Redox-Hypersensitive Organic Nanoparticles for Selective Treatment

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Redox-Hypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells Wei Zhang, Wenhai Lin, Qing Pei, Xiuli Hu, Zhigang Xie, and Xiabin Jing Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b01641 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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Chemistry of Materials

Redox-Hypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells Wei Zhang,†,‡ Wenhai Lin,†,‡ Qing Pei,†,‡ Xiuli Hu,† Zhigang Xie, *,† and Xiabin Jing† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT: The diselenide-containing fluorescent molecules (SeBDP) and antitumor drug paclitaxel (SePTX) were synthesized and used for constructing SeBDP nanoparticles (SeBDP NPs) and SePTX NPs in aqueous solution through nanoprecipitation method. Both SeBDP NPs and SePTX NPs exhibit high stability and excellent reduction-sensitivity. More interestingly, SeBDP and SePTX could co-assemble into uniform and spherical nanoparticles (co-NPs) with dual functions of fluorescence imaging and antitumor activity. These organic NPs could be internalized by different cells as revealed by confocal laser microscopy. Importantly, the co-NPs exhibited selectivity of cytotoxicities between cancerous and normal cells. The cellular proliferation inhibition toward tumor cells (including HeLa and MCF-7 cells) was obviously higher than that toward normal cells(BEAS-2B and L929 cells), which might be attributed to the increasing reactive oxygen species in cancer cells treated by diselenide-containing NPs. These results highlight the potential of developing diselenidecontaining organic molecules as molecularly tunable and sensitive nanoplatform for cancer treatment.

Owing to the advantages of precise molecular structure and multifarious synthesis chemistry, self-assembly of organic molecules has been used to fabricate various functional materials.1-3 The assembly can be realized through supramolecular chemistry, such as hydrophobic interactions of amphiphilic small molecules and π-π interactions between π-conjugated monomers.4,5 For example, Kasai et al. reported the fabrication of pure nanodrugs from podophyllotoxin (PPT) dimer, which was called carrier-free pure nanodrugs (PNDs).6 Since then, increasing work about the dimer-induced self-assembly of pure drugs has been reported.7-10 Recently, our group reported that a fluorescent dye of BODIPY (4, 4-difluro- 4bora-3a,4a-diaza-s-indacence) dimer could self-assemble into nanoparticles for cellular imaging.11 Inspired by these works, we hypothesized that the dimeric organic molecules could form nanoparticles via self-assembly under suitable conditions. As a necessary microelement for human body, selenium plays an important part in wide ranges of biological processes, such as the regulation of reduction and oxidation processes which are of great importance to cancer prevention.12-16 Selenium possesses many unique chemical properties owing to its special electronegativity and atomic radius. The bond energy of Se-Se is 172 KJ mol-1, which is weaker in comparison with that of C-C bond (364 KJ mol1) and S-S bond (240 KJ mol- 1).17 This difference makes it more sensitive to be oxidized and reduced than low va-

lence state sulfur compounds. A series of seleniumcontaining compounds were reported in the pharmacochemistry as antioxidants and stimuli-sensitive drug delivery carriers. For example, Xu’s group successfully introduced diselenide bonds into a series of amphiphilic polymers with different topology, which could self-assemble into micellar structures for drug delivery and enzyme mimics.18-21 However, there is few report about the diselenide-containing nanoparticles assembled from organic molecules. For the tumor treatment, it is challenging to develop drugs which can selectively kill cancer cells and show less toxicity towards normal cells. Scientists have made much effort to enhance tumor selectivity by using multifunctional nanomedicines.22-24 The most widely used strategy is the surface modification of vehicles with targeting moieties including peptides (e.g. RGD),25 small molecules (e.g. folate and galactose),26-28 and even short oligonucleotide (aptamer).29-32 In general, tumor tissues and cells have many unique characteristics, including lower pH values, higher reduction potential and ROS levels (oxidative stress), special biological enzymes, and tissue hypoxia, etc.33-35 Thus, another strategy is using “smart nanocarriers”, which can make quick response to relevant tumor microenvironment, such as lower pH value,36-39 high concentration of glutathione (GSH)40,41 and enzyme.42,43 However, all methods mentioned above usually need sophisticated and tedious synthesis and modification processes.

