A reduction and pH dual-sensitive polymeric vector for long-circulating and tumor-targeted siRNA delivery.

www.advmat.de www.MaterialsViews.com

COMMUNICATION

A Reduction and pH Dual-Sensitive Polymeric Vector for Long-Circulating and Tumor-Targeted siRNA Delivery Jingguo Li, Xingsu Yu, Yong Wang, Yuanyuan Yuan, Hong Xiao, Du Cheng, and Xintao Shuai* Owing to the distinct virtue in silencing onco-related genes, the emerging RNA interference technique has drawn great attention in cancer treatment over the past decade.[1–3] However, its clinical application potential is still far from being realized due to some intractable problems including the non-specific action, inability to cross cell membrane and other biological barriers, and nuclease degradation of the small interfering RNA (siRNA) molecules in vivo. Admittedly, highly effective and meanwhile safe carrier systems for siRNA delivery are crucial for this promising technique to be applied in clinical disease treatments.[4] To date, in addition to tremendous efforts in developing viral vectors for delivery of genetic medicines, a great number of nonviral polymeric vectors such as poly(ethylenimine) (PEI),[5] poly(L-lysine),[6] chitosan,[7] and polyamidoamine (PAMAM) dendrimers[8] have been intensively investigated as well. Upon forming a compact nanocomplex with these amine-contained cationic polymers, i.e., the so-called polyplex, the siRNA molecules can be protected from enzymatic degradation in vivo. Furthermore, the polyplex can be readily modified with a tumor targeting ligand to enable tumor cell-specific uptake and action. Since complexation of the anionic siRNA with cationic polymers is driven by electrostatic interactions, the molar ratio of polymer nitrogen to siRNA phosphorus, namely the N/P ratio, significantly influences the performance of polyplex both in vitro and in vivo. Based upon a literature survey of numerous works on polymeric vectors for nucleic acid delivery, one can easily find that a relatively high N/P ratio (usually above 10) is generally required for achieving high transfection efficiency.[9–13] The underlying reason is that only at high N/P ratios, the nucleic acids can be fully complexed to prevent the nuclease degradation, and meanwhile the polyplexes possess a positive surface prone to binding the negative cell membrane for enhanced endocytosis. However, the cationic polyplexes have intrinsically insurmountable problems hindering their in vivo applications. Firstly, they are apt to adsorb opsonin or other negatively J. Li,[+] Y. Wang, Y. Yuan, H. Xiao, Dr. D. Cheng, Prof. X. Shuai PCFM Lab of Ministry of Education School of Chemistry and Chemical Engineering Sun Yat-sen University Guangzhou 510275, China E-mail: [email protected] X. Yu,[+] Prof. X. Shuai Center of Biomedical Engineering Zhongshan School of Medicine Sun Yat-sen University Guangzhou 510080, China [+] These authors contributed equally to this work.

DOI: 10.1002/adma.201403877

Adv. Mater. 2014, DOI: 10.1002/adma.201403877

charged serum proteins in the bloodstream if PEG protection is insufficient, resulting in large aggregates with decreased capability of penetrating the leaky tumor vascular endothelium as well as short circulation time due to easy clearance by the mononuclear phagocytic system (MPS) after opsonization.[14,15] Secondly, non-specific cell uptake is inevitable because of their electrostatic interaction with all cell types. Thirdly, high cytotoxicity exists because cationic objects usually cause cellular membrane damage. On the contrary, polyplexes formed at low N/P ratios are negatively charged nanoparticles with uncomplexed nucleic acid chain moieties situated on their surface. Due to the negative surface charge, they are much less cytotoxic and more anti-biofouling than the cationic polyplexes formed at high N/P ratios, i.e., surface more resistant to non-specific adsorption of opsonin and other serum proteins. Despite their advantages, the anionic polyplexes can hardly be taken up by cells, and even worse, the uncomplexed nucleic acid fragments exposed on the surface are subject to nuclease degradation, both of which make their in vivo application highly problematic. Therefore, construction of a new generation of polyplexes combining the advantages while avoiding the disadvantages of the cationic and anionic ones has become a hot research topic nowadays in cancer siRNA therapy. So far, the most commonly adopted approach to achieve the goal utilizes a so-called ‘sheddable’ strategy. The polyplexes thus prepared usually contain a water soluble and microenvironment-sensitive coating which is stable during blood circulation but self-removable in tumor issue by responding to local stimuli such as decreased extracellular pH or upregulated matrix metalloproteinase.[16] In most events, these polyplexes were surface-decorated with an acid or enzyme-detachable polyethylene glycol (PEG) shell.[17,18] The sheddable polyplexes remain intact during circulation in bloodstream. However, when they accumulated in acidic tumor tissue (below pH 6.8) via a mechanism known as enhanced permeability and retention effect (EPR) stemming from the leaky vasculature and lack of lymphatic drainage,[19] the PEG layer departs to expose a cationic surface for electrostatic interaction with cancer cells that enhances endocytosis. Although great potential has been demonstrated, PEG shedding only decreases the surface charge of polyplex from a high positive value to a weak positive one, which means that the risk of non-specific cell uptake in normal tissues via electrostatic interaction may still exist. In this context, different strategies to construct polyplexes, which have a neutral or even negatively charged particle surface in bloodstream but can reverse to a cationic one in tumor tissue, are envisioned to provide further opportunity to balance the long circulation property and easy cancer cell uptake of polyplexes. It is well-known that PEI has been the most intensively investigated polymeric vector with unparallel properties in

