vis spectrophotometer. Fluorescence emission spectra were recorded with a Varian Cary Eclipse fluorescence spectrophotometer. Upconversion photoluminescence measurements were performed on a PTI Quantamaster spectrofluorometer. Malvern Zetasizer Nano-S dynamic light scattering (DLS) (Malvern Instruments, UK) was used to determine the hydrodynamic diameters and size distribution of nanoparticles. The morphology of the as-synthesized micelles was observed on field emission transmission electron microscopes (TEM, Tecnai G2 20 St). Thermogravimetric analysis (TGA) was carried out with a TGA Q500 V20.13 Build 39 instrument at the heating rate of 10 °C/min under air condition. Synthesis and Characterization of Amphiphilic PBAE. The (1-nitro-2,5phenylene) bis(methylene) diacrylate (NPBMDA) was synthesized according to the previous reported protocols12 with modifications (Scheme 2). 1H NMR (CDCl3): δ 7.52 (m, 3H, ArH), 6.42 (d, 2H, -CH=CH2), 6.11 (d, 2H, -CH=CH2), 5.91 (d, 2H, -CH=CH2), 5.31 (s, 4H, ArCH2O-). Light-responsive PBAE was synthesized from NPBMDA, PEG-acrylate, and amine-containing compounds via Michael addition reaction. (1nitro-2,5-phenylene) bis(methylene) diacrylate (292 mg ， 1 mmol),

methoxy PEG

acrylate (500 mg, 0.1 mmol) and dodecylamine (222 mg，1.2 mmol) were added into three-necked, 100-mL flask under nitrogen protection. The mixture was allowed to stirring at 100 °C for 12 h. Then the reaction was stopped by cooling to room temperature. 10 mL CHCl3 was added to dissolve the yellowish solid. Methoxy PEG acrylate (500 mg, 0.1 mmol) was added to consume the acrylate groups as end cap segments. The products were purified by precipitation in a large amount of ether followed by vacuum drying. The polymer was characterized by UV-vis spectra and 1H NMR. 1H NMR (CDCl3): δ 0.85(3H,-CH3), 1.25-1.38 (18H, -(CH2)9CH3), 2.13 (2H,N-CH2-(CH2)9-), 2.30-2.50 (4H, -CH2-COO- and 2H, -N-CH2-CH2-COO), 2.60-2.80 (6H, -N(CH2)2-), 3.38 (3H,-OCH3), 3.51(2H, -COO-CH2-CH2-PEG), 3.60-3.70 (CH2 in PEG repeat unit), 4.20-4.30 (2H, -COOCH2-CH2-PEG), 5.19 (4H, -CH2-bezene), 7.49 (3H, CH in benzene). Cy5 dyes conjugated PBAE polymers were synthesized via



a two-step route. (1-nitro-2,5-phenylene) bis(methylene) diacrylate (292 mg，1 mmol), methoxy PEG acrylate (500 mg, 0.1 mmol),

dodecylamine (222 mg，1 mmol) and 6-

amino-1-hexanol (234 mg, 0.2 mmol) were used to synthesize the hydroxyl groups containing PBAE copolymers, where Cy5-NHS was subsequently added to form the ester bonds for covalent conjugation onto the PBAE copolymers (Cy5-PBAE). Preparation and Characterization of NaYF4:Yb, Tm UCNPs. The NaYF4:Yb, Tm (0.5%) UCNPs were synthesized similar to a recently reported procedure.23 Briefly, OA-capped NaYF4:Yb, Tm UCNPs were synthesized via a two-step strategy using lanthanide oleate as the precursor. Rare earth chloride and 3-fold sodium oleate were mixed in a solvent (ethanol, distilled water, and hexane) at room temperature for 24 h. Rare earth oleate complexes were obtained by removing the hexane. Then NaF (4 mmol) was added into 1 mmol rare earth-oleate complexes in a solution of 6 mL of OA and 15 mL of ODE. The reaction was carried out at 70 °C with vigorously stirring for 1 h flushed with Ar, and heating up at 110 °C for another 1 h. Afterward, the solution was rapidly heated to 320 °C with Ar and maintained for 1 h. The reaction mixture was allowed to balance to room temperature. After centrifugation, the products were washed with ethanol for three times and dried. The upconversion luminescence (UCL) spectra before and after the surface modification by PBAE were recorded by a Varian Cary Eclipse fluorescence spectrophotometer with an external 980-nm laser as the excitation source. Preparation and Characterization of NIR-Sensitive Micelles (PBAE@UCNPs). The oleic acid modified UCNPs were dispersed in cyclohexane at 1 mg mL-1, followed by ultrasonic dispersion for 15 min. PEG-PBAE (5 mg) was dissolved in 1 mL chloroform, followed by addition of 1 mL UCNPs with rigorously stirring for 30 min. Afterward, 10 mL distilled water was added dropwise to the organic phase and emulsified for 10 min by pulsed sonication (100 W), followed by being stirred overnight at room temperature to evaporate the organic solvent until the solvent was transparent. The solution was then dialyzed against distilled water for 24 h (molecular weight cut-off= 3500) to remove excess polymers and unincorporated nanoparticles.24 The critical micellar concentration (CMC) of PBAE copolymer in water was estimated