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Thus, it is challenging for researchers to develop smart nanomaterials via a simple way.

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and Figure S1C, all the protons and the peak at m/z 389.98 were all agreed well with theoretical calculation.

Figure 1. (A) DLS results and (B) TEM image of SeBDP NPs. (C) Changes of the diameter and PDI as a function of time measured by DLS. (D) TEM image of SeBDP NPs after being stored for two weeks. Scheme 1. A schematic illustration of (A) the dimerinduced self-assembly of SeBDP, SePTX and the coassembly of the two dimers and (B) the cell endocytosis and disassembly upon the intracellular GSH of the nanoparticles. In the present work, reduction-sensitive diselenidecontaining fluorescent molecule SeBDP and antitumor drug SePTX were synthesized, and these two molecules could self-assemble into nanoparticles (SeBDP NPs and SePTX NPs) or co-assembly into co-NPs in the absence of any surfactants or adjuvants (Scheme 1). The co-NPs not only possess dual functions of fluorescence imaging and antitumor activity, but also exhibit high selectivity on tumor cells (human cervical carcinoma (HeLa) cells and human mammary tumor (MCF-7) cells).

RESULTS AND DISCUSSION Synthesis of 6,6'-diselanediyldihexanoic acid. The synthetic routes for the diselenide-containing acid were shown in Figure S1A (Supporting Information). Disodium diselenide (Na2Se2) was synthesized according to the literature methods.44 Then, Na2Se2 was treated with a tetrahydrofuran (THF) solution of 6-bromohexanoic acid under inert atmosphere at 50 oC overnight. After purification on a silica gel column, the 6,6'-diselanediyldihexanoic acid was obtained and characterized by proton nuclear magnetic resonance (1H NMR) and electrospray ionization mass spectrometry (ESI-MS). As shown in Figure S1B

Synthesis of SeBDP and SeBDP NPs. Passerini reaction is a kind of three-component reactions involving carboxylic acid, isocyanide, and aldehyde, which is highly efficient and tolerant.45,46 Our group has reported the synthesis of polymers via Passerini reaction.47,48 In this work, the target diselenide-containing BDP molecules were synthesized via the similar processes. 4,4-Difluoro-8-(4- isocyanophenyl)-3,5-dimethyl-4-bora-3a,4a-diaza-indacene (NC-BDP) was made by following the literature methods.49 Then NC-BDP, 6,6'-diselanediyldihexanoic acid and o-nitrobenzaldehyde were reacted in dichloromethane for 4 days at room temperature. The obtained SeBDP was characterized by 1H NMR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). As shown in Figure S2A, the signals at 8.57 ppm and 6.76 ppm for protons of imine and methane indicated that NC-BDP was successfully incorporated into SeBDP dimer. The peak at m/z 1309.4 in the MALDI-TOF MS spectrum (Figure S2B) further confirmed the successful synthesis of the fluorescent dimer. SeBDP could self-assemble into nanoparticles (SeBDP NPs) in aqueous solution via nano-precipitation method. The THF solution of SeBDP was added dropwise into deionized water under stirring and then dialyzed to remove the residual THF. The morphology and size distribution of SeBDP NPs were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS), respectively. As shown in Figure 1A, the average diameter of SeBDP NPs measured by DLS was 158 nm,


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and the polydispersity index (PDI) was 0.12. The TEM image (Figure 1B) indicated the isolated nanoparticles, whose size was close to the value obtained by DLS. The diameter and PDI measured by DLS almost kept unchanged even in two weeks (Figure 1C). Moreover, the morphologies of SeBDP NPs were maintained even after being stored for two weeks (Figure 1D). The results above demonstrated that SeBDP NPs could keep their stability in aqueous solution. As shown in Figure S3, SeBDP NPs also exhibited favorable stability in physiological environment as evidenced by the unchangeable particle size and PDI for up to 24 h by incubating the SeBDP NPs in PBS (pH 7.4) containing FBS (10 %) at 37 oC.