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

wileyonlinelibrary.com

1


COMMUNICATION

delivering nucleic acids.[2,20] In addition to the remarkable polyanion affinity and chain flexibility essential for siRNA complexation and condensation, PEI can exert a unique ‘proton sponge effect’ to escape from endosomal entrapment and release the complexed nucleic acids into cytosol. Owing to the multiamine structure and close neighborhood of linker amino groups, PEI retains substantial proton-buffering capacity at virtually any pH.[21] For example, the amine protonation degree of branched PEI increases to about 45% from approximately 20% when the solution pH decreases to 5 from 7.[22] This distinct feature of PEI has driven us to consider whether it can be utilized to design a surface charge reversible polyplex which is triggerable by environmental pH change. Aiming at a proof of concept demonstration, we attempted to prepare a polyplex, which has a negative surface charge at neutral pH of bloodstream but a positive one at lower pH of tumor extracellular space due to the strong proton-buffering capacity of PEI, for optimized siRNA delivery in vivo. As mentioned before, a major challenge herein is how to improve the stability of anionic polyplexes during blood circulation as they have surface exposure of uncomplexed siRNA fragments susceptible to nuclease degradation. To address this issue, we turned to the reduction-sensitive interlayer package strategy which has been applied to increase the bloodstream stability of anticancer drugloaded micelles in our recent work.[23] The designed surface charge-reversible polyplex comprised a pH-buffering core Figure 1. a) Formation of charge-reversible polyplex for long circulation, tumor-specific cell of PEI complexed with siRNA to generate uptake, and easy intracellular siRNA release. b) Schematic illustration of the performance of microenvironment pH-inducible charge the surface charge-reversible polyplex in vivo. reversal, a water soluble polyethylene glycol (PEG) corona and a reduction-sensitive disulfide-crosslinked Supporting Information, Scheme S1), and was characterized interlayer to stabilize the polyplex and prevent the encapsulated with 1H NMR, Fourier transform infrared spectroscopy (FTIR) siRNA from enzymatic degradation in blood circulation (neuand gel-permeation chromatography (GPC) analyses (see Suptral pH and non-reductive environment). Owing to the proton porting Information, Figure S1–6). The degrees of polymerizabuffering of PEI, the polyplex was negatively charged in bloodtion for the PEG, PAsp(MEA) and PEI blocks were 45, 5, and 58, stream (pH ≈ 7.4) for long circulation but positively charged respectively. The short PAsp(MEA) midblock was selected so that inside tumor tissue (pH ≈ 6.8) for easy cell uptake. When the the disulfide crosslinking could be readily broken up by GSH polyplex was internalized into tumor cells and entrapped inside to release siRNA inside cancer cells quickly. In comparison, the lysosomes (pH ≈ 5), it was turned into more positively charged PEG and PEI chains were much longer in order to have sufficient to facilitate lysosomal escape. Finally, the interlayer package colloidal stability and effective siRNA complexation, respectively. by disulfide crosslinking was untied and meanwhile deprotoUsing electrophoresis in agarose gel, we first confirmed that the nation of PEI was induced to release the encapsulated siRNA siRNA-complexing ability of copolymer is pH-dependent due while the polyplex was migrating into cytosol with neutral pH to the PEI buffering effect. It is well-known that complexation and enriched reducing agent glutathione (GSH, up to the milof siRNA with cationic polymers is driven by electrostatic neulimolar scale) (Figure 1). tralization, upon which siRNA loses negative charge essential for The triblock copolymer, PEG-PAsp(MEA)-PEI, of polyethylene its motion in electric field. Therefore, the mobility of polyplex glycol (PEG), 2-mercaptoethylamine (MEA)-grafted poly(Lin gel electrophoresis reflects the state of surface charge. Only negative polyplexes formed by insufficient siRNA complexation aspartic acid) (PAsp(MEA)), and polyethylenimine, for polyat low N/P values may migrate towards the anode. As shown in plex preparation was synthesized via multistep reactions (see

