Phototriggered N2-Generating Submicron Particles for Selective

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Photo-triggered N-generating submicron particles for selective killing of cancer cells Huiying Li, Wenbo Zhang, Ning He, Weijun Tong, and Changyou Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16362 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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ACS Applied Materials & Interfaces

Photo-triggered N2-generating submicron particles for selective killing of cancer cells Huiying Li,† Wenbo Zhang,† Ning He, † Weijun Tong*,† Changyou Gao*† MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University in Hangzhou, 310027, China.

Abstract: Killing of cancer cells by applying mechanical disruption is an appealing emerging strategy for cancer treatment in recent years. In this study, photo-responsive submicron particles based on diazo-resin (DZR) which are able to release N2 under UV irradiation were prepared through a polyamine-salt aggregation method. After surface modification with hyaluronic acid (HA), the particles could be internalized selectively by cancer cells and mainly located in lysosomes after 6 h incubation. The viability of cancer cells decreased obviously after cocultured with photo-responsive particles and UV irradiation due to the integrity damage of lysosomes by photo-triggered N2 generation and the subsequential increased reactive oxygen species.

Keywords: Diazo-resin; N2; Photo-responsive; Particles; Targeted delivery

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1. Introduction Cancer has been a major threat to human beings for a long time. Currently, chemotherapy is an important and effective strategy, in which better therapeutic effect can be achieved by combining delivery system of colloidal particles.

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However, cancer drug resistance and some severe side

effects may be induced after the cancer drugs are repeated administrated, which may significantly limit the efficacy of chemotherapy.

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Therefore, it is important to develop new

strategies for cancer treatment. Recently, utilizing the physicochemical property change of materials in specific location to disrupt cancer cells has been regarded as a potential way to treat cancers. For example, researchers tried to achieve cancer cells killing by designing the materials which can release gas under stimulation.

5, 6

Xia and co-workers

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fabricated liposomes

containing ammonium bicarbonate (NH4HCO3). After internalization, the liposomes could be thermally triggered to generate CO2 bubbles. And the transient cavitations of these bubbles could produce violent force on lysosomes to disrupt the membranes and release proteolytic enzymes into the cytosol, which finally result in cell necrosis. Zhao and co-workers 6 utilized the energy transfer from upconversion nanoparticles to photolytic [NH4][Fe4S3(NO)7] (Roussing’s black salt) to produce NO under 980 nm laser irradiation, and found that high concentration of NO can kill cancer cells directly while low concentration of NO combined with chemotherapy can overcome multi-drug resistance. Compared with temperature, light has the advantage of real-time high precision spatiotemporal control. Diazo-resin (DZR) is one of the classic sensitizers. Due to the instability of diazo group, DZR is sensitive to UV irradiation, and can release N2 under suitable conditions. Meanwhile, due to its positive charges, DZR can interact with molecules containing groups with negative charges such as carboxyl and sulfonic group via electrostatic interaction


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or species containing hydroxyl

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group via hydrogen bonding, thus DZR based photo-responsive thin films have been fabricated through layer-by-layer method. 7, 8 Due to the transformation of ionic bond to covalent bond after irradiation, stability of the multilayer film increased obviously. 7, 8 DZR has also been introduced into fabrication of colloidal particles. 9-11 Sukhorukov and co-workers

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prepared DZR-based

multilayer microcapsules through layer-by-layer assembly. Upon irradiation, the shell could be covalently cross-linked and encapsulation of small molecules was achieved. However, to the best of our knowledge the effect of photo-triggered N2 releasing from DZR-based colloidal particles inside cancer cells has not been explored. Hyaluronic acid (HA) is a natural biomacromolecule and widely exists in extracellular matrix, and its receptor CD44 is over-expressed by many cancer cells. 13, 14 Thus, colloidal particles surface modified with HA can achieve the targeting to cancer cells. 13 In this study, DZR-based photo-responsive particles which can generate N2 upon UV irradiation are prepared via a polyamine-salt aggregate method.

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Then the particle surface is

modified by HA to realize the selective internalization of particles by cancer cells. Following irradiation-triggered N2 generation inside the cells, the cancer cells are killed (Scheme 1). The mechanism of cell death is also investigated and proposed.


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Scheme 1. A schematic illustration showing the fabrication and UV response of (DZR-Cit)/PSS/PAH/HA particles and how the cancer cell is killed by their intracellular UV-triggered N2 release.