Figure 2. (A) UV-vis absorption and (B) fluorescence spectra of SeBDP NPs in water and SeBDP in DMF: H2O=1: 1 (v/v). Insets in (B) are photos of SeBDP in DMF: H2O=1: 1 (v/v) solution (left) and SeBDP NPs in water (right) under natural light and the illumination of UV light (365 nm). Figure 2 showed the UV-vis absorption and fluorescence spectra of the SeBDP NPs in water and SeBDP in DMF:H2O = 1: 1 (v/v), respectively. The maximum absorption of SeBDP NPs in water was centered at 507.5 nm, which was red-shifted by 7 nm relative to that of SeBDP. Bathochromic shift absorption of SeBDP NPs was attributed to the possible J-aggregate of SeBDP in aqueous solution, as reported for the aggregation of BODIPY.50 In Figure 2B, the maximum fluorescence wavelength was seen at 515 nm for SeBDP, but almost no fluorescence was seen for SeBDP NPs due to the aggregation-caused quenching (ACQ).51 The phenomenon of ACQ could be observed visually by photos under the 365 nm light irradiation. As shown in Figure 2B, SeBDP in DMF: H2O=1: 1 (v/v) emitted a strong yellow-green fluorescence while no fluorescence was observed for SeBDP NPs in water. Redox Sensitivity. To demonstrate the reductionsensitive disruption of SeBDP NPs, the size changes of SeBDP NPs were monitored by DLS in presence of GSH. As shown in Figur 3A, the size of SeBDP NPs increased from 157 nm to 2586 nm at 7 h with 10 mM GSH, which was due to the disruption of diselenide bond and aggregation of hydrophobic fragments into large agglomerates.52 It is well known that the concentration of GSH is approximately 1-10 mM in cytosol.53 Thus, we also tested the reduction-sensitive behaviors upon the treatment of 1 mM GSH. The size of SeBDP NPs increased from 157 nm to 1149 nm, indicating efficient reduction-sensitivity even

Figure 3. (A) Size changes of SeBDP NPs (42.5 µg mL-1) over time and (B) size distribution changes of SeBDP NPs (42.5 µg mL-1) after 7 h determined by DLS in the presence of different concentrations of GSH. with low concentration of reducing agents. As control, the change of size was negligible over 7 h in the absence of GSH, confirming the good stability of SeBDP NPs. These results confirmed that SeBDP NPs were reductionsensitive and could disassemble in the presence of reducing agents. Meanwhile, we also monitored the zeta potential changes along with the size changes. As shown Figure S4, the zeta potential of the nanoparticles gradually increased along with the incubation time, changing from 23.3 mV to -1.7 mV. Subsequently, we examined whether the reductive behaviors could still be kept in serum containing solution with GSH. As shown in Figure S5, the size of NPs increased from 220 nm to 1718 nm and obvious flocculent suspension could be found, which indicate the efficient sensitivity of the formed NPs. Cellular Uptake. The biocompatibility of nanomaterials is vital for their biomedical applications. The in vitro cytotoxicities of SeBDP NPs toward HeLa cells were quantified by methyl tetrazolium (MTT) viability assay. Figure S6 showed the viability of cells incubated with various con-

Figure 4. Confocal microscopy images of HeLa cells (A) pretreated with GSH (10 mM) for 2 h, (B) without any pretreatment and (C) pretreated with NEM (1 mM) for 15 min, then treated with SeBDP NPs (2 µg mL-1) for 2 h. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), BDP fluorescence in cells (green), and overlays of both images. Scale bar: 20 μm.



