Multifunctional organic nanoparticles with aggregation-induced emission (AIE) characteristics for targeted photodynamic therapy and RNA interference therapy

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Nanoparticle-based theranostic platforms that allow incorporation of both imaging and therapeutic reagents into one single probe are strongly desired for cancer diagnosis and treatment, which facilitate concurrent image-guided diagnosis and therapies. 1Among various theranostic platforms, photodynamic therapy (PDT) has been increasingly recognized as an attractive approach for cancer treatment because the PDT treatment regulated by a beam of light has distinct advantages such as precise controllability, minimal invasive nature and high spatiotemporal accuracy. 2The mechanism of PDT is that the photosensitizers are able to generate toxic reactive oxygen species (ROS) after light irradiation to kill tumor cells.Additionally, the fluorescence from photosensitizers could also facilitate imaging-guided theranostic process.However, traditional fluorescent photosensitizers generally suffer from π-π stacking due to the intrinsic hydrophobic and rigid planar molecular structures, further leading to aggregation caused quenching (ACQ) and significant decrease in ROS generation. 3Recently, we have demonstrated that a class of fluorogens with aggregation-induced emission (AIEgens) characteristics could act as effective fluorescent materials for theranostic applications. 4The propeller-shaped AIEgens are generally non-emissive in solution but become highly emissive upon aggregation caused by the restriction of intramolecular rotations, which block the non-radiative pathway and activate the radiative channels for energy dissipation. 5As such, the AIEgen-based photosensitizers are excellent key components for PDT with the expectation to yield bright emission and high phototoxicity upon loading into nanocarriers. 6This is opposite to the traditional ACQ photosensitizers, which offer quenched fluorescence and reduced phototoxicity in the nanoparticle (NP) format.

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Another challenge in PDT is that the cancer cells could respond to ROS stress by upregulating the level of vascular endothelial growth factor (VEGF) to suppress cellular senescence by inducing angiogenesis.
8 As a result, the resistance process in tumors treated with PDT will attenuate its therapeutic effect.To address this issue, small interfering RNA-vascular endothelial growth factor (siVEGF) that has been widely used to suppress VEGF expression for inhibition of tumor growth and metastasis in cancer treatments has been used to provide a promising solution. 9However, the delivery of short small interfering RNA (siRNA) remains a key challenge in the development of RNA interference therapeutics because of the poor stability of siRNA in biological environment. 10Recently, it has been found that the stability of siRNA against enzymatic degradation could be improved when siRNA conjugated poly(ethylene glycol)-lipid forms nanocarriers. 11Considering the above mentioned facts, a new multifunctional theranostic platform based on AIE photosensitizers with the ability for siRNA delivery will offer synergistic effect to achieve improved therapeutic outcome in image-guided PDT.
Typically, intersystem crossing occurs from the singlet excited state to the triplet state upon excitation of the photosensitizers, which is followed by reaction with molecular oxygen to yield ROS, including singlet oxygen ( 1 O 2 ). 16To achieve satisfied therapeutic outcome, photosensitizers with a high 1 O 2 yield is desirable.17 Therefore, the 1 O 2 quantum yield of cRGD-siVEGF-TTD NPs (Φ TTD ) was quantified using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) as the indicator and Rose Bengal (RB) as the standard photosensitizer (the 1 O 2 quantum yield for Φ RB is 75% in water). 18 the presence of 1 O 2 , ABDA could undergo oxidation to yield endoperoxide, resulting in a decrease of ABDA absorption. 19Under white light irradiation, the absorbance of ABDA solution with cRGD-siVEGF-TTD NPs decreased gradually with prolonged irradiation time (Fig. S3 ESI †), indicating the generation of 1 O 2 from cRGD-siVEGF-TTD NPs in the solution.The absorbance of ABDA in the presence of photosensitizer (cRGD-siVEGF-TTD NPs or RB) at 378 nm before and after irradiation was defined as A 0 and A, respectively.The plot of ln(A 0 /A) against time gives straight lines in Fig. 1C.From the slopes, the decomposition rate constants (K TTD ) of cRGD-siVEGF-TTD NPs and RB (K RB ) could be calculated as 0.0058 and 0.0072, respectively.The integrations of the optical absorption in the wavelength range of 400-800 nm for RB (A RB ) and cRGD-siVEGF-TTD NPs (A TTD ) were 21.09 and 18.68, respectively.The 1 O 2 quantum yield of cRGD-siVEGF-TTD NPs was calculated to be 68.2%.The in vitro release profiles of siVEGF from cRGD-siVEGF-TTD NPs were also studied in 1× PBS buffer with or without GSH.The concentration of GSH solution is 10 mM, mimicking intracellular reductive conditions. 20It is known that the concentration of GSH in intracellular compartments is significantly higher than that in extracellular plasma and the difference in GSH concentration between cytoplasm and extracellular space provides a great potential in achieving highly efficient delivery and controlled release of siVEGF that was conjugated on the surface of cRGD-siVEGF-TTD NPs though disulfide bonds. 21As shown in Fig. 1D, faster release of siVEGF was triggered in the presence of GSH, suggesting that 31% and 73% of the siVEGF were released in the GSH-supplemented buffer after 6 and 72 h, respectively.However, only 18% and 47% of the siVEGF were released in buffer without GSH after 6 and 72 h, respectively.These data revealed that the effective intracellular release of siVEGF could be expected in cell cytoplasm upon internalization.