2





COMMUNICATION Figure 2. a) Effect of N/P ratio on the electrophoretic mobility of siRNA complexed with mPEG-PAsp(MEA)-PEI in agarose gel at pH 6.8 and 7.4. b) Transmission electron microscopy (TEM) images of surface charge-reversible polyplex (RP, N/P 3, pH 7.4) stained with uranyl acetate. The scale bars are 500 nm and 50 nm (inset), respectively. c,d) Effect of the pH value (7.4 vs 6.8) on the zeta potential (c) and particle size (d) of the three polyplexes (mean ± SD, n = 3).

Figure 2a, the copolymer was more potent in complexing siRNA at pH 6.8 than at pH 7.4 as more manifest electrophoresis was observed in the N/P range between 1 and 3. In addition, N/P 3 appeared to be the threshold for copolymer to fully neutralize the negatively charged siRNA at pH 6.8. At this N/P value, complete retardation of siRNA migration was achieved at pH 6.8 whereas weak bands running into the gel were shown at pH 7.4, implying conversion of surface charge from negative to positive along with decreasing the solution pH. At both pH 6.8 and 7.4, electrophoretic bands were displayed at N/P lower than 3 but not detected at N/P above 3, which indicates that the studied polyplexes were negatively charged at N/P below 3 but positively charged at N/P above 3, regardless of the switching between the two values of pH. Based on the electrophoresis data, we chose N/P 1, 3, and 5 to form the always-negative (NP group), surface charge-reversible (RP group) and always-positive (PP group) polyplexes at pH 7.4 and 6.8, respectively. These polyplexes were prepared by complexing siRNA at pH 5, followed by crosslinking the interlayer upon disulfide formation and then adjusting the solution pH to 7.4 (Figure 1a). The abbreviations NP, RP, and PP refer to the crosslinked polyplexes unless otherwise indicated. A high degree of crosslinking reaching 95.86% conversion of thiol to disulfide was achieved according to the measurement of sulfhydryl content using Ellman's reagent.[24] Disulfide crosslinking of the PAsp(MEA) interlayer appeared crucial for obtaining stable polyplexes with small size


(Table S1, Supporting Information). Moreover, the three interlayer-crosslinked polyplexes all showed fairly narrow size distribution (polydispersity < 0.05) at both pH 7.4 and 6.8 (Table S1 and Table S2, Supporting Information). Because siRNA can be more sufficiently complexed and condensed at higher N/P ratio, the size of prepared polyplexes decreased appreciably with an increase in N/P ratio (Figure 2d) as previously reported.[25,26] For all the three investigated N/P ratios, the interlayer-crosslinked polyplexes are obviously smaller than their interlayer non-crosslinked counterparts at pH 7.4. It is assumed that there are two underlying causes for the appreciably decreased size of polyplexes by interlayer crosslinking. Firstly, the polyplexes were prepared by complexing siRNA at pH 5.0, crosslinking the interlayer and adjusting the solution pH to 7.4. Due to the proton-buffering effect of PEI, complexation between the polymer and siRNA became less sufficient when the solution pH was increased to 7.4 from 5, leading to nanoparticle expansion which is restrainable by the crosslinked interlayer. Secondly, the interlayer of non-crosslinked polyplexes should be more outstretched than that of the crosslinked polyplexes. Nuclease degradation of siRNA in polyplexes was performed at pH 7.4 to assess the role of interlayer crosslinking in siRNA protection. As shown in Figure S7 in the Supporting Information, for the surface charge-reversible polyplex (RP group, N/P 3), interlayer crosslinking appeared essential for preventing siRNA from nuclease degradation. In this case,