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by fluorescence spectroscopy using pyrene as a probe. 20 μL of pyrene acetone solution (20 μg/mL) was added to a 4-mL vial, and then acetone was allowed to evaporate to dry. 4 mL of aqueous solution containing 1x10-4-0.128 mg mL-1 of PBAE copolymers were added to the vials, respectively. The final concentration of pyrene in each sample solution was 0.1 μg/mL. The excitation (300-360 nm) and an emission wavelength of 395 nm were applied with the excitation and emission bandwidths set at 5 nm. The ratios of the peak intensities at 338 nm to 334 nm (I338/I334) were recorded and plotted versus polymer concentrations. NIR-responsive disassembly of the PBAE@UCNPs was measured using a dynamic dialysis method. 1 ml of aqueous solution containing 2 mg mL-1 of PBAE@UCNPs was added to the vials, and then was irradiated with NIR (980 nm, 1 W cm-2). After several hours, the sizes of PBAE@UCNPs were determined by the DLS. In Vitro DOX Release from PBAE@UCNPs-DOX Micelles. For preparation of drug-loaded nanoparticles, 1 mg DOX (deprotonated, dissolved in 100 μL chloroform), 1 mg UCNPs and 5 mg PBAE were added to the organic phase before the formation of micelles by ultrasonic emulsification. The drug-loading content (%) and drug-loading efficiency (%) were defined according to the eq 1, 2. The concentration of DOX was 0.1 mg mL-1 in in vitro drug release experiments. 3 mL PBAE@UCNPs-DOX micelles were treated with NIR irradiation (980 nm, 1 W cm-2) for five minutes, followed by dialysis (Mw cut-off = 3.5 kDa) in 17 mL PBS (0.01 M, pH = 7.4 or 5.0) at 37 °C. 3 mL solution was taken to record DOX absorbance at 480 nm, and the same amount of PBS solution was used to re-fill it. A standard curve of DOX absorbance was used to determine DOX concentrations in the samples. weight of loaded drug + weight of loaded drug

Drug ― loading content (%) = weight of copolymer

weight of loaded drug

∗ 100%

Drug ― loading efficiency （%） = weight of feeding drug ∗ 100%

(1)

(2)

Cytotoxicity of PBAE@UCNPs-DOX Micelles. MCF-7 cells were used to investigate cell inhibition of PBAE@UCNPs-DOX micelles upon NIR light. MCF-7 cells were cultured with DMEM supplemented with 10% FBS, 1.0x105 U L-1 penicillin



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(Sigma) and 100 mg L-1 streptomycin (Sigma) at 37 °C in 5 % CO2. The cytotoxicity of the PBAE@UCNPs-DOX micelles was determined using a MTT cell proliferation kit (Biotium Inc.). MCF-7 cells were seeded into a 96-well tissue culture plate at a density of 10000 cells per well and were incubated at 37 °C with 5% CO2. The growth medium was replaced with fresh medium after 24 h. PBAE@UCNPs-DOX, free DOX or blank micelles were added to cells (six wells/sample). After 980-nm NIR irradiation (1 W cm-2, 5 min), cells were cultured for 48 h. The medium was removed and 200 μL DMSO was added to dissolve the formazan. The absorbance was recorded with an ELISA plate reader at a test wavelength of 570 nm (reference wavelength 630 nm). The cell growth inhibition of samples was calculated as eq 3: 𝐼sample ― 𝐼blank

(3)

Cell Viability (%) = 𝐼control ― 𝐼blank * 100%

where Isample, Iblank and Icontrol represent the intensity determined for cells treated with different samples, micelles solutions and for control cells (untreated), respectively. Cell Uptake and Intracellular Drug Distribution. The intracellular delivery of DOX was examined with confocal laser scanning microscopy by incubating MCF-7 cells with PBAE@UCNPs-DOX or free DOX with or without NIR (980 nm). MCF-7 cells were seeded at a density of 2 x 105 cells/slip in cover slips and cultured for 24 h. During the culture, cells were irradiated with NIR laser (980 nm) for 10 min (twice). The cells were washed with PBS, followed by adding 1 mL of 4% paraformaldehyde/PBS. Cell staining was carried out following the manufactures’ protocols, followed by cell imaging with confocal laser scanning microscopy. Pearson correlation coefficient was defined as eq 4: 𝑅(𝑟) =

