Synthesis of Water-Soluble Iridium ( III )-Containing Nanoparticles for Biological Applications

Water-soluble nanoparticles (Ir/PGlc-NP, Ir/β-1,3-glucan-NP) based on water-soluble glycopolymers (PGlc), β-1,3-glucan polysaccharide, and conjugated phosphorescent Ir (III) complexes were successfully synthesized by self-assembly. The obtained nanoparticles have good spherical morphological characterization with a mean diameter of 50 nmmeasured by TEM. Ir/PGlc-NP and Ir/β-1,3-glucan-NP showed the same emission maxima at 565 nm in aqueous solution and both caused effective apoptosis and death of HepG2 and Hela cells after being irradiated at 445 nm for 30min in vitro. Fluorescence cellular imaging was conducted by confocal laser scanning microscopy (CLSM) using HepG2 cells as the model cell in which the nanoparticles had successfully entered into the cytoplasm with high brightness. Furthermore, after injecting the nanoparticles into live mice in vivo, the realtime fluorescence imaging as well as the nanoparticles distribution in organs at 24 hours after administration indicated that these nanoparticles can serve as fluorescent imaging contrast for further biological applications.


Introduction
In recent years, water-soluble fluorescent -conjugated nanoparticles (WCPNs) have received much attention in bioimaging [1][2][3][4], drug-delivery, and sensing applications due to their high fluorescent brightness, excellent biocompatibility, and photostability [5][6][7].The preparation of WCPNs was mainly focused on self-assembly method, because this method could retain the physical properties of the polymers.Our group has got some achievements in this field.For example, Lu et al. developed kinds of water-soluble nanoparticles using oligos (p-phenyleneethynylene) (OHOPEL) and three water-soluble polymers (PEG, PAA, and PG) by noncovalent bond self-assembly [8].Zhang et al. synthesized types of water-soluble fluorescent nanoparticles, and the constituent conjugated polymer chains of them showed without distinct aggregation [9].Other groups like Moon et al. [10][11][12] demonstrated that the aggregation structure of the conjugated polymer nanoparticles can be changed by different organic acids treatment and that the water-soluble nanoparticles can be fabricated via controlled aggregation of semiflexible poly(phenylenebutadiynylene) with linear polysaccharide.
Phosphorescent Ir (III) complexes have attracted considerable interest during the past decade because of their excellent properties when used as energy receptor in organic light-emitting devices, chemical detection, and protein detection.Shi et al. [13] designed and synthesized phosphorescent conjugated polyelectrolyte with different contents of Ir (III) complex and demonstrated the applications of conjugated polyelectrolytes (CPEs) in time-resolved luminescent biodetection and fluorescence lifetime imaging.Compared with various organic fluorescent dyes units, Ir (III) complexes show unique advantages, for example, long fluorescent lifetime, large Stokes shift, high phosphorescent quantum yield, excellent photostability, tunable wavelengths, and easy modification of ligand structures [14][15][16].These characteristics make Ir (III) complexes the most successful candidates in optical sensing and bioimaging applications in complicated media.Our group has systematically studied conjugated polymers containing Ir (III) complexes for years in the application of biosensing, optical oxygen sensing, and photodynamic therapy [15][16][17][18][19]. Therefore, it is reasonable to introduce different amounts of the Ir (III) complex into the conjugated polymers backbone as the energy acceptor to acquire different phosphorescent nanoparticles.
Polysaccharide refers to any of a class of carbohydrates whose molecules contain chains of monosaccharide molecules.In recent years, Twomey et al. [11] utilized hyaluronic acid (HA) as hydrophilic group to obtain coreshell nanoparticles, and HA has specific binding ability with cell surface receptors.Glycopolymer is a synthetic polymer with pendant carbohydrate and various reports have concerned glycopolymer-based nanoparticles due to the amphiphilic properties of glycopolymer [20][21][22][23].In this work, we reported that different water-soluble phosphorescent nanoparticles containing the Ir (III) complexes were obtained when -conjugated polymers were treated with glycopolymer (poly(glucose)) or polysaccharide (-1,3glucan) based on self-assembly.Because of the amphiphilic structures, the polymers formed nanoparticles with the size around 50 nm in aqueous solution without additional decoration.Moreover, we studied the nanoparticles from the point of morphological characterization, optical properties, and cellular imaging.Finally, we analyzed the distribution of Ir/PGlc-NP and Ir/-1,3-glucan-NP in different organs of the mice at 24 h after administration.This study provided a possibility for further investigation on the application of photodynamic therapy.

Preparation of Core-Shell Nanoparticles.
The strategy for fabricating WCPNs was based on self-assembly method; typically, iridium complex (Ir (ppy) 3 ) and water-soluble glycopolymer (PGlc) were solubilized in DMF (0.1 mg/mL), respectively, and then 5 mL DMF solution was quickly injected into 5 mL water under ultrasonication.After being dialyzed against water for 2 days, getting the pale yellow clear aqueous solutions, the nanoparticle was denoted as Ir/PGlc-NP.Similarly, Ir/-1,3-glucan-NP nanoparticles formed via the self-assembly of Ir (ppy) 3 and polysaccharide (glucan).

Instrumentation.
Hydrodynamic radius of the core-shell nanoparticles was measured by dynamic light scattering (DLS) using particle size analyzer (ALV/5000E) at a fixed scattering angle of 90 ∘ .UV-visible spectra were recorded with a Shimadzu UV-3150 UV-VIS-NIR spectrophotometer.Transmission electron microscopy (TEM) experiments were conducted using a Philips CM 120 electron microscope operated with accelerating voltage of 80 kV.A small drop of a water-soluble nanoparticles solution was added to a carbon-coated copper grid.Drying for several hours, TEM measurements can be performed.Photoluminescent spectra (PL) were obtained on an FLS 920 spectrofluorophotometer which was purchased from Edinburgh Instruments.Confocal laser scanning microscope (CLSM) images were taken using a Leica TCS SP5 microscope.was clear that the average hydrodynamic radius ( ℎ ) mostly ranged from 10 to 100 nm.Table 1 gave the detailed data about  ℎ and PI, which revealed that the average diameter was about 60 nm with narrow size distribution as PI of around 0.2.