2. Experimental Section 2.1 Materials Poly(allylamine hydrochloride) (PAH, Mw ~ 58 kDa), poly(sodium 4-styrenesulfonate) (PSS, Mw ~ 70 kDa), acridine orange (AO), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2yl)-2,3-diphe-nyltetrazodium bromide (MTT) were purchased from Sigma-Aldrich. Cyclohexyl isocyanide

and

fluoresceinamine

isomer

(AF)

were

purchased

from

Acros.

4-

Diazodiphenylamine sulfate was obtained from TCI. Lyso Tracker®Red and Rhodamine 123 were obtained from Invitrogen. Paraformaldehyde, zinc chloride (ZnCl2), sulfuric acid and trisodium citrate (Na3Cit) were purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium hyaluronate (HA, Mw ~ 150 kDa) was purchased from Lifecore Biomedical. All chemicals were


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used as received. Reactive oxygen species assay kit (containing 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 mM) and Ros up 50 mg mL-1) was purchased from Beyotime Biotechnology. DZR was synthesized according to a previously reported method.

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The water

used in all experiments was prepared via a Millipore Milli-Q purification system and had a resistivity higher than 18 MΩ cm-1. 2.2 Fabrication of DZR-based photo-responsive submicron particles DZR-based photo-responsive particles were prepared by mixing DZR solution (10 mL, 8 mg mL-1) and Na3Cit solution (25 mL, 28.83 mM) under ultrasonication for 10 s. After 1 min, PSS solution (1 mL, 2 mg mL-1) was added into the mixture under ultrasonication. The formed (DZRCit)/PSS particles were washed with water three times through centrifugation. Then, (DZRCit)/PSS particles (0.5 mL) were sequentially incubated in PAH (5 mL) and HA (5 mL) or AFlabeled HA (HA-AF

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) solution (1 mg mL-1, 0.5 M NaCl) for 15 min under constant shaking.

The excess polyelectrolytes were removed by three centrifugation/washing cycles with water. Finally, (DZR-Cit)/PSS/PAH/HA or (DZR-Cit)/PSS/PAH/HA-AF particles were obtained and dispersed in water, and stored at 4 ˚C in dark. 2.3 Photo-response of (DZR-Cit)/PSS/PAH/HA particles To check the photo-response of the obtained particles, (DZR-Cit)/PSS/PAH/HA particles suspension (300 µL, 10 µg mL-1) was placed in 96-well plates and irradiated by light (365 nm, 30 mw cm-2, 3 min). The UV-vis spectra before and after irradiation were measured. In order to better mimic the real intracellular response of particles, the particles were further dispersed in a cell lysis solution. In detail,

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HepG2 cells were dispersed in phosphate-buffered saline (PBS,

0.1 M, pH = 7.4) with a concentration of 1×106 cells mL-1. After ultrasonication for 20 min, the solution was centrifuged and the supernatant was collected, which was treated as a cell lysis


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solution. Dispersed in the cell lysis solution (10 µg mL-1), the particles suspension was irradiated (365 nm, 30 mw cm-2, 3 min) and the UV-vis spectra were measured. Meanwhile, to further confirm the N2 generation ability of (DZR-Cit)/PSS/PAH/HA particles, the digital images of particles suspension (3 mg mL-1) in cuvette before and after irradiation were also taken. 2.4 Cell culture experiments 2.4.1 Cell culture HepG2 and NIH 3T3 cells were purchased from the Cell Bank of Typical Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured with a regular growth medium consisting of high-glucose Dulbecco's modified eagle media (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U mL-1 penicillin, and 100 µg mL-1 streptomycin, and incubated in a 5% CO2 incubator at 37˚C and 100% humidity. 2.4.2 Intracellular distribution of (DZR-Cit)/PSS/PAH/HA-AF particles HepG2 cells were first seeded on a 20-mm glass bottom cell culture dish with a density of 5 ×104 cells per well and incubated overnight at 37 ˚C. Then, (DZR-Cit)/PSS/PAH/HA-AF particles (50 µg) were added into the medium. After 6 h co-culture, the cells were washed with PBS three times, and then Lyso Tracker®Red was added (diluted 1000 times by cell culture medium). The cells were cultured in dark for 30 min. After removal of cell culture medium, the cells were washed with PBS three times and observed under confocal laser scanning microscopy (CLSM). 2.4.3 Intracellular response of (DZR-Cit)/PSS/PAH/HA particles and its influence on cell viability The cell viability before and after intracellular photo-triggered response of particles were determined by MTT assay. Briefly, 1×104 HepG2 or NIH 3T3 cells in 100 µL medium were


