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Development of a Light-Controlled Nanoplatform for Direct Nuclear Delivery of Molecular and Nanoscale Materials Ya-Xuan Zhu, Hao-Ran Jia, Guang-Yu Pan, Nathan W. Ulrich, Zhan Chen, and Fu-Gen Wu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13672 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Journal of the American Chemical Society

Development of a Light-Controlled Nanoplatform for Direct Nuclear Delivery of Molecular and Nanoscale Materials

Ya-Xuan Zhu,†,§ Hao-Ran Jia,†,§ Guang-Yu Pan,† Nathan W. Ulrich,‡ Zhan Chen,*,‡ and Fu-Gen Wu*,†

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering,

Southeast University, 2 Sipailou Road, Nanjing 210096, P. R. China ‡

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan 48109, United States

1 ACS Paragon Plus Environment

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ABSTRACT Research on nanomedicines has rapidly progressed in the last few years. However, due to the limited size of nuclear pores (9−12 nm), the nuclear membrane remains a difficult barrier to many nucleus-targeting agents. Here, we report the development of a general platform to effectively deliver chemical compounds such as drug molecules or nanomaterials into cell nuclei. This platform consists of a polyamine unit polyhedral oligomeric silsesquioxane (POSS), a hydrophilic polyethylene glycol (PEG) chain, and a photosensitizer rose bengal (RB), which can self-assemble into nanoparticles (denoted as PPR NPs). Confocal fluorescence imaging showed that PPR NPs mainly located in lysosomes after cellular internalization. After mild light irradiation, however, PPR NPs effectively disrupted lysosomal structures by singlet oxygen (1O2) oxidation and substantially accumulated on nuclear membranes, which enabled further disruption of the membrane integrity and promoted their final nuclear entry. Next, we selected two chemotherapeutic agents (10-hydroxycamptothecine and docetaxel) and a fluorescent dye DiD as payloads of PPR NPs and successfully demonstrated that this nanocarrier could efficiently deliver them into cell nuclei in a light-controlled manner. In addition to molecular compounds, we have also demonstrated that PPR NPs could facilitate the nuclear entry of nanomaterials, including Prussian blue NPs as well as gold nanorods. Compared to traditional strategies for nuclear delivery, this highly controllable nanoplatform avoids complicated modification of nucleus-targeting ligands and is generally applicable to both molecular compounds and nanomaterials.

KEYWORDS: nuclear delivery, lysosomal escape, photosensitizer, chemotherapeutics, nanomaterials


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INTRODUCTION Nanomaterials have been extensively designed into effective drug delivery systems (DDSs), with excellent performance including the enhanced tumor tissue accumulation/retention, reduced systemic side effects, and increased cellular internalization.1−5 However, in many DDSs, the nanocarriers typically alter the intrinsic subcellular fate of their payloads,6 which is an enormous challenge for the delivery of diagnostic and/or therapeutic materials into targeted locations. In some cases, therapeutic agents cannot effectively induce cell death unless they enter the nucleus. For example, doxorubicin (Dox) is a DNA-toxin which damages DNA structure and inhibits the topoisomerase activity in cell nucleus.7 However, the clinically approved nanoagent Doxil (liposomal Dox) is mainly entrapped in lysosomes after cellular internalization with only 0.4% Dox entering the nucleus.8 Therefore, it is important to develop a general platform to efficiently deliver nucleus-targeted materials to cell nuclei, which is the focus of this research. To overcome the problem of efficient nuclear delivery, the designed platform must meet the following criteria: (1) it should have a high amount of cellular uptake, (2) it is able to accumulate in the perinuclear region, and (3) it is capable of facilitating the migration of diagnostic/therapeutic materials into the nucleus.9 It is well known that the cellular internalization of nanoparticles (NPs) can be enhanced if these NPs are coated with targeting ligands or carry positive charges on their surfaces.2 Most of these NPs enter cells via the clathrin-dependent endocytosis which leads to the subsequent endo/lysosomal trafficking for enzymatic degradation.10 It is crucial for the nucleus-targeted NPs to escape from the lysosomes so that they can successfully enter the cell nucleus. Therefore, in addition to