3

www.advmat.de

COMMUNICATION

www.MaterialsViews.com

significant siRNA degradation could be avoided by interlayer crosslinking. In contrast, interlayer crosslinking in the alwaysnegative polyplex (NP group, N/P 1) was evidently less effective in preventing siRNA from enzymatic degradation. Furthermore, the always-positive polyplex (PP group, N/P 5) offered siRNA essential protection against nuclease degradation even without crosslinking the interlayer. The various efficacies of interlayer crosslinking in siRNA protection against nuclease can be attributed to the different complexation states of siRNA in three polyplexes at pH 7.4. In the present study, siRNA was complexed at pH 5.0 and then the interlayer was crosslinked at the same pH (Figure 1). A distinct feature of this approach is that interlayer crosslinking may effectively restrain the siRNA segments that will be liberated at pH 7.4 inside the core of polyplex and hence hindering their contact with nuclease. However, the uncomplexed siRNA segments which already existed at pH 5 may still escape encapsulation of the crosslinked interlayer and therefore are accessible to nuclease at pH 7.4. In this context, at N/P 5 the polyplex can offer siRNA efficient protection against nuclease regardless of interlayer crosslinking, because siRNA was completely complexed even at pH 7.4 and hence inaccessible to nuclease. At N/P 1, siRNA complexation was highly insufficient even at pH 5, which means that a great deal of uncomplexed siRNA segments could escape the protection of interlayer crosslinking and thereby are susceptible to nuclease degradation at pH 7.4. At N/P 3, siRNA was completely complexed at pH below 6.8 and partially complexed at pH 7.4. In this case, interlayer crosslinking at pH 5 restrained all siRNA, including still complexed and newly freed siRNA segments at pH 7.4, inside the polyplex core and thus effectively prevented them from enzymatic degradation. Since the size of an anticancer nanomedicine has a significant effect on its pharmacokinetics and its ability to accumulate in tumor tissue through the EPR effect,[19] size stability, in addition to siRNA protection capability in blood circulation, is important for the polyplex to be applied in vivo. Therefore, we investigated whether the surface charge-reversible polyplex which well protected siRNA from enzymatic degradation at pH 7.4 may maintain size stability in the presence of blood serum. As shown in Figure S8 in the Supporting Information, due to the negatively charged surface at pH 7.4, the surface chargereversible polyplex with interlayer crosslinking maintained its initial size around 155 nm in pH 7.4 buffer containing 10% fetal bovine serum (FBS) over the experimental time. On the contrary, despite their satisfactory siRNA protection, the alwayspositive polyplex rapidly showed size increase regardless of interlayer crosslinking before leveling off within 10 min, probably because the positively charged surfaces are apt to interact with anionic proteins abundantly existing in blood serum. These results implied that only the surface charge-reversible polyplex may protect siRNA from degradation and meanwhile maintains initial size stability in the blood circulation, which further rationalizes our polyplex preparation strategy. Direct zeta potential measurement of the interlayercrosslinked polyplexes with dynamic light scattering (DLS) obtained data which are supportive of the aforementioned results and discussions. As shown in Figure 2c, the polyplexes at three different N/P ratios all displayed surface charge increase following a decrease of the solution pH value, due