Results and Discussion
TEM Analysis.The size and morphology of the Ir/PGlc-NP and Ir/-1,3-glucan-NP in solid state were studied using TEM.The pictures, obtained by freeze drying, were shown in Figure 2. The pictures showed the two sample nanoparticles have similar structure.
In Figure 2, both the Ir/PGlc-NP and the Ir/-1,3-glucan-NP samples showed good spherical morphology with average diameters of ∼50 nm.Because the density of coordination complex Ir (ppy) 3 and PGlc/-1,3-glucan was the same and consequently the same contrasted to the background, there was no evidence to distinguish them from the pictures.However, the diameters observed were smaller than those obtained from DLS (⟨ ℎ ⟩ = 33 nm) mostly because the nanoparticles were shrunk during the process of freeze drying.The DLS and TEM results show that the nanoparticles (Ir/PGlc-NP and Ir/-1,3-glucan-NP) have spherical morphology, with the water-soluble polymer (PGlc and -1,3glucan) as the shell and Ir (ppy) 3 as the core.Also, diameter of nanoparticle (Ir/PGlc-NP and Ir/-1,3-glucan-NP) was about 50 nm, which was much smaller than our previous reports [8,9].

Optical Properties.
The photoluminescence (PL) emission spectra of Ir (ppy) 3 in THF, Ir/PGlc-NP, and Ir/-1,3-glucan-NP in aqueous solution are shown in Figure 3.The photoluminescence emission spectra of nanoparticles and Ir (ppy) 3 in THF were similar without distinct blueshift or red-shift and their emission maximum was around 565 nm.Compared with other nanoparticles with conjugated polymers as the core, which can induce PL changes for aggregation, our nanoparticles showed good optical stability.This proved that, after self-assembly of the particles formed in water, there was no significant change.

Cellular Imaging.
In order to demonstrate the ability of Ir/PGlc-NP and Ir/-1,3-glucan-NP as a probe for These results further demonstrated the nanoparticles can be used in cell imaging.

Photocytotoxicity Test.
In order to prove the application potential of Ir/PGlc-NP and Ir/-1,3-glucan-NP in photodynamic therapy (PDT), next the photodynamic cytotoxicity experiment of these two nanoparticles against HepG2 and Hela cells was evaluated by traditional MTT assay [15].The nanoparticles with a series of concentrations (2.5, 5, 10, 20, and 50 g) were tested in the dark and under light irradiation at 445 nm for 30 min.As shown in Figures 5(a In this study, Ir/PGlc-NP or Ir/-1,3-glucan-NP was injected into mice via the tail vein and then we took observation of the mice after 24 h.At 24 h after administration, the mice were killed to isolate the organs including liver, lung, heart, kidney, and spleen for fluorescence imaging and the images were shown in Figure 6.The pictures were obtained using optical and X-ray small animal imaging system and the colors with the order of red, orange, yellow, green, and blue in them represented the successive decrease in fluorescence intensity.

Conclusions
In conclusion, we reported the formation of two different water-soluble core-shell nanoparticles based on selfassembly.Morphological characterization of the nanoparticles was obtained by DLS and TEM which demonstrated that the nanoparticles were spherical in shape (∼50 nm in diameter).The photophysics properties of Ir/PGlc-NP and Ir/-1,3-glucan-NP were systematically analyzed by PL emission spectra.In aqueous solutions, Ir/PGlc-NP and Ir/-1,3glucan-NP showed almost the same absorption and emission characteristics because of the similar molecular structure and nature properties.Upon coincubation of Ir/PGlc-NP and Ir/-1,3-glucan-NP with HepG2 cells, strong green fluorescence signals were mainly observed in cytoplasm of the cells.In photocytotoxicity experiment, with MTT assay nanoparticles caused effective apoptosis and death of HepG2 and Hela cells after irradiation at 445 nm for 30 min.Furthermore, we observed the real-time fluorescence imaging of the live mice in vivo after injecting nanoparticles into them and the distribution of the Ir/PGlc-NP and Ir/-1,3glucan-NP in organs from the pictures at 24 after administration.Our results indicated the potential applications of the nanoparticles used as fluorescence probe for live tissue imaging.

3. 1 .
Morphological Characterization.First, dynamic light scattering (DLS) was used to characterize the sample solutions, and the diameter distribution was shown in Figure1.It

Figure 6 :
Figure 6: Fluorescence images of the organs of the mice after intravenous injection of nanoparticles via the tail: (a) Ir/PGlc-NP; (b) Ir/-1,3glucan-NP at 24 h after administration.

Figures 6 (
a) and 6(b) indicated that the distribution of the Ir/PGlc-NP and Ir/-1,3-glucan-NP in organs was similar and the fluorescence intensity in the organs was clearly visualized.The fluorescence intensity distribution suggested that most of the nanoparticles have a tendency to be enriched in the liver; meanwhile, other organs like lung and kidney still have obvious fluorescence intensity distribution.The in vivo studies indicated that Ir (III) complexes packaged with two different water-soluble substances (PGlc, -1,3-glucan) had no difference in the distribution in organs and that these two nanoparticles can serve as fluorescent drug tracer and fluorescent probe for different organs.