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centrations of SeBDP NPs for 24 h. SeBDP NPs didn’t show obvious cytotoxicities on cell proliferation with the concentration up to 25 μg mL-1. We could come to the conclusion that SeBDP NPs could be used as safe nanomaterials for cell imaging. The cellular uptake and reduction-response of SeBDP NPs were carried out by confocal laser scanning microscopy (CLSM). HeLa cells were firstly pretreated with 10 mM GSH or 1 mM N-ethylmaleimide (NEM) according to the reported protocol,54,55 and then incubated with 2 μg mL-1 of SeBDP NPs for 2 h. The untreated cells incubated with the same dose of NPs were used as control (Figure 4B). As expected, the strongest intracellular green fluorescence was observed in the cytoplasm for the GSH pretreated cells in Figure 4A, indicating the effective internalization of SeBDP NPs. In contrast, little fluorescence was observed in pretreated with NEM (Figure 4C), an efficient modifier to react with biological thiol groups.55 This results demonstrated that SeBDP NPs could rapidly dissociate in cells in the presence of intracellular reducing agents. We also used flow cytometry to quantitatively examine the cellular uptake of the nanoparticles. As shown in Figure S7, most of HeLa cells were able to internalize NPs because of the increased green fluorescence for cells treated with SeBDP NPs. Synthesis of SePTX NPs and co-NPs NPs. Recently, carrier-free pure nanodrugs have drawn much attention, in which native pharmacological drugs are converted into nanoscale formulation.56 Kasai’s group demonstrated that prodrugs of the commercial antitumor agents (e.g. SN-38 and PPT) with dimeric structures could form stable nanoparticles.6,8 Inspired by their works, we synthesized the diselenide-containing PTX dimer via the condensation reaction, which was confirmed by 1H NMR (Figure S8) and MALDI-TOF MS. The peak at 2061.62 was ascribed to [M+H]+ of SePTX, which was close to the theoretical mo-

Figure 5. (A) TEM image and (B) DLS results of co-NPs. Changes of diameter and PDI of co-NPs (C) in water and (D) in PBS with FBS (10 %) over different times measured by DLS.

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lecular weight of SePTX. SePTX also could self-assemble into nanoparticles (SePTX NPs) in aqueous solution via reprecipitation method. The SePTX NPs possessed spherical morphology with diameter of 120 nm and low size distribution as revealed by TEM and DLS in Figure S9A and Figure S9B. The diameter and PDI of SePTX NPs measured by DLS were almost unchanged over half a month (Figure S9C). SePTX NPs also exhibited excellent stability in PBS (pH 7.4) containing FBS (10 %) at 37 oC for 24 h (Figure S9D). The reduction-induced dissociation of the SePTX NPs was confirmed in the presence of GSH as shown in Figure S10. Due to the structural similarities of SeBDP and SePTX, we speculated that they could co-assembled into nanoparticles containing both fluorescent chromophore (BDP) and anti-tumor drug (PTX), which could be used as potential imaging and therapeutic agents. The coassembling nanoparticles (co-NPs) were obtained after the mixture of SeBDP and SePTX (SePTX: 1.4 mg and SeBDP: 0.2 mg) in THF solution was dropped into water. In order to further demonstrate the structure of the coassembly, we used the scanning electronic microscopy (SEM) and energy dispersive spectrometer (EDS) to qualitatively analysis the distribution of the element (Se and B). As shown in Figure S11A, spherical nanoparticles with the diameter around 150 nm could be seen. Not all Se elements superpose with the B elements (Figure S11B). The overlapping regions of Se and B are ascribed to the SeBDP (containing both Se and B element), while the remaining regions are assigned to the SePTX (only containing Se element). These results validate partly the co-assembly of the two small organic molecules (SeBDP and SePTX). The final concentrations of SeBDP and SePTX were 118 μg mL-1 and 17.5 μg mL-1, respectively, which were determined by high-performance liquid chromatography (HPLC) with UV-vis spectrophotometer (as described in the Experimental Section). Figure 5A showed isolated spherical particles with an average diameter of 190-200 nm. The average size of co-NPs measured by DLS was 195.2 nm and the PDI was 0.087 (Figure 5B). The co-NPs also maintained

Figure 6. (A) Size and PDI changes of co-NPs resulting from reduction-induced disassembly in the presence of 10 mM GSH. Inside: Photos of co-NPs in the absence and presence of 10 mM GSH after 0.5 h. (B) The size changes of co-NPs in the presence of different concentrations of GSH. Inside: Changes of size distribution of co-NPs with the treatment of GSH after 7 h.