4


to the fact that more amino groups of PEI were protonated at lower pH value. However, whether a reversion of surface charge would occur or not depends significantly on the N/P ratio being applied to form the polyplex. Among the three studied polyplexes, only the polyplex of N/P 3 showed a surface charge reversion from negative to positive when the solution pH was decreased to 6.8 from 7.4 (−3.21 mV at pH 7.4; +3.97 mV at pH 6.8). In contrast, the polyplex formed at N/P 1 only showed a zeta potential change from −12.42 mV to −6.03 mV following this pH decrease, which indicates that the number of newly protonated amino groups upon pH decrease have not been enough to neutralize all the free phosphate groups of siRNA. As to the polyplex of N/P 5, since it was already positively charged at pH 7.4 due to the excess amount of protonated amino groups of PEI over the phosphate groups of siRNA, protonation of more amino groups of PEI upon pH decrease further increased the positive surface charge. The interlayer crosslinked polyplexes exhibited fairly good size stability against pH change to 7.4 from 6.8 (Figure 2d). In the TEM measurement, the surface charge-reversible polyplex showed uniform size distribution at pH 7.4 (Figure 2b). In addition, a core–shell structure was indicated. Complexation of siRNA with PEI resulted in highly compact polyplex core, inducing the significant darkening effect in the TEM image. More direct evidence for the core–shell structure of polyplex was obtained by selectively decorating the interlayer of polyplex with small Au nanoparticles. A core–shell structure featuring a round core circled by Au dots was shown in the TEM image (Supporting Information, Figure S11). In addition, the three polyplexes showed fairly low cytotoxicity at polymer concentrations up to 1 mg/mL at pH 7.4 (Supporting Information, Figure S10a), as assessed in human lung adenocarcinoma A549 cell line (Cold Spring Biotech Corp, Shanghai, China). These polyplexes exhibited very good blood compatibility as well (Supporting Information, Figure S10b). It was noted that, during the course of the present study, a PEI/poly(PEG-His-PEG-Glu) binary mixture was used to form a siRNA polyplex that changed surface charge from negative to positive following pH decline, resulting in enhanced cell uptake in vitro. Mixing of PEI with pH-responsive histidine was found to be vital for obtaining the pH-inducible charge reversal.[27] By contrast, surface charge-reversible polyplex was successfully prepared without using other pH-responsive polymers in addition to PEI in the present study, since interlayer crosslinking endowed the otherwise unstable polyplex with significantly increased stability. Biological studies were then conducted in the human lung adenocarcinoma A549 cell line and animal model to evaluate the potential of surface charge-reversible polyplex in anticancer siRNA therapy. We first examined whether the cell uptake of polyplex can be prevented at neutral pH whereas promoted at lower pH of tumor tissue (below 6.8) utilizing the pH-inducible surface charge reversal strategy.[28] The A549 cells were incubated for 12 h with the Cy3-labeled polyplexes at pH 7.4 and pH 6.8 respectively, and then visualized under laser confocal scanning microscope. As shown in Figure 3a, the always-negative polyplex (NP group) could hardly enter the cells at both pH 7.4 and 6.8. On the contrary, the always-positive polyplex (PP group) was effectively taken up by the cells at both pH 7.4 and 6.8. Unlike the NP and PP group polyplexes, the surface