∑𝑖(𝑆1𝑖 ― 𝑆1𝑎𝑣𝑒𝑟) ∙ (𝑆2𝑖 ― 𝑆2𝑎𝑣𝑒𝑟) ∑𝑖(𝑆1𝑖 ― 𝑆1𝑎𝑣𝑒𝑟)2 ∙ ∑ (𝑆2𝑖 ― 𝑆2𝑎𝑣𝑒𝑟)2

(4)

𝑖

where S1 is the intensity value of each pixel of the first channel, and S2 is the intensity value of each pixel of the second channel. S1aver and S2aver are the average values of pixel intensity of the first channel and the second channel, respectively. Upconversion

Luminescence

Microscopy.

Upconversion

luminescence

microscopy was rebuilt on an inverted fluorescence microscope (Nikon Ti-S) with an


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external CW 980-nm diode laser. MCF-7 cells were incubated with PBAE@UCNPs at 37 °C. Afterward, the cells were fixed with 2.5% formaldehyde (1 mL well-1) for 10 min at 37 °C, and then washed with PBS three times. A Mouse Model of Breast Cancer. All the animal experiments were performed following the guidelines approved by the Animal Care and Use Committee of CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety. Female Balb/c mice (4-5 weeks, 13 ± 2 g) were obtained from Beijing WeiTongLiHua Animal Co. Ltd. The subcutaneous breast cancer tumor model was established as the following procedure. Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate at 10 mL/kg. Aftereward, 107 4T1 cells were subcutaneously injected. Biodistribution of Drugs in Mice. When the tumor was palpable (about two weeks after tumor inoculation), intravenous injections of PBAE@UCNPs-DOX-Cy5 or free Cy5 (Ex/Em = 646/662 nm) were carried out. The tumor was exposed to NIR (980 nm, 1.5 W/cm2) for 10 min. At the time points of 1 h, 6 h, 24 h post injection, the fluorescence images of the living mice were taken by Lumina III in vivo imaging system. At 24 h post injection, tumors were excised and then imaged by an ex vivo imaging system after being washed twice with PBS. Antitumor Effect in Mice. Five days after 4T1 cell inoculation, intravenous injection of the drugs was performed through the tail veins of mice (n = 5 per group). The treatment was carried out twice a week for two weeks. The tumor region was irradiated with NIR (980 nm, 1.5 W/cm2, 10 min). The body weight and

tumor

volume were monitored every other day. Two days post the final administration, mice were sacrificed and tumors were dissected.

RESULTS AND DISCUSSION Synthesis and Characterization of PBAE Copolymers. For targeted drug delivery, it is expected that disassembly of nanocarriers occurs rapidly upon diverse stimuli. To this end, we designed and synthesized amphiphilic PEG-PBAE containing the lightresponsive moiety in each repeating unit of PBAE backbones. The light-sensitive



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polymer was synthesized by Michael-type reaction of three monomers, NPBMDA, dodecylamine and methoxy PEG acrylate. The Michael-type addition has been widely used for the polymerization of PBAE and poly(amido amine)s with advantages including the formation of degradable polymers backbones, mild reacting condition and versatile functionalization.25 To make amphiphilic PBAE, we adopted dodecylamine as hydrophobic side groups that would facilitate the subsequent hydrophobic interaction with oleic acid layer on the UCNPs. The PEG chain can dramatically enhance the stability of the nanocomposites in aqueous media.

Scheme 2. Synthesis routine of (2-nitro-1,3-phenylene) bis(methylene) diacrylate, light-sensitive PBAE and proposed degradation mechanism of PBAE in response to UV irradiation.

Table 1 Characterization of chemical structure of the PBAE copolymers. Sample

PBAE5000

PEG : ONB:

Yield

Mnb

dodecylamine

(%)

(kDa)

25%

7.54

Feed

Obtaineda

1/10/12

1/5/5


PDIb

CMCc (μg mL-1)

1.91

30

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PBAE2000

1/10/12

1/8/8

33%

6.48

1.38

39

a Determined by 1H NMR measurement. b Determined by GPC measurement. c Determined by fluorescence spectrometry using pyrene as probe.