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the excellent stability in water (Figure 5C) or in PBS containing FBS (10 %) (Figure 5D), indicating the coassembling did not affect the stability.

cence from BDP and the red fluorescence from Lyso Tracker red, indicating the accumulation of co-NPs in lysosomes.

The reduction-sensitive behaviours of the co-NPs were investigated through the changes of size and morphology. As shown in Figure 6A, the increase in diameter of co-NPs from 143 nm to 2261 nm was observed after adding GSH in half an hour. The PDI also increased from 0.16 to 0.39, which confirmed the disassembly of co-NPs. The co-NPs solution changed from clear to muddy, and a large number of precipitates were found (photos in Figure 6A). Furthermore, the spherical nanoparticles became irregular, and large agglomerates could be seen after 0.5 h upon the treatment of 10 mM GSH (Figure S12). Addition of different concentrations of GSH could result in the disparate size changes. As shown in Figure 6B, the size of co-NPs increased from 145.6 nm to 2045 nm in the presence of 10 mM GSH for 7 h, and could increase from 145.6 nm to 308.6 nm with 1 mM GSH. These results confirmed that reduction-sensitivity could still be maintained after coassembly.

Cytotoxicitieds of co-NPs toward the tumor cells (HeLa and MCF-7 cells) and the normal cells (BEAS-2B and L929 cells) were evaluated by MTT assays. Comparing with the free PTX, co-NPs have shown lower cytotoxicity as shown in Figure 8A-C and Figure S10, which might be attributed to the rapid diffusion of PTX into cytoplasm other than endocytosis of co-NPs. Interestingly, obvious cell cytotoxicities were induced by co-NPs toward cancer cells compared with normal cells. More than 75 % of HeLa cells and 60 % of MCF-7 cells were killed at the concentration of 5 μg mL-1 of PTX. In contrast, no more than 25 % of BEAS-2B cells and 20 % of L929 cells were dead, as shown in Figure 8C and Figure S14. The IC50 values were about 0.83 μg mL-1 and 3.03 μg mL-1 toward HeLa cells and MCF7, which were actually much smaller than that of the normal cells (Figure 8D and Table S2). These results indicated the co-NPs possessed good selectivity on tumor cells.

Cellular uptake of nanoparticles is important for exerting their function. The internalization of co-NPs by HeLa, MCF-7, BEAS-2B and L929 cells were evaluated by CLSM. These cells were incubated with co-NPs (SeBDP: 2 μg mL-1) for 0.5 h and 2 h, respectively. The cell nuclei were dyed with DAPI. As shown in Figure 7A and Figure S13, green fluorescence was observed in the cell plasma, indicating that the co-assembled nanoparticles could be endocytosed by both cancer and normal cells. Moreover, the green fluorescence in cells increased obviously along with the increasing incubation time. The way of endocytosis was studied by colocalization of the nanoparticles and lysosomes labelled with red fluorescence (Lysosome Tracker red). As shown in Figure 7B, the observed yellow colocalization area was the overlay of the green fluores-

As an essential regulator of reactive oxygen species (ROS) in vivo, selenium is considered that it will undergo a series of sophisticated biological processes. It might be reduced to low-valence-state (Se-2 or HSe-) by thiolcontaining compounds or be oxidized to high-valencestate (SeO32- or SeO42-) by O2 and H2O2. During these processes, ROS will be generated continuously, which are crucial intermediates in selenium-induced apoptosis.57,58 Thus, in order to study the mechanism for selectivity of the selenium-containing nanomedicines, the intracellular concentration of ROS was quantified by using the 2’,7’dichlorofluorescence diacetate (DCFH-DA) fluorescence