criteria (1) and (2), there is typically another criterion: the designed platform needs to be capable of escaping lysosomes easily. Various approaches have been developed to release the entrapped drug carriers in lysosomes to the cytosol,11 such as pore formation in the lysosomal membrane induced by cationic amphiphilic peptides,12 endo/lysosome rupture caused by the “proton sponge” effect,13−16 pH-responsive disassembly of nanocarriers,17−20 photo-induced disruption of endo/lysosomes,21−29 and so forth.30−34 In addition, the lysosomal escape triggered by light can be synergetic with other methods to enhance the escape efficiency.35−39 Unfortunately, even though the nanoagents can escape from endo/lysosomes, their nuclear entry is still largely encumbered due to the presence of nuclear pore complexes (NPCs) with a pore size of around 9−12 nm,40 which, in most cases, only allows the free passage of small molecules. To facilitate the nuclear entry, nanocarriers are designed with positive surface charge to enhance their electrostatic interaction with the nucleus41−47 or modified with nuclear localization sequence (NLS) peptides to be recognized by importin in the cytoplasm for nuclear translocation.48−54 Even with these developed strategies, the nucleus uptake efficiency of nanomaterials remains low, especially when the size of NPs exceeds that of NPC. For instance, NLS-modified gold NPs (~35 nm) were mainly trapped by the nuclear membrane with rare nuclear internalization.40 Moreover, the poor cellular uptake efficiency of NLS-tagged NPs still remains a challenging issue.55 Recently, it has been reported that the nanoparticle shape is also an important factor that affects the nuclear access of nanomaterials and only appropriately shaped nanoparticles can achieve satisfactory drug release inside the nuclues.56 Consequently, there is an imperative need to design a new generally applicable nuclear delivery platform capable of addressing the abovementioned challenges to effectively


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deliver theranostic materials into cell nuclei for disease treatments. Herein, we rationally fabricated a novel pH-responsive and light-triggerable nanocarrier platform for direct nuclear delivery via increased cell uptake, effective lysosomal escape, and augmented

nuclear

membrane

accumulation

and

permeation.

In

our design,

a

multiamine-containing polyhedral oligomeric silsesquioxane (POSS) molecule is chemically conjugated with a hydrophilic polyethylene glycol (PEG) chain and several hydrophobic rose bengal (RB, a photosensitizer) molecules to form the final PEG-POSS-RB (abbreviated as PPR) conjugate. The as-prepared amphiphilic PPR conjugates can self-assemble into NPs (denoted as PPR NPs) in aqueous solutions and are capable of hydrophobic drug encapsulation (Scheme 1a). Without hydrophobic drug loading, the PPR NPs can achieve enhanced cellular uptake due to their positive surface charges (carried by the amine groups), increased singlet oxygen (1O2) generation under light irradiation in response to lysosomal acidic conditions, and efficient lysosomal escape upon light irradiation via the photochemical disruption of lysosomes (due to the generated 1O2, Scheme 1b). More importantly, the released PPR NPs are able to accumulate on nuclear membranes and augment the permeability of the nuclear membrane by inducing lipid peroxidation, upon further light exposure. To demonstrate that PPR NPs can be employed as a generally applicable nanocarrier

for

nuclear

delivery,

two

chemotherapeutic

agents

including

10-hydroxycamptothecine (HCPT) and docetaxel (DTX), and a commercial fluorescent dye DiD, were all separately loaded into the hydrophobic core of PPR NPs. After being triggered by light, PPR NPs successfully delivered their payloads into cell nuclei. Furthermore, it is feasible to use PPR NPs (together with light irradiation) to facilitate the nuclear entry of



nanomaterials such as Prussian blue (PB) NPs and gold nanorods (Au NRs) which typically cannot cross the NPCs. To the best of our knowledge, the present work represents the first example of using a light-activatable strategy for the nuclear delivery of molecular compounds and nanomaterials.

Scheme 1. (a) Schematic Illustrating the Formation of PPR NPs and PPR/HCPT NPs. (b) Schematic Illustration of the pH-Responsive and Light-Triggerable Nuclear Delivery Strategy Using PPR/HCPT NPs as an Example.