A

B d,e

b a

O O

j k O

c

NO2

O

N O

f

n

O

i

O

O 133

g h

Figure 1.

(A) 1H-NMR of light-sensitive diacrylate monomer (1-nitro-2,5-phenylene)

bis(methylene) diacrylate (NPBMDA). (B) 1H-NMR spectrum of copolymer PEGPBAE in CDCl3.

1H-NMR

results indicated that structures of the PBAE were slightly different from

the predicted structures (Figure 1), which may result from the different reactivity of amine with acrylate in NPBMDA and methoxy PEG acrylate. Elevated reaction temperature was employed to increase the reactivity of amine groups in dodecylamine to obtain the grafted structure. The chemical structures were confirmed by calculating the integrals of peak g, peak h and peaks d, e in 1H-NMR spectra. The phenyl groups and the dodecyl groups had almost the same equivalent proportion, which suggested the successful synthesis of PBAE backbone. Each PEG chain was in correspondence with ~5 BAE repeat units, and the calculating molecular weight of copolymer was ~



7570 Da. GPC-light scattering was also employed to analyze the molecular weight and the polydispersity of the copolymer (Figure S1), which was in accordance with the result calculated from 1H NMR characterization. In PBAE2000 that was synthesized from PEG (Mn = 2 kDa), the degree of polymerization was higher than that of PBAE5000, and was attributed to the higher activity of amine group in PEG2000 (Table 1). All the data demonstrated the successful preparation of the copolymer with defined structure and moderate molecular weight distribution. The amphiphilic copolymer PBAE can self-assemble to form micellar nanoparticles in aqueous media. The morphology and the average size of the self-assembled nanoparticles were investigated by TEM and DLS. The average size analyzed by TEM (~60 nm) was slightly smaller than that determined by DLS (~80 nm), presumably because DLS determined the hydrodynamic radius of nanoparticles, instead of the dry nanoparticles observed by TEM. The stability of nanoparticles underwent high dilution was critical for the application of nanomedicines in vivo. The CMC value was defined as the concentration corresponding to crossing point of the tangent to rapidly changing portion of the curve with the horizontal tangent through the points at lower concentrations (Figure S2).26 The CMC of PBAE5000 and PBAE2000 was about 30 μg mL-1 and 39 μg mL-1, respectively (Table 1), indicating that the nanocomposites would remain stable upon extremely high dilution (e.g. after i.v. injection). We chose PBAE5000 in the following experiments. Preparation and Characterization of PBAE@UCNPs. Recently, UCNPs with various shapes, sizes, inorganic chemical components and architectures have been synthesized and explored for the broad applications to optogenetics, disease diagnosis and theranostics.27,

28

Due to radiationless deactivation, the content of Tm3+ in the

nanoparticles usually does not exceed 0.5%.29 As such, the content of UCNP in this study was set according to this threshold. The obtained UCNPs were coated by oleic acid for superior mono-dispersion in organic media, which is a common method for stabilization of metal and metal oxide nanoparticles. After addition of the synthesized PBAE amphiphilic copolymer, the dodecane chains were intercalated into the layer of oleic acid via hydrophobic interaction, while the PEG chains dispersed the


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nanoparticles in aqueous medium. The obtained water-soluble nanoparticles had a hierarchical surface structure denoted PBAE@UCNPs. Typical DLS results indicated a uniform and narrow size distribution of the PBAE@UCNPs micelles (Figure 2). As summarized in Table 2, the diameters of micelles were about 114.9 nm for PBAE@UCNPs and about 171.3 nm for DOX-loaded PBAE@UCNPs. TEM micrograph showed UCNPs had a size of about 30-40 nm (Figure 2). After selfassembled with copolymers, several UCNPs were clustered in one nanoparticle with a spherical morphology and moderate size distribution (Figure 2). The size change of micelles in response to 980-nm NIR light in pH 7.4 PBS buffer was also examined by DLS. Remarkably, both the size reduction and aggregation of the light-sensitive PBAE@UCNPs micelles were observed, as the appearance of multiple peaks upon NIR irradiation (Figure 2D). The new peaks that appeared after 980-nm NIR irradiation were most likely due to PBAE degradation that resulted in the formation of small fragments and loose micellar aggregation (Figure S3).

Figure 2. (A) Size distribution of PBAE micelles determined by DLS, and the insert graph is a typical TEM micrograph of PBAE micelles. Scale bar = 100 nm. (B) A typical TEM micrograph of oleic acid@UCNPs. Scale bar = 50 nm. (C) Size



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distribution of PBAE@UCNPs determined by DLS, and the insert graph is a typical TEM micrograph of PBAE@UCNPs nanocomposites. Scale bar = 100 nm. (D) Size change of light-sensitive PBAE@UCNPs micelles in response to 980-nm NIR light determined by DLS.