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seeded into a well of 96-well plate and incubated overnight. The experimental groups were divided into five groups, UV: irradiation only (365 nm, 30 mw cm-2, 3 min, same for all the groups with irradiation); 1: particles only; 2: irradiated immediately after adding particles and then co-cultured for 6 h; 3: co-cultured with particles for 6 h, and then irradiated; 4: co-cultured with particles for 6 h, washed off free particles, and then irradiated. After washed with PBS three times, the cells were maintained in fresh medium containing 0.5 mg mL-1 of MTT for another 4 h at 37 ˚C. The dark blue formazan crystals generated by the mitochondria dehydrogenase were dissolved with DMSO (150 µL), and the absorbance at 570 nm was measured by a microplate reader (Model 680, BioRad). The data were normalized to that of the particles and irradiation-free cells. 2.4.4 Measurement of intracellular reactive oxygen species (ROS) The oxidation-sensitive probe DCFH-DA was employed to determine the intracellular ROS level. 19 HepG2 cells were seeded on 24-well plates at a density of 8 × 104 cells per well and allowed to adhere for 16 h. The cells treated with Ros-up reagent (4 µg mL-1) for 30 min and the untreated cells were used as positive and negative controls, respectively. Other samples were described as below, UV: irradiation only (365 nm, 30 mw cm-2, 3 min, same for all the groups with irradiation); particles: co-cultured with particles for 6 h without irradiation; particles + UV: co-cultured with particles for 6 h, washed off free particles, and then irradiated. All groups were then incubated with serum-free media containing 10 µM DCFH-DA at 37 ˚C for 30 min in dark. Then the cells were washed with PBS, trypsinized, collected and analyzed by flow cytometry (FACS caliber, BD) at an excitation wavelength of 488 nm. 2.4.5 Integrity characterization of lysosomes after intracellular response of (DZRCit)/PSS/PAH/HA particles


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HepG2 cells were first seeded on a 20-mm glass bottom cell culture dish at a density of 5 × 104 cells per well and incubated overnight at 37 ˚C. Then, the samples were divided into four groups. A: control; B: only irradiation (365 nm, 30 mw cm-2, 3 min, same for all the groups with irradiation); C: co-culture with particles 6 h (10 µg PAH/HA/(PSS DZR-Cit) particles per 1×104 cells); D: co-culture with particles for 6 h (10 µg PAH/HA/(PSS DZR-Cit) particles per 1×104 cells), washed off free particles, and then irradiated. After washed by PBS, the samples were incubated with 6 µM AO in cell culture medium for 15 min in dark. After washed with PBS for three times, the cells were observed under CLSM (Excitation wavelength 488 nm; emission wavelength 515-545 nm (Green) and 610-640 nm (Red)). 2.5 Characterizations The UV-vis spectra were measured with a Shimadzu UV2550 spectrophotometer. Scanning electron microscopy (SEM) images were recorded on a field emission SEM instrument (HITACHI S-4800) at an acceleration voltage of 3 kV. A drop of particles suspension was placed on a silicon wafer, and then dried in air overnight. Transmission electron microscopy (TEM) images were recorded by a JEM-1230 TEM instrument at an acceleration voltage of 120 kV. A drop of sample suspension was placed onto a carbon film-coated copper grid, and dried naturally. Confocal laser scanning microscopy (CLSM) images were taken by TCS SP5 CLSM (Leica, Germany) equipped with a 20×/0.7 NA immersion objective. 2.6 Statistical analysis All values are presented as mean ± standard deviations (SD). Statistical analyses were applied using the Student’s t-test and one-way analysis of variance to determine statistical significance (p < 0.05).


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3. Results and discussion 3.1 Fabrication of photo-responsive (DZR-Cit)/PSS/PAH/HA particles DZR was synthesized via a condensation reaction between 4-diazodiphenylamine sulfate and parafoemaldehyde 16 (Figure S1A), which showed good solubility with an average molecular weight (Mn) of 2.5 kDa and PDI of 1.22. It was completely decomposed within 40 s (Figure S1B), producing N2 bubbles in the solution (Figure S2). Please see support information (SI) for more details. By simply mixing a cationic polyamine with a multivalent anionic salt in aqueous solution at ambient conditions, polyamine–salt aggregates (PSA) can be formed and further transformed into colloidal particles with controlled structures and functions.