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RESULTS AND DISCUSSION Design and Characterization of PPR NPs. POSS, a three-dimensional cubic nanocage, is one of the smallest silica-like nanostructure. Unlike conventional silica nanoparticles which are hard to chemically modify, a POSS molecule can have covalently bonded, reactive, functionalities at each of the eight corners, making it highly suitable for further modification.57 In addition, with its excellent stability and biocompatibility, POSS has been widely used for bioimaging,58,59 biodetection,60 and drug delivery.61−63 More importantly, the 7 ACS Paragon Plus Environment


rigid three dimensional structure of POSS can prevent the interaction between the photosensitizers (PSs) chemically linked to the eight corners of the molecule, thereby significantly enhancing the fluorescence intensity and singlet oxygen generation of the PSs for improved therapeutic performance.63 In this work, we took full advantage of the merits of POSS to fabricate a smart pH-responsive and light-triggerable nanocarrier platform for effective cell nucleus entry. The successful synthesis of PPR was confirmed by Fourier transform infrared (FTIR) spectroscopy (Figure S1) and 1H nuclear magnetic resonance (NMR) spectroscopy (Figure S2 and S3). NMR data showed that the molar ratio of PEG:POSS:RB was estimated to be 1:1:1.7. This result was further supported by UV–vis spectroscopy (Figure S4), which indicated that the average molar ratio of PEG:POSS:RB was 1:1:1.8. The PPR NPs retained the characteristic absorption peak of free RB with a slight red shift, possibly due to the change in the molecular conformation of RB after chemical conjugation. The loading rate of RB in PPR NPs was 23.8 wt% which is higher than that of most reported PDT nanoconstructs (generally below 10 wt%).64 The PPR NPs in aqueous solution had an average hydrodynamic diameter of 83.6 ± 21.7 nm, as measured with dynamic light scattering (DLS) (Figure 1a). The transmission electron microscopy (TEM) image (Figure 1a, inset) confirmed the spherical structure of the PPR NPs. The smaller size of PPR NPs (~75 nm) revealed by TEM as compared with the hydrodynamic diameter value revealed by DLS was possibly due to the presence of the hydration layer on the surface of the NPs. The zeta potential of PPR NPs at neutral pH was measured to be 12.8 ± 3.6 mV. By treating the PPR NPs with different chemicals, i.e., Triton X-100 (to


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disassociate the hydrophobic interaction in complexes), urea (to form competitive hydrogen bonds), and NaCl (to exert electrostatic shielding effect), we further proved that the different components in the NPs were self-assembled via hydrophobic interaction, since the complexes were effectively disassociated by Triton X-100, but not urea or NaCl (Figure S5). We then measured the fluorescence spectra of free RB and PPR NPs in phosphate buffered saline (PBS) solutions (pH = 7.4). As shown in Figure 1b, although RB molecules were incorporated into PPR NPs, their fluorescence intensity was even higher than that of free RB, confirming that the rigid cubic structure of POSS can restrict the conformational flexibility of grafted RB molecules and thus weaken their π–π stacking interaction, and subsequently reduce their self-quenching effect. In addition, the 1O2 generation of RB in PPR NPs was also evaluated using singlet oxygen sensor green (SOSG) as an indicator, and the results indicated that the grafted RB had a similar 1O2 generation rate as compared to free RB (Figure 1c), demonstrating that the incorporation of RB in NPs did not influence its 1O2 generation capability. This is important because 1O2 could disrupt the lysosomal membranes for PPR NPs to escape from the lysosomes (see more details later). Successful Cellular Entry of PPR NPs. A prerequisite of successful nucleus entry for nanocarriers is a drastic cellular uptake of these nanocarriers. Therefore, we first investigated the cellular entry and cellular fate of the as-designed PPR NPs in human lung cancer A549 cells. Confocal fluorescence images (Figure 1d) revealed that the PPR NPs were considerably internalized by cells, when the incubation time increased. In contrast, no obvious intracellular fluorescence signal was observed in free RB-treated cells even after incubation for 12 h. The enhanced cellular uptake of PPR NPs was mainly ascribed to their positive surface charges.