TGA analysis was conducted to study the composition of OA-capped UCNPs nanocomposites and PBAE@UCNPs micelles. The surface of as-prepared UCNPs was coated by an oleate layer as the surfactant. As shown in Figure S4, the OA-capped UCNPs showed the weight loss was ~17 wt%. This indicated that the weight loss of OA-layer covered on the surface of UCNPs was approximately 17 wt%. The TGA curve of PBAE@UCNPs micelles displayed two stages of weight loss in the range of room temperature to 900 °C. The first weight-loss stage was about 10 wt% in the range of 25–200 °C, which came from the water loss. The second weight-loss stage was about 69 wt% in the range of 200–900 °C, which may result from the combustion of the PBAE and OA layer. The total weight loss of the PBAE@UCNPs was approximately 79 wt%, which was much higher than that of the OA-capped UCNPs. These results suggested that PBAE copolymer had been assembled on the surface of OA-capped UCNPs.

Table 2 Characterization of PBAE@UCNPs and PBAE@UCNPs-DOX.

Sample

PBAE@UCNPs PBAE@UCNPsDOX

Diametera (nm)

PDIa

Drug

Drug

CMCb

Loading

Loading

(mg/mL)

Contentc

Efficiencyc

(%)

(%)

114.9

0.172

0.03

N/A

N/A

171.3

0.178

N/A

11

75

a Size and PDI of PBAE@UCNPs and PBAE@UCNPs-DOX were determined by DLS. b Determined by fluorescence spectrometry using pyrene as probe. c Determined by UV-vis absorbance measurement.


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As the optical property is crucial for this nanosystem, the light-responsive behavior of PBAE copolymer in cholorform solution (0.2 mg mL-1) was investigated by UV−vis spectroscopy (Figure 3A). A characteristic absorbance peak at ~290 nm increased in a function of UV irradiation time, suggesting that o-nitrobenzyl groups were gradually cleaved from copolymers by UV irradiation. Figure 3B exhibited the UCL spectra of the UCNPs before (green line) and after (pink line) the modification with PBAE, and absorption spectrum of the PBAE (blue line). When irradiated with 980-nm laser, the UCNPs emitted at several wavelengths within the UV−vis region. The corresponding typical peaks also appeared in the sample of PBAE@UCNPs, indicating UCNPs were successfully entrapped inside the nanoparticles. It was notable that the characteristic absorption region of PBAE was approximately 250-400 nm, and one characteristic emitting peak of UCNPs was at ~350 nm. We thus speculated that the cleavage of onitrobenzyl groups from the backbones of the PBAE could be achieved by emission of UCNPs at 350 nm. As shown in Figure 3C, the absorption peaks ranging from about 250 nm to 350 nm increased along with NIR irradiation time, which was similar to the results of Figure 3A. These results showed that the nitrosobenzaldehyde produced by UV cleavage could also be seen in PBAE@UCNPs upon NIR, suggesting UCNPsmediated conversion of NIR to UV and cleavage of PBAE.



Figure 3. (A) UV-vis spectra of PBAE polymer upon UV at 365 nm. (B) Emission spectra of the composites upon NIR. (C) Time-resolved UV-vis spectra of PBAE@UCNPs upon NIR. (D) Zeta potentials of PBAE@UCNPs at different pH values with or without NIR irradiation (λ = 980 nm, 2 W cm-2 for 10 min).

To evaluate the pH response of PBAE@UCNPs, we investigated the zeta potentials of nanoparticles dispersed in the buffer solutions with different pH values (pH 7.4, 6.5, 5.5, 4.5) (Figure 3D). These pH values represent the characteristic pH range from the physiological environment, tumor microenvironment, to endo/lysosomal milieu. The zeta potentials of PBAE@UCNPs changed from slightly negative (–2.94 mV) at pH 7.4 to positive (+1.3 mV) at pH 6.5 (Figure 3D), indicating that the pKb value of nanoparticles was ~ 6.5. This result was in agreement with the buffer capacity test of PBAE polymers as shown in Figure S5. The zeta potentials of the nanoparticles continuously increased to +5.76 mV at pH 5.5 and +7.29 mV at pH 4.5 (Figure 3D). In