15, 20

Herein, the positively

charged DZR was interacted with trivalent citric acid (Na3Cit) to result in aggregates. To overcome their poor stability,

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another layer of PSS was adsorbed atop the particle surface,

obtaining the (DZR-Cit)/PSS particles. Furthermore, a bilayer of PAH/HA was assembled to achieve the selectivity of particles for cancer cells. The assembly was monitored by the change of zeta-potential (Figure S3). The negatively charged (DZR-Cit)/PSS particles became positive after PAH assembly. Assembly of HA decreased the zeta-potential, but the particle surface remained positive. After incubated in cell culture medium containing 10 % FBS, the particles turned to be slightly negative due to the adsorption of proteins. Furthermore, the bright green fluorescence emitting from (DZR-Cit)/PSS/PAH/HA-AF also confirmed the successful assembly of HA as well (Figures S4 and S5 for details). By using the fluorescence calibration curve, the amount of HA on the particles was determined to be about 13% (w/w). And after incubation in cell culture medium for 6 h, the loss of HA was only 10% of the assembled amount (Please see SI for experimental details). Hence the HA modification was stable under cell culture conditions.


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The (DZR-Cit)/PSS/PAH/HA particles had good colloidal stability as well. After co-incubation in cell culture medium containing 10% or 50% fetal bovine serum (FBS) at 37 oC for 6 h, the appearance of the size and zeta potential of the particles (Table S1) were not changed significantly, suggesting no obvious aggregation or disruption. Elemental analysis (Table S2) revealed that the weight ratio of DZR in

the particles was 31% according to the percentage of N element. Therefore, 1 g (DZRCit)/PSS/PAH/HA particles are estimated to generate about 33 mL N2 maximally. The particles in a dry state were spherical with an average size of about 700-800 nm. (Figure 1A, B). Their surface was quite rough (Figure 1A, inset). The rough surface is beneficial for their cellular uptake by cells because the particles surface roughness can promote their adhesion on the cell membrane and the engulfment likely due to the large interaction area and adhesive strength between the particles and biomembranes.22-24The particles had a solid internal structure and a thin film

coating on the surface, demonstrating the successful assembly of multilayers (Figure 1B). UVirradiation did not bring obvious change in size and morphology of the particles (Figure 1C) as reported.

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After irradiation in water or cell lysis solution, the peak at about 370 nm almost

disappeared (Figure 2A), which is in accordance with the decomposition of DZR. Bubbles were floating on the surface or adhered on the cuvette inner wall as well (Figure 2B, indicated by arrows). The photo-triggered N2-generation ability of particles was also evaluated by ultrasound imaging. The particles were dispersed in the cell lysis solution in an Ependorf tube to mimic the intracellular environment. No gas bubbles were observed before light irradiation, while a large number of bubbles appeared when the sample was irradiated (Figure S6). The photo-response of (DZR-Cit)/PSS/PAH/HA particles was also confirmed by FTIR (Figure S7，Please see SI for more details), demonstrating the transformation of electrostatic interaction into covalent bond as a consequence of high reactivity of the generated carbocations (Figure 2C). Hence, the DZR in the particles remains the photo-responsive characteristic and still can generate N2, and the


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intracellular species such as proteins and enzymes do not interfere with the photo-responsive property of such particles.

Figure 1. SEM (A, C) and TEM images of (DZR-Cit)/PSS/PAH/HA particles before (A, B) and after (C) UV irradiation (365 nm, 30 mw cm-2, 3 min). The insets in A and C are the magnified images of the particles.

Figure 2. (A) The UV-vis spectra of (DZR-Cit)/PSS/PAH/HA particles in different media before and after UV irradiation for 3 min. H2O-0, H2O-3 means irradiation of particles incubated in water for 0 or 3 min, respectively. Cell lysates-0, cell lysates-3 means irradiation of particles incubated in cell lysis solution for 0 or


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3 min , respectively. (B) Digital images of particle suspension before (left) and after irradiation (right). The arrows indicate the bubbles floating on the surface or adhered on the cuvette inner wall. (C) Scheme representation of chemical structure changes of particles upon UV irradiation.

3.2 Cancer cell targeting The particles with cancer cell targeting ability can actively distinguish cancer cells from healthy ones, thus greatly enhance their cellular uptake in cancer cells, improve the therapeutic effect, and reduce side effects. HA can integrate with receptors over-expressed by many cancer cells, thus can be used as a targeting ligand to cancer cells.