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We also carried out flow cytometry experiments to quantitatively evaluate the cellular uptake efficiency of PPR NPs. As shown in Figure S6, the internalized PPR NPs gradually increased and reached a plateau at 12 h of incubation, which was approximately 8-fold higher than that of free RB. The cellular entry of NPs is only the first step for NPs to enter cell nuclei. Whether NPs can enter the cell nucleus is related to where the NPs are located after entering cells, which is in turn related to their cellular internalization pathways. To study the internalization mechanism of PPR NPs, A549 cells were pretreated with low temperature or a series of uptake inhibitors to block specific internalization pathways, and then incubated with PPR NPs. Confocal images demonstrated that the uptake of PPR NPs was significantly slowed down in cells after treatment of 4°C or NaN3 (which can inhibit energy-dependent endocytosis), indicating that the uptake process was energy-dependent endocytosis (Figure 1e). For the cells treated with methyl-β-cyclodextrin

(MβCD,

to

inhibit

lipid

raft-mediated

endocytosis),

5-(N,N-dimethyl)amiloride hydrochloride (amiloride, to inhibit macropinocytosis-dependent endocytosis), chlorpromazine hydrochloride (CPZ, to inhibit clathrin-dependent endocytosis), and genistein (to inhibit caveolae-dependent endocytosis), respectively, we can see that the addition of CPZ effectively suppressed the uptake of PPR NPs by > 30%, while the other three inhibitors had little effect on the uptake process (Figure S7). The above results suggest that PPR NPs entered cells mainly through clathrin-dependent endocytosis. It has been reported that clathrin-dependent endocytosis ultimately transports internalized NPs into lysosomes.10 This matched the observed distribution pattern of PPR NPs in A549 cells, indicating that the PRR NPs were located in lysosomes after cellular internalization. To


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further confirm this, PPR NPs-treated cells were co-stained with LysoTracker Green (a commercial fluorescent probe for lysosomes). Confocal images showed that the distribution of the intracellular fluorescence signals emitted by PPR NPs overlapped almost completely with that of LysoTracker Green (Figure 1f). The overlap coefficient (OLC) and Pearson’s correlation coefficient (PCC) were calculated to be 0.83 and 0.80, respectively, further confirming the lysosomal localization of PPR NPs.

Figure 1. Characterization and cellular fate of PPR NPs. (a) DLS result of PPR NPs in a PBS solution. Inset: TEM image of PPR NPs (scale bar = 100 nm). (b) Fluorescence spectra of free RB and PPR NPs (3 µg/mL RB in both samples) in PBS solutions. (c) 1O2 generation of free RB and PPR NPs (3 µg/mL RB in both samples) in PBS solutions under 532 nm laser irradiation (8 mW/cm2) as reflected by the fluorescence intensity changes of SOSG. (d) Confocal fluorescence images of A549 cells after incubation with free RB or PPR NPs (3 µM RB in both samples) for different time periods. Scale bar = 25 µm. (e) Confocal fluorescence images A549 cells after incubation with PPR NPs (3 µM RB) in the absence (control) and 11 ACS Paragon Plus Environment


presence of 4°C or NaN3 treatment. Scale bar = 10 µm. (f) Confocal fluorescence images of A549 cells stained by LysoTracker Green after incubation with PPR NPs (3 µM RB) for 2 h. Scale bar = 10 µm.

pH-Responsive and Light-Triggered Lysosomal Escape of PPR NPs. To enter the cell nucleus, it is necessary for NPs trapped in lysosomes to be efficiently released into the cytoplasm. We then investigated the capability of PPR NPs to escape from the lysosomes by light irradiation. Light irradiation could trigger RB to generate highly oxidative 1O2, which could effectively cause the rupture of lysosomal membranes and then induce lysosomal disruption for PPR NPs to escape.24,26 Acridine orange (AO) staining was performed to evaluate the integrity of lysosomes. Upon excitation, AO emits intense red fluorescence in acidic lysosomes, while it generates green fluorescence in cytoplasm and nuclei. Before light exposure, a large number of red dots were observed, validating the presence of intact lysosomes (Figure 2a). The red fluorescence signal disappeared after exposing the sample to mild light irradiation (2 mW/cm2, 3 min) from a white LED, which indicated the effective photodynamic disruption of the lysosomal structure. It is worth noting that the environment inside lysosomes is highly acidic with an internal pH around 5.11 We found that in an acidic environment (e.g., an acetic acid/sodium acetate buffer with pH = 5.0), PPR NPs produced a much larger amount of 1O2 compared to that in a PBS solution with pH = 7.4 (Figure 2b). The low pH environment in lysosome greatly facilitated the RB component in PRR NPs to generate more 1O2 upon light irradiation to disrupt lysosomal membranes so that NPs can effectively escape from the lysosomes.