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addition, the zeta potentials of the nanoparticles upon NIR irradiation were also determined. The zeta potentials of PBAE@UCNPs changed from slightly positive (+1.08 mV) at pH 7.4 to +6.57 mV at pH 6.5, +10.1 mV at pH 5.5, and +13.1 mV at pH 4.5 (Figure 3D). These results suggested that PBAE@UCNPs were pH-sensitive. Moreover, the results also indicated that the surface of nanoparticles exhibited relatively more number of positive charges upon NIR irradiation, which would facilitate the endo/lysosomal escape. In Vitro Drug Release of DOX-Loaded PBAE@UCNPs. DOX-loading content of PBAE@UCNPs-DOX (feeding weight ratio of PBAE:UCNPs:DOX=5:1:1) was ~8%, and DOX loading efficiency was ~57%. NIR- and pH-sensitive DOX release from the PBAE@UCNPs-DOX micelles was investigated by UV. Figure 4A shows the cumulative release profiles of PBAE@UCNPs-DOX micelles at different pH values (5.0 and 7.4) with or without NIR. About 31% of the encapsulated DOX at pH 7.4 was slowly released in 72 h. At pH 5.0, DOX release from the micelles was increased, and about 53% of DOX was released in 72 h (Figure 4A). As expected, DOX release from PBAE@UCNPs-DOX micelles upon NIR irradiation was accelerated at pH 7.4 with 19 % DOX released in the first 24 h and up to 39 % released in 72 h (Figure 4A). At pH 5.0, NIR induced approximately 31% of DOX release in the first 24 h and up to 66% DOX was released in 72 h. These results suggested that DOX release from the nanoparticles was both NIR- and pH-sensitive.

Figure 4. (A) Cumulative DOX release of PBAE@UCNPs-DOX micelles at different pH values with or without NIR irradiation. (B) Cell viability of PBAE@UCNPs micelles at pH=7.4 with or without NIR. Data are presented as mean ± SD (n = 6). (C)



Cell viability of 1) free DOX, 2) PBAE@UCNPs, 3) PBAE@UCNPs with NIR, 4) PBAE@UCNPs-DOX, and 5) PBAE@UCNPs-DOX with NIR at pH 7.4. X-axis indicates DOX concentrations. PBAE@UCNPs micelles with or without NIR were used as blank controls to compare the cytotoxicity of DOX-loaded nanoparticles and corresponding nanocarriers. Data are presented as mean ± SD (n = 6).

Cytotoxicity of PBAE@UCNPs-DOX. MCF-7 cells were used to evaluate the cytotoxicity of the nanoparticles by using MTT assay. Cells were treated by free DOX, PBAE@UCNPs or PBAE@UCNPs-DOX micelles with or without NIR. As shown in Figure 4B and 4C, PBAE@UCNPs micelles and their degradation products upon NIR showed minimal cytotoxicity, indicating these nanocarriers were generally biocompatible. The half-inhibitory concentration (IC50) of free DOX was about 2 μg mL-1, while IC50 of the equivalent DOX concentration for PBAE@UCNPs-DOX micelles at pH 7.4 was about 54 μg mL-1 (Figure 4C). Upon NIR irradiation (λ = 980 nm, 2 W cm-2, 10 min), PBAE@UCNPs-DOX showed remarkably enhanced effects of cell growth inhibition. For example, the percentage of cell viability for PBAE@UCNPs-DOX (equivalent DOX concentration of 1 μg mL-1) was reduced from ~88% to ~18 % by NIR (Figure 4C). The enhanced cellular inhibition of PBAE@UCNPs-DOX by NIR may be due to NIR-induced fast release of DOX from the micelles, which is in line with the results shown in Figure 4A. Cell Uptake and Intracellular Drug Distribution. The intracellular delivery of DOX was examined with confocal laser scanning microscopy and upconversion luminescence microscopy in MCF-7 cells. As shown in Figure 5, UCNPs displayed blue fluorescence in cells upon NIR, which is in agreement with the results determined in vitro by fluorescence spectrometer. From the overlaid images, the numbers of cells containing UCNPs were increased over time (Figure 5), suggesting time-dependent cellular uptake of UCNPs. In addition, we asked whether UCNPs were still associated with PBAE inside cells. Cy5 dye was conjugated to PBAE for cell imaging. As shown in Figure S6, blue fluorescence of UCNPs was generally colocalized with red fluorescence of Cy5-PBAE at 1 h and 3 h. After 6-h incubation, separation of blue


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fluorescence from red fluorescence was found (Figure S6), which might be resulting from the slow hydrolysis of PBAE in cells.