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To demonstrate the targeting

delivery property, carcinoma HepG2 cells and normal NIH 3T3 cells were co-cultured with the (DZR-Cit)/PSS/PAH/HA-AF particles, respectively. As shown in Figure S4, after 6 h, obvious green fluorescence was observed in HepG2 cells due to the high level of CD44 receptors on their surface. As a sharp contrast, almost no fluorescence could be seen on the NIH 3T3 cells, demonstrating low cellular uptake of the particles. In order to verify the selective receptor mediated internalization of HA-coated particles, a blocking control study was performed. HepG2 cells were co-incubated with the particles and free HA. The free HA molecules can block the CD44 receptors expressed on the cell surface. In such a case, the internalization of particles by HepG2 cells was significantly decreased (Figure S5), revealing that the strong targeting ability of the particles is attributed to the specific ligand-receptor interaction. 3.3 Intracellular photo-response and cytotoxicity After internalization, most substances are transported to lysosomes first,

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and then resided

in other cytocompartments or cytosols depending on the properties of materials. The colocalization study showed that the green and red fluorescence were mostly overlapped (Figure 3), displaying that after co-culture for 6 h most of the particles have been transported into


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lysosomes. Before reaching lysosomes, the particles may be transported via endocytic vesicles. The submicron particles (700-800 nm) are unlikely to go into organelles with smaller size, such as mitochondria and ribosomes. Very few particles may exist in cytosol due to escape from lysosomes. The photo-responsive (DZR-Cit)/PSS/PAH/HA particles thus can release N2 inside lysosomes upon UV irradiation. As shown in Figure 4A, B, the viability of cells only treated with particles or UV irradiation showed no obvious difference. On the contrary, when irradiated after co-culture with the particles for certain time, the viability of cancer cells decreased significantly depending on the feeding dose and incubation time. However, if the cells were irradiated immediately after addition of particles, no obvious cell death was found (Figure 4A, B, Group 2). Moreover, when the incubation time was prolonged to 6 h (and hence the particles were largely internalized), removal of the non-internalized particles before UV irradiation or not had no obvious difference on the cell viability (Figure 4A, B, Groups 3 and 4). These results demonstrate that only those particles being internalized into cells and irradiation under UV can result in cell death as a result of N2 release. As a sharp contrast, when NIH 3T3 cells were similarly incubated with the particles (10 µg particles per 1×104 cells) for 6 h and irradiated with UV light, the cell viability had no obvious difference from other groups (Figure 4C), which is in good agreement with the low internalization of particles by normal cells (Figure S4) and thereby no obvious gas generation inside cells.


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Figure 3. CLSM images of HepG2 cells after being incubated with (DZR-Cit)/PSS/PAH/HA-AF particles for 6 h. The feeding ratio was 10 µg particles per 1×104 cells. (A) (DZR-Cit)/PSS/PAH/HA–AF particles (green), (B) lysosomes (red), (C) merge image of (A) and (B), (D) merge image of (C) and the bright-field image. Scale bar is 25 µm.

Figure 4. Relative cell viability of (A, B) HepG2 and (C) NIH 3T3 cells under UV treatment (365 nm, 30 mw cm-2, 3 min) with (A) different feeding ratios of (DZR-Cit)/PSS/PAH/HA particles and (B) co-culture time. Samples were divided into five groups, UV: only irradiation, 1: co-culture with particles without irradiation; 2: irradiated immediately after adding particles and then co-culture for 6 h; 3: co-culture with particles 6 h, and then irradiated; 4: co-culture with particles for 6 h, washed off free particles, and then irradiated. The values were normalized to those of the particle and irradiation-free cells. (D) Intracellular ROS level of HepG2 cells. Untreated cells and the cells treated with ROS-up reactant for 30 min were used as negative and positive controls, respectively. UV: only UV irradiation, particles: co-culture with particles without irradiation, particles+UV: co-culture with particles for 6 h, washed off free particles, and then irradiated. * and ** indicates significant difference at p < 0.05 and p < 0.01 levels, respectively.


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Xia and co-workers

5

have demonstrated that during the formation, swelling and finally

collapse of bubbles, a disruptive force could be produced similar as the cavitation effect. If the force acts on lysosomes, it may disrupt the membranes, resulting in release of proteolytic enzymes into the cytosol and finally cell necrosis. This explanation can be applied onto the current system too. To study the integrity of acidic organelles, acridine orange (AO) was used to stain the cells. It emits red fluorescence in lysosomes, and green fluorescence in cytosols and nuclei.

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When the membrane of lysosomes is disrupted, the red fluorescence will disappear.

Figure 5 showed that the red fluorescence could be observed in the control group and the cells treated with UV irradiation or particles only. However, when the cells were incubated with particles for 6 h and irradiated with UV, the red fluorescence almost disappeared, companying with the cell morphology change from well spreading to shrinkage. Hence, one can conclude that the released N2 bubbles can disrupt the membrane of lysosomes, leading to the death of cancer cells.