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We hypothesize that the enhanced 1O2 generation of PPR NPs in acidic conditions may be due to the protonation of the remaining unreacted amine groups on POSS, and thus the electrostatic repulsion between protonated amine groups can compete with the hydrophobic force between RB molecules, which weakens the self-quenching effect of RB to a certain extent. This would increase the lifetime of RB excited states, facilitating both 1O2 generation and fluorescence emission. In addition, the remaining unreacted amine groups could have a “proton sponge” effect inside the acidic lysosomes, which synergistically promoted the rupture of lysosomes. To verify the presence of unreacted amine groups in PPR NPs, a ninhydrin-based assay was carried out according to the previously reported protocol.65 The result revealed that 1 mg PPR NPs contained 0.68 µmol amine groups. Next, we compared the fluorescence intensity of PPR NPs at pH 5.0 and 7.4. It was found that PPR NPs emitted stronger fluorescence in a buffer with a lower pH value (Figure 2c), further confirming the weakened self-quenching effect of RB. An increase in the hydrodynamic size of PPR NPs from 83.6 nm at pH 7.4 to 94.8 nm at pH 5.0 was observed (Figure S8), indicating the loosened nanostructure of PPR NPs in an acidic environment, reducing the close contact of RB molecules and further avoiding the RB self-quenching. To further confirm the role of amine groups in the enhancement of 1O2 generation of PPR NPs under acidic conditions, RB was directly grafted with NH2-PEG5k via the carboxyl–amine reaction to form PEG-RB conjugate, which could also self-assemble into NPs, but with no amine functionality, and an average size of 37 nm (Figure S9). Similar to PPR NPs, PEG-RB NPs were also distributed in lysosomes after internalization (Figure S10). However, different from the PPR NPs, the PEG-RB NPs failed to effectively disrupt the integrity of lysosomes under the same



irradiation condition, as demonstrated by the intense red fluorescent dots (the labeled lysosomes) of A549 cells after AO staining (Figure S11). These results strongly suggest the importance of the remaining additional amine groups introduced by POSS in PPR NPs, which facilitated the light-triggered lysosomal rupture of the NPs in a pH-responsive manner so that NPs could effectively escape from the lysosomes. Light-Driven Intracellular Redistribution of PPR NPs from Lysosomes to Nuclei. Next, we carefully studied the subcellular distribution of PPR NPs after lysosomal escape. A549 cells were incubated with PPR NPs (3 µM RB) for 2 h and then stained by Hoechst 33342 (to label cell nuclei). Confocal imaging was performed before and after white light irradiation (2 mW/cm2, 3 min). As shown in Figure 2d, vacuolated structures labeled by red fluorescent dyes (the RB moieties in PPR NPs) were observed after light exposure, possibly indicating the enlarged and swollen lysosomes under 1O2-induced oxidative stress. This observation confirms the light-induced lysosomal disruption. Interestingly, we also noticed an obvious red fluorescence distribution at the boundary of the blue fluorescent nucleus, which implied that PPR NPs could be substantially accumulated on the nuclear membrane. To verify the corresponding co-localization efficiency between PPR NPs and the nucleus, we performed fluorescence intensity profile analysis in the areas marked by white lines in Figure 2d, and the results shown in Figure 2e, confirmed that the position of the maximum fluorescence intensity of PPR NPs (marked by the arrows) almost overlapped with the periphery of the maximum fluorescence area of Hoechst 33342 after light irradiation, which is a unique character of the PPR NPs. We also studied the redistribution behavior of PEG-RB NPs after light irradiation. Even though a fraction of PEG-RB NPs leaked out from lysosomes into other places in the


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cytoplasm, no nuclear membrane accumulation was observed (Figure S12). It has been well demonstrated that a number of amine-containing cationic polymers, such as poly(ethylene imine) (PEI), could be used for nucleic acid delivery due to their nuclear localization property.66,67 Hence, we propose that the nucleus-tropic property of PPR NPs should share the same mechanism as that of PEI-type polymers.