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Please do not adjust margins The targeting effect of cRGD-siVEGF-TTD NPs was investigated using MDA-MB-231 cancer cells with overexpressed α v β 3 integrin as the target cells, while MCF-7 and SK-BR-3 cancer cells with low expression level of α v β 3 integrin were used as the negative controls. 22The fluorescence signals of NPs in MDA-MB-231, MCF-7 and SK-BR-3 cells were recorded under confocal microscope after incubation with cRGD-siVEGF-TTD NPs at 5 μg mL -1 of TTD for 4 h.
As shown in Fig. 2, strong red fluorescence from the internalized NPs in the cytoplasm was clearly observed in MDA-MB-231 cells (Fig. 2A).On the contrary, the MCF-   To evaluate the cytotoxicity of TTD, cRGD-TTD NPs without siVEGF were fabricated from the same nanoprecipitation method using DSPE-PEG-Mal as the encapsulation matrix.MTT results indicated that the viability of MDA-MB-231 cells was kept at above 90% after incubation with cRGD-TTD NPs at 10, 20 and 30 μg mL -1 of TTD for 48 h, indicating the low cytotoxicity of cRGD-TTD NPs to cells (Fig. S8, ESI †).The synergistic effect of PDT and RNAi therapy from cRGD-siRNA-TTD NPs was further evaluated by MTT assay.SK-BR-3, MCF-7 and MDA-MB-231 cells were incubated with cRGD-siRNA-TTD NPs at 5 μg mL -1 of TTD for 4 h.The free NPs were washed away and the cells were exposed to 0.2 W cm 2 light irradiation for 10 minutes, followed by incubation in fresh medium for 24 and 48 h, respectively.The results suggested that the cRGD-siVEGF-TTD NPs showed obvious cytotoxicity to MDA-MB-231 cells and the viability of MDA-MB-231 cells without light illumination This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins decreased to 61% after 48 h, which was mainly caused by delivered siVEGF though RNA interference.Exposure to light irradiation resulted in a further decrease of the viability of MDA-MB-231 cells to 25% after 48 h.Noteworthy is that the viability of MDA-MB-231 cells treated with cRGD-TTD NPs without surface siVEGF remained 70% after the same PDT treatment while the light irradiation itself did not cause damage to cell viability (Fig. S9, ESI †).These data confirmed the synergistic effect of RNA interference and PDT effect, which could lead to more effective therapeutic outcome.From Fig. 3B, it could also be seen that the viability of MDA-MB-231 cells after cRGD-siVEGF-TTD NP treatment was significantly lower as compared to that of SK-BR-3 and MFC-7 cells under the same experimental conditions, revealing the targeted therapeutic effect of cRGD-siVEGF-TTD NP to integrin-overexpressed cancer cells.
In conclusion, a new multifunctional AIE NP based siRNA vector was successfully developed for combined image-guided PDT and RNAi therapy to targeted cancer cells.The platform shows bright fluorescence and could effectively generate ROS under light irradiation.VEGF siRNA conjugated on the surface of NPs has been successfully transfected to cancer cells to downregulate VEGF mRNA and protein expressions.Cell viability studies showed that the siVEGF-TTD NPs could selectively and efficiently kill the α v β 3 integrin overexpressed cancer cells with synergistic effect between PDT and RNA interference.In future, AIE photosensitizers with efficient absorption in long wavelength will be promising to explore the potential of such multifunctional theranostic platform in in-vivo research.

Figure 1 .
Figure 1.(A) Transmission electron microscopy (TEM) image of cRGD-siVEGF-TTD NPs.The scale bar is 200 nm.(B) UV-vis absorption and emission (λex = 494 nm) spectra of cRGD-siVEGF-TTD NPs in water.The inset shows a photo of NPs in water under UV lamp.(C) The decomposition rates of ABDA in the presence of cRGD-siVEGF-TTD NPs and RB; A0 and A are the absorbance of ABDA in the presence of the photosensitizers at 378 nm before and after irradiation, respectively.(D) Time course of release profiles of siVEGF from cRGD-siVEGF-TTD NPs in in 1× PBS buffer with or without GSH at 37 °C.
7 and SK-BR-3 cells showed much weaker fluorescence, indicating the higher internalization efficiency of cRGD-functionalized NPs in MDA-MB-231 cells.This observation was consistent with the mean fluorescence intensity of each cell quantitatively analyzed by ImageJ from confocal images of MDA-MB-231, MCF-7, and SK-BR-3 cells (Fig. S4 ESI †).To evaluate the ROS productivity by cRGD-siVEGF-TTD NPs after cancer cell uptake, a cell permeable fluorescent reagent (CellROX ® deep red reagent) was employed to detect the ROS generation under light irradiation.In the presence of ROS, the CellROX® deep red reagent could be rapidly oxidized to exhibit strong red fluorescence.As shown in Fig. 2B, strong red fluorescence was observed from the cell cytoplasm, suggesting efficient ROS generation from the cRGD-siVEGF-TTD NPs.The NP-treated cells without light irradiation were used as control.No fluorescence could be observed from the cells under confocal microscope with the same parameters (Fig. S5, ESI †), further confirming that the red fluorescence is only from the oxidized CellROX® deep red reagent in the presence of ROS.

Figure 3 .
Figure 3. (A) The relative VEGF protein level in culture medium of cRGD-siVEGF-TTD NPs (5 μg mL -1 of TTD) treated MDA-MB-231 cells and VEGF mRNA level determined from the lysate of MDA-MB-231 cells.Controls were shown in black and set to be 100%.(B) Viability of MDA-MB-231, MCF-7 and SK-BR-3 cells after incubation with cRGD-siVEGF-TTD NPs (5 μg mL -1 of TTD) for 4 h followed by light irradiation (0.20 W cm -2 , 10 min) and further incubation in fresh medium for 24 and 48 h.Data present mean values ± standard deviation, n = 3.