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Figure 5. Observation of lysosome disruption of HepG2 cells being induced by (DZR-Cit)/PSS/PAH/HA particles upon UV irradiation . The cells were treated with (A) DMEM without irradiation, (B) irradiation only, (C) 10 µg (DZR-Cit)/PSS/PAH/HA particles per 1×104 cells, (D) 10 µg (DZR-Cit)/PSS/PAH/HA particles per 1×104 cells followed by UV irradiation (365 nm, 30 mw cm-2, 3 min). Upper row: overlay images of the green and red channels; middle row: red channel; bottom row: green channel. The cells were further incubated with 6 µM AO for 15 min before CLSM measurement. Ex: 488 nm; Em: 515-545 nm (green) and 610-640 nm (red). Scale bar is 25 µm.

Reactive oxygen species (ROS) can cause cell death. Rise of the intracellular ROS level has been regarded as the main reason for the toxicity of many nanoparticles, 29 which can be utilized to achieve cancer cell killing in photodynamic therapy. 30, 31 By measuring the ROS generation of cells after different treatments (Figure 4D), it is clear that the ROS level of the cells which internalized particles increased obviously after UV irradiation, while the value of cells without UV irradiation kept at a low level. Also, the irradiation only did not cause obvious generation of ROS. As an organelle containing numerous hydrolases, lysosome has been found to play an important role in cell death. 32, 33 Here, during intracellular response the membrane of lysosomes is destroyed, and enzymes will be released into the cytosol, which affect the normal physiological activities of cells, 34, 35 leading to the generation of ROS and subsequent cell death. Mitochondria play an essential role in cell metabolism, which can provide energy for cells. Mitochondrial membrane potential (MMP) is a key indicator reflecting whether the mitochondria are in good cindition or not. In order to investigate if the mitochondria function can be affected by the ROS generation, Rhodamine 123 was utilized to detect the MMP of the cells after different treatments. If the mitochondria are damaged with ROS, the MMP would decrease. And thus the binding ability of the mitochondria with Rhodamine 123 will be reduced, resulting in weakened fluorescence. Results showed that after cells were incubated with particles for 6 h and


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irradiated with UV, the MMP decreased obviously, compared with the cells without treatment, with UV irradiation only or incubated with particles (Figure S8). Therefore, the ROS generation do obviously affect the mitochondria function, which may be an important factor to cause cell death. 4. Conclusion Photo-responsive submicron particles based on DZR were fabricated via a polyamine-salt aggregate method. Surface modification with HA endowed the particles with cancer cell targeting ability. Such particles were spheres with an average size of 700-800 nm. They had rough surface and could release N2 upon UV irradiation. After co-cultured with particles and subsequent UV irradiation, the viability of carcinoma HepG2 cells was significantly lowered down due to the damage of the integrity of lysosomes and release of various enzymes, and enhanced ROS generation. By contrast, the viability of normal NIH3T3 was not affected due to the very low uptake ability of HA-coated particles. ASSOCIATED CONTENT Supporting Information. Synthesis

of

diazo-resin

(DZR),

photo-response

of

DZR

solution,

synthesis

of

fluoresceinamine-labeled sodium hyaluronate (HA), determination of HA amount on the particles and test of HA stability, cancer cell targeting ability of (DZR-Cit)/PSS/PAH/HA-AF particles, competition assay to assess CD44 receptor-mediated cellular uptake and measurement of mitochondrial membrane potential. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author * Email: [email protected] (W. Tong). * Email: [email protected] (C. Gao) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest Acknowledgements This study is financially supported by the Natural Science Foundation of China (21374101 and 51120135001), the Key Science Technology Innovation Team of Zhejiang Province (2013TD02), and the Fundamental Research Funds for the Central Universities (2016QNA4033).

References (1)

MacDiarmid, J. A.; Mugridge, N. B.; Weiss, J. C.; Phillips, L.; Burn, A. L.; Paulin, R. P.;

Haasdyk, J. E.; Dickson, K.-A.; Brahmbhatt, V. N.; Pattison, S. T.; James, A. C.; Al Bakri, G.; Straw, R. C.; Stillman, B.; Graham, R. M.; Brahmbhatt, H., Bacterially Derived 400 nm Particles for Encapsulation and Cancer Cell Targeting of Chemotherapeutics. Cancer cell 2007, 11, 431445. (2)

Ashley, C. E.; Carnes, E. C.; Phillips, G. K.; Padilla, D.; Durfee, P. N.; Brown, P. A.;


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Hanna, T. N.; Liu, J.; Phillips, B.; Carter, M. B.; Carroll, N. J.; Jiang, X.; Dunphy, D. R.; Willman, C. L.; Petsev, D. N.; Evans, D. G.; Parikh, A. N.; Chackerian, B.; Wharton, W.; Peabody, D. S.; Brinker, C. J., The Targeted Delivery of Multicomponent Cargos to Cancer Cells by Nanoporous Particle-supported Lipid Bilayers. Nat. Mater. 2011, 10, 389-397. (3)