Figure 7. Representative CLSM images of (A) HeLa and BEAS-2B cells incubated with co-NPs for 0.5 h and 2 h, respectively. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), BDP fluorescence in cells (green), and overlays of both images. Scale bar: 20 μm. (B) Colocalization images of HeLa cells treated with co-NPs for 2 h. The images from left to right show cell nuclei stained by DAPI (blue), BDP fluorescence in cells (green), Lyso Tracker fluorescence (red) in lysosomes and overlays of three images. Scale bar: 20 μm.

Figure 8. In vitro cytotoxicities of different concentrations of PTX and co-NPs toward (A) HeLa cells, (B) MCF7 cells and (C) BEAS-2B cells incubated for 48 h. (D) Comparison of cytotoxicities in vitro toward tumor cells (HeLa and MCF-7 cells) and normal cells (BEAS-2B cells) after being incubated with co-NPs for 48 h. Statistical significance analysis was assessed by SPSS via one-way ANOVA test; P≤0.01 were considered statistically highly significant and were denoted as “**”; P≤0.001 were considered statistically highly significant and were denoted as “***”.



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method in HeLa and BEAS-2B cells. DCFH-DA is a commonly used fluorescence sensor for ROS, which can be rapidly oxidized to 2’,7’-dichlorofluorescein (DCF) with green fluorescence, and the fluorescence intensity is proportional to the concentration of ROS.59 As shown in Figure S15-S17, the fluorescence intensity of HeLa and MCF-7 cells treated with the SePTX NPs are much stronger than that of the control group. SePTX was used to instead of co-NPs due to the same green fluorescence of co-NPs and DCFH-DA under excitation at 488 nm. There are almost no obvious changes of the DCF fluorescence intensity in the normal cells (L929 or BEAS-2B cells), validating that the selenium-containing nanoparticles could up-regulate the intracellular ROS in cancer cells. Thus, we concluded that the selectivity between normal cells and tumor cells was mainly contributed to the higher selenium-induced ROS in cancer cells, which was consistent with the results of Xu’s group.60 Very recently, a similar strategy of reducing glutathione levels in cancer cells was used to enhance the photodynamic therapy and preferential killing of cancer cells.61,62 This selenium bonds-based selective treatment of cancer cells without any additional targeting agents is valuable for the development of effective clinical antitumor drugs.

CONCLUSION In this work, we reported the synthesis of reductionsensitive diselenide-containing dimers (SeBDP and SePTX) and corresponding organic nanoparticles formed by supramolecular self-assembly. It is the first example of dimer-induced self-assembly from selenium-containing organic molecules. The obtained nanoparticles showed excellent reductive sensitive behaviors and good selectivity between tumor cells and normal cells in absence of targeting molecules. This highly selective cytotoxicities of nanoparticles is promising in future cancer therapy. The diselenide-based self-assembling nanoparticle represents a new and important synthetic development in the design of functional nanomaterials.

ASSOCIATED CONTENT Supporting Information. Experimental details and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author

(16) Preedy, V. R., Selenium: Chemistry, Analysis, Function and Effects. Royal Society of Chemistry: 2015.

* corresponding authors: [email protected] Funding Sources

(17) Kildahl, N. K., Bond Energy Data Summarized. J. Chem. Educ. 1995, 72, 423-424.

This work was supported by the National Natural Science Foundation of China (Project No. 51522307 and 51373167).

(18) Xu, H.; Cao, W.; Zhang, X., Selenium-containing Polymers: Promising Biomaterials for Controlled Release and Enzyme Mimics. Acc. Chem. Res. 2013, 46, 1647-1658.

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Redox-Hypersensitive Organic Nanoparticles for Selective Treatment

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