Figure 2. Lysosomal escape and nuclear membrane distribution of PPR NPs. (a) Observation of lysosomal disruption of A549 cells treated with PPR NPs before and after white light irradiation (2 mW/cm2, 3 min) using AO staining. AO generates red fluorescence in acidic lysosomes and emits green fluorescence in cytosol and nucleus. Scale bar = 20 µm. (b) 1O2 generation of PPR NPs in the buffer solutions at pH 5.0 and 7.4 under laser irradiation (532 nm, 8 mW/cm2). (c) Fluorescence spectra of PPR NPs in the buffer solutions at pH 5.0 and 7.4. (d) Confocal fluorescence images of A549 cells stained by Hoechst 33342 after 15 ACS Paragon Plus Environment


incubation with PPR NPs (3 µM RB) for 2 h before and after white light irradiation (2 mW/cm2, 3 min). Scale bar = 10 µm. (e) The corresponding fluorescence intensity profiles of the areas marked by white lines in the confocal images in (d). The green-shaded areas indicate the nuclear regions.

It is worth noting that a part of PPR NPs could be detected inside the nucleus after light exposure (Figure 2e), demonstrating the nuclear entry of the NPs. In view of the much larger size of PPR NPs than NPCs, the PPR NPs cannot pass through nuclear membranes in a regular manner. We believe that the significant accumulation of PPR on nuclear membranes disrupted the membrane integrity upon light irradiation, therefore allowing PPR NPs to enter nuclei freely. In this regard, we deduced that PPR NPs could serve as a unique but general nanoplatform for nuclear entry. First, this process was activated by external light rather than internal stimuli, which is highly controllable. In addition, the mechanism of this strategy is based on the enhanced permeability of cell membranes caused by lipid oxidation by 1O2 and can cause irreversible damage to nuclear membranes, which is difficult for cancer cells to develop resistance to. Last but not least, the PPR NPs may facilitate not only chemical compounds but also nanoparticles to enter cell nuclei without the tedious conjugation steps of targeting ligands. PPR NPs-Assisted Nuclear Delivery of Hydrophobic Drugs/Dyes. To test whether the PPR NPs can help the nuclear delivery of molecular compounds, several hydrophobic molecular cargos were chosen for this purpose since these molecules can be encapsulated within the hydrophobic cores of the PPR NPs via hydrophobic interactions. First, HCPT was


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selected as a model. HCPT is a DNA-topoisomerase I inhibitor and has been extensively used as an anticancer drug. It is extremely difficult to deliver free HCPT to the cell nucleus where it exerts therapeutic effect, possibly due to its extremely poor solubility in aqueous solutions.68 Here, we constructed a HCPT-loaded PPR NPs (denoted as PPR/HCPT NPs) via hydrophobic encapsulation. The as-prepared NPs had an average size of 92.3 nm (Figure S13), and the drug encapsulation ratio (ER%) and drug-loading coefficiency (DL%) of HCPT were measured to be 81.6% and 8.2 wt%, respectively. The release behavior of HCPT from PPR/HCPT NPs (Figure S14) suggests that the nanoconstruct has acceptable stability. Next, we investigated whether the PPR/HCPT NPs could effectively enter cancer cells. A549 cells were incubated with free HCPT or PPR/HCPT NPs for different incubation time periods, followed by confocal fluorescence imaging. The intracellular fluorescence of free HCPT was considerably weak, demonstrating its poor cellular internalization (Figure S15). In contrast, the cellular uptake of PPR/HCPT NPs was very high at 2 h, and the uptake amount further increased as the incubation time increased to 6 and 12 h. The internalization efficiency of free HCPT and PPR/HCPT NPs was further determined by measuring the fluorescence intensity of HCPT in each cell using the ImageJ software. As predicted, the PPR/HCPT NPs showed a significantly higher (~6-fold) uptake efficiency than that of free HCPT, as shown in Figure S16. We also noticed that the fluorescence signal of RB coincided with that of HCPT, indicating the integrity of the PPR/HCPT nanoconstruct after cellular internalization. The distribution of PPR NPs in lysosomes was not altered after HCPT encapsulation, as confirmed by the colocalization between RB/HCPT and the commercial lysosome dye LysoTracker Green (Figure 3a).