Yashchenok, A. M.; Delcea, M.; Videnova, K.; Jares-Erijman, E. A.; Jovin, T. M.;

Konrad, M.; Möhwald, H.; Skirtach, A. G., Enzyme Reaction in the Pores of CaCO3 Particles upon Ultrasound Disruption of Attached Substrate-Filled Liposomes. Angew. Chem., Int. Ed. 2010, 49, 8116-8120. (4)

Lee, S.-M.; Lee, O.-S.; O'Halloran, T. V.; Schatz, G. C.; Nguyen, S. T., Triggered Release

of Pharmacophores from Ni(HAsO3)-Loaded Polymer-Caged Nanobin Enhances Pro-apoptotic Activity: A Combined Experimental and Theoretical Study. ACS Nano 2011, 5, 3961-3969. (5)

Chung, M. F.; Chen, K. J.; Liang, H. F.; Liao, Z. X.; Chia, W. T.; Xia, Y.; Sung, H. W., A

Liposomal System Capable of Generating CO2 Bubbles to Induce Transient Cavitation, Lysosomal Rupturing, and Cell Necrosis. Angew. Chem., Int. Ed. 2012, 51, 10089-10093. (6)

Zhang, X.; Tian, G.; Yin, W.; Wang, L.; Zheng, X.; Yan, L.; Li, J.; Su, H.; Chen, C.; Gu,

Z.; Zhao, Y., Controllable Generation of Nitric Oxide by Near-Infrared-Sensitized Upconversion Nanoparticles for Tumor Therapy. Adv. Funct. Mater. 2015, 25, 3049-3056. (7)

Sun, J. Q.; Wu, T.; Liu, F.; Wang, Z. Q.; Zhang, X.; Shen, J. C., Covalently Attached

Multilayer Assemblies by Sequential Adsorption of Polycationic Diazo-resins and Polyanionic Poly(acrylic acid). Langmuir 2000, 16, 4620-4624. (8)

Sun, J. Q.; Wu, T.; Sun, Y. P.; Wang, Z. Q.; Zhang, X.; Shen, J. C.; Cao, W. X.,

Fabrication of a Covalently Attached Multilayer via Photolysis of Layer-by-Layer Selfassembled Films Containing Diazo-resins. Chem. Commun. 1998, 1853-1854.


19


(9)

Page 20 of 23

Zhu, H. G.; McShane, M. J., Macromolecule Encapsulation in Diazoresin-based Hollow

Polyelectrolyte Microcapsules. Langmuir 2005, 21, 424-430. (10) Yi, Q.; Sukhorukov, G. B., Single-Component Diazo-resin Microcapsules for Encapsulation and Triggered Release of Small Molecules. Part. Part. Syst. Charact. 2013, 30, 989-995. (11) Pastoriza-Santos, I.; Scholer, B.; Caruso, F., Core-shell Colloids and Hollow Polyelectrolyte Capsules Based on Diazoresins. Adv. Funct. Mater. 2001, 11, 122-128. (12) Yi, Q.; Sukhorukov, G. B., Photolysis Triggered Sealing of Multilayer Capsules to Entrap Small Molecules. ACS Appl. Mater. Interfaces 2013, 5, 6723-6731. (13) Yu, M.; Jambhrunkar, S.; Thorn, P.; Chen, J.; Gu, W.; Yu, C., Hyaluronic Acid Modified Mesoporous Silica Nanoparticles for Targeted Drug Delivery to CD44-overexpressing Cancer Cells. Nanoscale 2013, 5, 178-183. (14) Sun, H.; Benjaminsen, R. V.; Almdal, K.; Andresen, T. L., Hyaluronic Acid Immobilized Polyacrylamide Nanoparticle Sensors for CD44 Receptor Targeting and pH Measurement in Cells. Bioconjugate Chem. 2012, 23, 2247-2255. (15) Bagaria, H. G.; Wong, M. S., Polyamine–salt Aggregate Assembly of Capsules as Responsive Drug Delivery Vehicles. J. Mater. Chem. 2011, 21, 9454-9466. (16) Cao, W. X.; Zhao, C.; Cao, J. W., Synthesis and Characterization of 3Methoxydiphenylamine-4-diazonium Salt and Its Diazoresin. J. Appl. Polym. Sci. 1998, 69, 1975-1982. (17) Harada, H.; Takahashi, M., CD44-dependent Intracellular and Extracellular Catabolism of Hyaluronic Acid by Hyaluronidase-1 and-2. J. Biol. Chem. 2007, 282, 5597-5607. (18) Benjaminsen, R. V.; Sun, H.; Henriksen, J. R.; Christensen, N. M.; Almdal, K.; Andresen,