Figure 5. The cellular uptake images of PBAE@UCNPs by upconversion luminescence microscopy. To study the intracellular distribution of the nanoparticles, MCF-7 cells co-cultured with Cy5-labeled PBAE@UCNPs micelles were examined by confocal laser scanning microscopy. Lysosomes and mitochondria displayed green fluorescence after the cells were stained with Lysotracker and Mitotracker, respectively, while Cy5-labeled PBAE@UCNPs displayed red fluorescence. After incubation for 2 h, Cy5PBAE@UCNPs with or without NIR irradiation were both found colocalized with lysosomes at low levels (Pearson correlation coefficient = 0.33 (NIR) and 0.47 (No NIR) by Image J) (Figure 6A), suggesting the lysosomal escape of protonated nanoparticles. It is crucial for intracellular drug delivery that the nanomedicines can avoid the degradation and exocytosis by lysosomes.30 With the aid of NIR, more nanoparticles



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were released from lysosomes (Figure 6A), presumably because of NIR-facilitated protonation of the nanoparticles as shown in Figure 3D. Besides lysosomes, the colocalization of mitochondria with the nanoparticles was also analyzed. We found that Cy5-PBAE@UCNPs

nanoparticles

were

substantially

colocalized

with

the

mitochondria (Pearson correlation coefficient = 0.85 (NIR) and 0.78 (no NIR)) (Figure 6B). As we know, the mitochondrion-targeting nanoparticles typically possess two characteristic properties, i.e., high hydrophobicity and positive charges.18 According to the results of buffer capacity and zeta potentials (Figure 3D and Figure S5), we speculated that NIR-mediated removal of PEG would expose the amine groups of PBAE that were protonated in endo/lysosomes. In addition to positive charges on the nanoparticles, the hydrophobicity of UCNP and the alkyl layer of PBAE@UCNPs synergistically contributed to the colocalization of PBAE@UCNPs with mitochondria. Mitochondria are one of the most important cellular organelles for maintaining cell activity, and DNA can be also found in mitochondria.31 As a DNA-binding drug, DOX may be able to interact with mitochondrial DNA and consequently interfere with mitochondrial function. Therefore, mitochondrion-targeting delivery of DOX to tumor cells may enhance the therapeutic efficacy of DOX, and development of mitochondrion-targeting nanocarriers is necessary. Nevertheless, DOX-induced mitochondrial damage in healthy cells could elicit severe adverse effects, including cardiomyopathy.32 To minimize the side effects, excellent tumor targeting of the nanocarriers might be a prerequisite to targeted delivery of DOX to mitochondria.


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Figure 6. Confocal laser scanning microscopy for intracellular distribution of Cy5PBAE@UCNPs in MCF-7 cells. Cells were incubated with Cy5-PBAE@UCNPs with/without NIR irradiation for 2 h at 37 °C, followed by staining with Lysotracker or Mitotracker. (A) The colocalization of Cy5-PBAE@UCNPs with lysosomes. (B) The colocalization of Cy5-PBAE@UCNPs with mitochondria. The scale bar is 10 μm.

To examine intracellular drug delivery, MCF-7 cells were treated with free DOX or PBAE@UCNPs-DOX micelles with or without NIR irradiation. After immunostaining with F-actin, the intracellular drug distribution was observed by confocal laser scanning microscopy, and the fluorescence intensity of intracellular DOX was analyzed. As



shown in Figure 7, free DOX was mainly distributed in the nuclei of MCF-7 cells at three time points. The intensity of intracellular DOX fluorescence was increased over time. For the PBAE@UCNPs-DOX, several red fluorescent dots were seen in cells after only 15 min of incubation with the micelles (Figure 7). After 2-h and 24-h incubation, more red dots were found both in cytosol and in nuclei, indicating sustained release of DOX from the nanoparticles. Upon NIR, the intracellular delivery of DOX was considerably enhanced (Figure 7), according to the quantitative analysis of the intracellular DOX fluorescence in three groups (Table S1). Furthermore, the colocalization of DOX fluorescence with mitochondria was also validated by confocal laser scanning microscopy for NIR-irradiated PBAE@UCNPs-DOX in MCF-7 cells (Figure S7). Therefore, NIR/pH dual-sensitive nanocarriers reported in this study may provide a superior tool for intracellular delivery of DOX.

Figure 7. Confocal images of MCF-7 cells treated with free DOX (a, d, g), PBAE@UCNPs-DOX micelles (b, e, h) and PBAE@UCNPs-DOX micelles with NIR (c, f, i) for 15 min (a, b, c), 2 h (d, e, f) and 24 h (g, h, i). The images of DOX (red), nuclei (blue) and F-actin (green) were analyzed. The magnification is 40 times.