We then explored the feasibility of light-triggered nuclear delivery of HCPT by PPR NPs. As shown in Figure 3b, PPR/HCPT NPs were efficiently released from lysosomes into cytoplasm after white light irradiation (2 mW/cm2, 3 min). In addition, both HCPT and PPR were clearly observed inside the white dotted circles which represented the boundaries of cell nuclei. These results unambiguously demonstrated the successful nuclear delivery of HCPT with the help of PPR NPs by augmenting the permeability of nuclear membranes under light irradiation. The improved therapeutic efficacy of PPR/HCPT NPs after nuclear entry was confirmed by cell counting kit-8 (CCK-8) assays (Figure 3c and 3d) and flow cytometric analysis (Figure S17) via the dual staining of annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). As expected, PPR/HCPT NPs plus light irradiation exhibited the highest cancer cell killing efficiency. The synergistic effect of PDT and chemotherapy for PPR/HCPT NPs was demonstrated by the combination index (CI) value which was smaller than 1 (Figure S18). In marked contrast, HCPT-loaded PEG-RB NPs (PEG-RB/HCPT NPs) which were also entrapped in lysosomes (Figure S19) only exhibited moderate therapeutic efficiency (Figure S20) with the CI value even larger than 1 (Figure S18), which was mainly attributed to their ineffective lysosomal escape and difficulty of nuclear entry.


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Figure 3. Confocal fluorescence images and in vitro combination therapy of PPR/HCPT NPs. (a) Confocal fluorescence images of A549 cells stained by LysoTracker Green after incubation with PPR/HCPT NPs for 12 h. Scale bar = 10 µm. (b) Confocal fluorescence images of A549 cells treated with PPR/HCPT NPs before and after light irradiation. The images in the “Magnified” row come from the dotted squares in the “After irradiation” row. The white dotted circles represent the area of cell nuclei. Scale bar = 10 µm. (c) Viabilities of A549 cells after incubation with free RB, free HCPT, PPR NPs, or PPR/HCPT NPs at different concentrations of RB (0.1, 0.2, 0.3, 0.5, 0.8, and 1.0 µM) for 24 h. The cell viabilities were determined by CCK-8 assay. (d) Viabilities of A549 cells after incubation with free RB, free HCPT, PPR NPs, or PPR/HCPT NPs under white light irradiation (2 mW/cm2, 5 min). All these cells were then incubated for 24 h before CCK-8 assay.



To further prove that the PPR NPs are generally applicable to facilitate the nuclear entry of other molecular drugs or chemical compounds, PPR NPs were incorporated with another commonly used anticancer drug DTX and a hydrophobic fluorescent dye DiD, which is a commercial red fluorescent dye for cell membrane labeling. Because DTX is non-fluorescent, we could not directly track its subcellular localization. However, the DTX loaded PPR NPs (PPR/DTX NPs) displayed a similar trend of drug activity as PPR/HCPT NPs (Figure S21). We also investigated the cellular fate of DiD after being loaded in PPR NPs. In this study, DiD was employed as a general model of hydrophobic compounds. As shown in Figure S22, after being encapsulated in the hydrophobic core of PPR NPs, DiD molecules were observed in the lysosomes rather than cell membranes, showing completely different cellular localization from that of free DiD. Similarly, light irradiation treatment also triggered the lysosomal escape as well as nuclear entry of the loaded DiD, further proving the feasibility of developing PPR NPs as a nanoplatform to realize the nuclear entry of hydrophobic drugs or compounds. PPR NPs-Assisted Nuclear Entry of Nanoparticles. To further demonstrate the generality of the nuclear delivery strategy, we proceeded to investigate the feasibility of delivering nanomaterials into cell nuclei by using PPR NPs as a precursor. Generally, it is extremely difficult for NPs larger than 39 nm to cross the NPC and enter the cell nucleus.69 Our aim was to use the nucleus membrane disruption ability of PPR NPs under light irradiation to compromise the integrity of the nuclear membrane so that NPs can enter nuclei (Figure 4a). As a proof-of-concept, we selected PB NPs (~50 nm) and PEG-modified Au NRs (10 nm × 100 nm) as two typical representatives of NPs for nuclear delivery with the help of PPR NPs.