20

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


T. L., Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements. ACS Nano 2011, 5, 5864-5873. (19) Curtin, J. F.; Donovan, M.; Cotter, T. G., Regulation and Measurement of Oxidative Stress in Apoptosis. J. Immunol. Methods 2002, 265, 49-72. (20) Zhang, P.; Yang, H.; Wang, G.; Tong, W.; Gao, C., Polyamine/salt-assembled Microspheres Coated with Hyaluronic Acid for Targeting and pH Sensing. Colloid. Surface B 2016, 142, 223-229. (21) Katas, H.; Alpar, H. O., Development and Characterisation of Chitosan Nanoparticles for siRNA Delivery. J. Controlled Release 2006, 115, 216-225. (22) Song H., Nor Y. A., Yu M. H., Yang Y. N., Zhang J., Zhang H. W., Xu C., Mitter N. Yu C. Z., Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition. J. Am. Chem. Soc. 2016, 138, 6455−6462. (23) Niu Y. T., Yu M. H., Hartono S. B., Yang J., Xu H. Y., Zhang H. W., Zhang J., Zou J., Dexter A., Gu W. Y., Yu C. Z., Nanoparticles Mimicking Viral Surface Topography for Enhanced Cellular Delivery. Adv. Mater. 2013, 25, 6233–6237. (24) Zhang W. B., Li H. Y., Qin Y., Gao C. Y., Self-Assembled Composite Microparticles With Surface Protrudent Porphyrin Nanoparticles Enhance Cellular Uptake And Photodynamic Therapy. Mater. Horiz. 2017, DOI: 10.1039/c7mh00420f. (25) Mercer, J.; Helenius, A., Virus Entry by Macropinocytosis. Nat. Cell Biol. 2009, 11, 510520. (26) Hillaireau, H.; Couvreur, P., Nanocarriers' Entry into the Cell: Relevance to Drug Delivery. Cell. Mol. Life Sci. 2009, 66, 2873-2896. (27) Chen, H.; Tian, J.; He, W.; Guo, Z., H2O2-activatable and O2-Evolving Nanoparticles for


21


Page 22 of 23

Highly Efficient and Selective Potodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547. (28) Chen, H.; Xiao, L.; Anraku, Y.; Mi, P.; Liu, X.; Cabral, H.; Inoue, A.; Nomoto, T.; Kishimura, A.; Nishiyama, N.; Kataoka, K., Polyion Complex Vesicles for Photoinduced Intracellular Delivery of Amphiphilic Photosensitizer. J. Am. Chem. Soc. 2014, 136, 157-163. (29) Zhang, W. J.; Jiang, P. F.; Chen, Y.; Luo, P. H.; Li, G. Q.; Zheng, B. T.; Chen, W.; Mao, Z. W.; Gao, C. Y., Suppressing the Cytotoxicity of CuO Nanoparticles by Uptake of Curcumin/BSA Particles. Nanoscale 2016, 8, 9572-9582. (30) Dolmans, D.; Fukumura, D.; Jain, R. K., Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380-387. (31) Zhao, E. G.; Deng, H. Q.; Chen, S. J.; Hong, Y. N.; Leung, C. W. T.; Lam, J. W. Y.; Tang, B. Z., A Dual Functional AEE Fluorogen as a Mitochondrial-specific Bioprobe and an Effective Photosensitizer for Photodynamic Therapy. Chem. Commun. 2014, 50, 14451-14454. (32) Kroemer, G.; Jaattela, M., Lysosomes and Autophagy in Cell Death Control. Nat. Rev. Cancer 2005, 5, 886-897. (33) Paquet, C.; Sane, A. T.; Beauchemin, M.; Bertrand, R., Caspase- and Mitochondrial Dysfunction-dependent Mechanisms of Lysosomal Leakage and Cathepsin B Activation in DNA Damage-induced Apoptosis. Leukemia 2005, 19, 784-791. (34) Zdolsek, J. M.; Olsson, G. M.; Brunk, U. T., Photooxidative Damage to Lysosomes of Cultured Macrophages by Acridine-orange. Photochem. Photobiol. 1990, 51, 67-76. (35) Bachor, R.; Shea, C. R.; Gillies, R.; Hasan, T., Photosensitized Destruction of Human Bladder-carcinoma Cells Treated with Chlorin E6-conjugated Microspheres. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 1580-1584.


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Figure for TOC


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Phototriggered N2-Generating Submicron Particles for Selective

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