In Vivo Drug Distribution and Antitumor effects in mice. To examine drug distribution in vivo, Cy5 was conjugated to the nanodrugs via amide reaction. After


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intravenous injections of free Cy5 or Cy5-labeled nanoparticles, the fluorescence at tumor region was examined with in vivo imaging system. As shown in Figure 8A, the nanoparticles accumulated in tumors over time, whereas little free Cy5 dye could be seen in tumors even at 24 h post injection. Upon NIR irradiation, slightly more nanoparticles accumulated in tumors (Figure 8A). This was presumably ascribed to NIR-induced hyperthermia and consequently dilated tumor blood vessels, as suggested by previous studies.10, 11 At 24-h post injection, tumors and other organs were excised and DOX fluorescence in tumors was examined. As shown in Figure 8B, DOX mainly accumulated in tumors, while little DOX was found in healthy organs. Since NIR could increase DOX release from the nanocarriers (Figure 4A), DOX accumulation in tumors was considerably enhanced by NIR (Figure 8B). These results suggested that NIR could facilitate the tumor targeting of DOX in mice. We next examined the antitumor effects of the nanodrugs in tumor-bearing mice. As shown in Figure 8C, the nanodrugs with NIR could potently inhibit tumor growth in mice, whereas the body weight of mice remained almost unaffected (Figure 8D). After the mice were sacrificed, tumors were excised and weighed. As shown in Figure 8E, tumors from PBAE@UCNPs-DOX+NIR group were significantly smaller than tumors from the control groups (Figure 8F). These results indicated a potent antitumor effect of PBAE@UCNPs-DOX in mice with the aid of NIR. Recently, Dai et al.33 have suggested that nanodrugs might be mainly taken up by tumor-associated macrophages. Consequently, decrease in tumor weights might be ascribed to the death of tumorassociated macrophages, instead of cancer cells.33 Further investigation is required to distinguish the killing of cancer cells from that of tumor-associated macrophages by the nanodrugs.



Figure 8. (A) Images of tumor-bearing mice treated with 1 = free Cy5; 2 = PBAE@UCNPs-DOX-Cy5; 3 = PBAE@UCNPs-DOX-Cy5 with NIR. The white


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circles highlight the region of tumors. (B) Representative ex-vivo fluorescence images of tumors and major organs of mice upon different treatments. (C) Tumor growth profiles and (D) body weight of the mice under different conditions. (E) Photographs of tumors from Ⅰ) PBS+NIR, Ⅱ) Free DOX+NIR, Ⅲ) PBAE@UCNPs-DOX, Ⅳ) PBAE@UCNPs-DOX+NIR. (F) Average weights of tumors collected from mice at the end of various treatments. * P < 0.05, ** P < 0.01.

CONCLUSION In summary, we have developed NIR/pH dual-responsive nanocarriers for intracellular DOX delivery. With the aid of UCNPs, NIR induced the degradation of the micelles, resulting in downsized nanoparticles and enhanced cellular uptake. When the nanodrugs were uptaken in endo/lysosomes, the protonation of amino groups increased the positive charges on the surface of the nanoparticles, which facilitated the lysosomal escape of the nanoparticles and subsequent mitochondrial targeting. This work provides a promising nanoplatform that combines light-sensitive polymers and upconversion nanomaterials for enhanced intracellular drug delivery.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: GPC curve of PEG5000-PBAE copolymer, the critical micelle concentration (CMC) of PBAE5000 micelles, TEM image, TGA curves of the OA-capped UCNPs nanoparticles and PBAE@UCNPs nanocomposites, titration curve of PBAE5000 copolymer, cellular uptake images of Cy5-PBAE@UCNPs, colocalization of DOX with mitochondria, quantitative analysis of intracellular DOX fluorescence (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]

Author Contributions §K.W.

and M.Z. contributed equally to this work.

Notes J.C., W.K. Y.H. applied a China invention patent related to near infrared-responsive nanocarriers.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21304098, 21574136, 21390411), CAS Youth Innovation Promotion Association Program (2015008), Hundred Talents Program of CAS.


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For Table of Contents Use Only

Near Infrared/pH Dual-Sensitive Nanocarriers for Enhanced Intracellular Delivery of Doxorubicin Kaikai Wen,†,‡,§ Mengxue Zhou,†,‡,§ Huiru Lu,† Ying Bi,† Lifo Ruan,†,‡ Jun Chen*,†,‡, and Yi Hu,*,†,‡


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pH Dual-Sensitive Nanocarriers for Enhanced

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