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Recently, PB NPs have attracted considerable attention in biomedical applications because of their excellent biocompatibility and biosafety,70 great stability in human blood serum,71 as well as enzyme-like activity.72−74 In addition, PB NPs are also widely employed in cancer theranostics as multifunctional contrast agents,75−77 photothermal agents,78−82 and drug carriers.78,79 In this study, the subcellular localization of PB NPs was monitored by dark-field imaging, which is advantageous for the imaging of materials with strong light scattering signal and structured surface.83 As displayed in Figure 4b, PB NPs could enter cells and were distributed throughout the cytoplasm. Although a fraction of PB NPs could be found at the perinuclear region, no scattering signal from PB NPs was observed inside the nucleus (stained blue by Hoechst 33342). The blockage of nuclear entry of PB NPs was due to their large size, as compared with the pore size of NPC. The PB NPs-treated cells were then further incubated with PPR NPs for 2 h, followed by mild light irradiation. Large white dots (the light scattering signals from PB NPs) were observed in the nucleus. As designed, the PPR NPs damaged nuclear membranes under light irradiation, which greatly helped PB NPs to enter cell nuclei. The capability of PPR NPs to realize the nuclear entry of nanomaterials was further supported by employing Au NRs as another model. Au NRs are a representative of gold nanomaterials which have been widely investigated in cancer therapy (e.g., radiotherapy and photothermal therapy) due to their good biocompability and low toxicity.84−88 The subcellular distribution of Au NRs was also monitored by dark-field imaging. As shown in Figure 4b, using confocal fluorescence imaging and dark-field imaging, we can see that most Au NRs were distributed in cytoplasm before light irradiation but strong light scattering signal of Au NRs was observed in cell nuclei after PPR NPs were irradiated with light. The



confocal images of PB NPs- and Au NRs-treated A549 cells were shown in Figure S23, which revealed that the nanoparticles could not enter nuclei after light irradiation without the assistance of PPR NPs. Collectively, the above results demonstrate that PPR NPs could serve as a light-triggerable precursor for the nuclear entry of nanomaterials, which may promote their therapeutic effect.

Figure 4. (a) Schematic diagram of light-promoted nuclear entry of nanoparticles larger than nuclear pores assisted by PPR NPs. (b) Confocal images of A549 cells treated by PB NPs and


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Au NRs before and after light irradiation (2 mW/cm2, 3 min). The cell nuclei were stained blue by Hoechst 33342 using confocal fluorescence imaging. the PB NPs and Au NRs were shown as white dots using dark-field imaging. Scale bar = 10 µm.

CONCLUSION In this work, we rationally designed a general nanoplatform which can facilitate hydrophobic molecules as well as nanomaterials to enter cell nuclei in a light-controlled manner. This nanoplatform (termed as PPR NPs) is formed by the self-assembly of POSS-PEG-RB molecules consisting of POSS, PEG, and RB moieties. It was shown that PPR NPs achieved enhanced cellular uptake benefiting from their positive surface charges and could be entrapped by lysosomes after cellular internalization. More importantly, the protonation of amine groups in acidic conditions caused electrostatic repulsion inside the nanoconstruct, which weakened the self-quenching effect of RB molecules and thus enhanced their 1O2 generation upon light irradiation. In doing so, the lysosomal structure suffered severe rupture under 1O2 oxidative stress, leading to the release of PPR NPs. Subsequently, in view of the polyamine property of POSS, PPR NPs could substantially accumulate on nuclear membranes. With further light irradiation, the integrity of nuclear membranes was compromised by 1O2 oxidation, allowing the nuclear entry of PPR NPs. The unique mechanism of the strategy makes PPR NPs suitable as a general platform for nuclear delivery. As a proof-of-concept, PPR

NPs

were

loaded

separately

with

hydrophobic

molecules

including

two

chemotherapeutics (HCPT and DTX) and the fluorescent dye DiD, and successfully realized the nuclear delivery of these molecular compounds with the help of light irradiation. In



addition, we also adopted PPR NPs as a precursor to trigger the nuclear membrane disruption upon light irradiation and assisted other nanomaterials such as PB NPs and Au NRs to enter nuclei. This light-controlled nuclear delivery strategy avoids traditional chemical modification of targeting ligands and is generally applicable to not only molecular compounds but also nanomaterials, which may shed new light on the development of more intelligent DDSs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials, methods, and supporting figures S1−S23 (PDF)

AUTHOR INFORMATION Corresponding Authors *(F.G.W.) E-mail: [email protected]. *(Z.C.) E-mail: [email protected].

Author Contributions §

Y.X.Z. and H.R.J. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS


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This work was supported by grants from the National Natural Science Foundation of China (21673037), Natural Science Foundation of Jiangsu Province (BK20170078), Fundamental Research Funds for the Central Universities, Innovative and Entrepreneurial Talent Recruitment Program of Jiangsu Province, and Six Talents Peak Project in Jiangsu Province (2015-SWYY-003). Z.C. thanks the University of Michigan for the support.

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