COMMUNICATION

interaction with cells in normal tissue can be effectively avoided. Consistent results were obtained in gene silencing experiments in vitro (Figure 3b). The firefly luciferaseexpressing A549 cells were treated with polyplexes bearing siRNA targeting luciferase gene. At both pH 7.4 and 6.8, down-regulation of luciferase expression was effective with the always-positive polyplex (PP group) but non-effective with the always-negative polyplex (NP group). In contrast, gene silencing with the RP group polyplex was pH-dependent, namely effective at pH 6.8 but non-effective at pH 7.4. Then, delivery of polyplexes in nude mice bearing luciferase-expressing A549 tumor xenograft was investigated. The triblock polymer inside polyplexes were labeled with a near-infrared (NIR) dye Cy7.5 for in vivo fluorescence imaging. As shown in Figure 4a and Figure S13a in the Supporting Information, the surface charge-reversible polyplex (RP group) accumulated much better than the always-positive and always-negative polyplexes (PP and NP groups) after tail-vein injection. Mice displayed the strongest fluorescence in tumor site 5 h after receiving the RP, and similar post-injection timecourse-dependent tumor accumulation was also observed for the PP injection. In contrast, Cy7.5 fluorescence in tumor site was not observed in mice receiving the NP at all experimental time points. Tumor accumulation study using Cy7.5-labeled siRNA obtained similar results. It appears that the biodistribution of siRNA is similar to that of the triblock copolymers (Supporting Information, Figure S14 vs Figure 4a and Figure 3. a) Laser confocal microscopic images of A549 cells incubated with different polyFigure S13a). It is reasonable that labeling plexes at pH 6.8 and 7.4. Incubation time: 12 h. Blue fluorescence: nuclei stained with Hoechst 33342. Red fluorescence: Cy3-labeled siRNA. Green fluorescence: lysosomes stained with siRNA instead of triblock polymer does not LysoTracker Green DND. b) Luciferase expression in firefly luciferase-expressing A549 cells make appreciable difference considering treated with polyplexes bearing siRNA targeting luciferase gene at pH 6.8 and 7.4 in vitro. Dose: that the crosslinked interlayer prevents 100 nM siRNA (0.15 µg per well). The data are mean ± SD (n = 3). *P < 0.01, compared with polyplex from dissociation in bloodstream. RP group of pH 7.4. *P < 0.01, compared with NP group of pH 6.8. Two factors might have contributed to the extremely poor tumor accumulation of the NP. Firstly, although nanomedicines may passively accumucharge-reversible polyplex (RP group) behaved very differently at late in tumor sites via the enhanced permeability and retentwo values of pH. That is, its cell uptake was negligible at pH 7.4 tion (EPR) effect,[19] the large particle size at pH 7.4 (around but effective at pH 6.8. Furthermore, LysoTracker staining indicated that the polyplex particles were likely located in the 490 nm) might have still impeded the NP group polyplex to lysosomal compartments at the incubation time of 12 h. Appareffectively cross the leaky tumor vasculature. Secondly, cell ently, the positive surface charge of polyplex facilitated its endouptake study have demonstrated that the NP group polyplex cytosis via electrostatic interaction with the negatively charged was negatively charged (about −6 mV) and thus could not be cell membranes. Considering the acidity of solid tumor intereffectively endocytosed by A549 cells at pH 6.8 mimicking stitial space (pH below 6.8), it is reasonable to envision that the acidic tumor microenvironment (Figure 3a). Therefore, the surface charge-reversible polyplex can effectively enter the even if entered the extravascular extracellular space of tumor tumor cells once it accumulated in tumor tissue via the EPR tissue, sequestration of the NP in there could be still much effect.[19] Moreover, the surface charge reversion strategy may less efficient. Based on its poor performances in protecting siRNA against nuclease degradation, entering tumor cells at be propitious to the reduction of side effects in vivo because the tumor acidic pH and accumulating in tumor sites after tail-vein polyplex is negatively charged at pH 7.4 and thus non-specific




5

www.advmat.de

COMMUNICATION

www.MaterialsViews.com

Figure 4. a) In vivo fluorescence images showing enhanced tumor accumulation of RP vs PP and NP after tail-vein injection into nude mice bearing A549 subcutaneous xenograft (Dose: 1.2 mg polymer/kg body weight, polymer labeled with Cy7.5). b) The clearance rate of polymer measured from the Cy7.5 fluorescence intensity of the orbital blood after tail-vein injection of polyplexes into the BALB/c mice. Dose: 1.2 mg polymer/kg body weight. Data are mean ± SD (n = 3). #*P < 0.01, compared with PP group at the same experimental time point.

injection, the NP group polyplex can be excluded from being a desirable system for in vivo application. Unlike the always-negative polyplex (NP) which has large particle size, both the PP and RP possess small sizes and the size of PP is even smaller than that of the RP (75 nm vs 155 nm at pH 7.4), which are propitious to tumor accumulation via EPR effect. To understand why the RP accumulated better in tumor site than the PP if the initial size of polyplex can be excluded herein as an underlying cause, we checked the clearance rate of three polyplexes from blood after tail-vein injection, by determining the Cy7.5 fluorescence intensity of the orbital blood at different postinjection time points (Figure 4b). The results indicate that the RP and NP circulated significantly longer than the PP. For instance, at the time point of 3 h, more than 50% of the PP was cleared from blood. However, only less than 30% of the RP and NP were eliminated at this time point. Apparently, the short circulation time accounts for the less efficient tumor accumulation of the PP. In addition, in the previous section, we have demonstrated that the RP was stable in serum-containing media, whereas the PP interacted with negatively charged serum proteins to result in large aggregates (Supporting Information, Figure S8). Therefore, another underlying cause for the poor tumor accumulation of the PP compared to the RP might be that in bloodstream it formed large aggregates which were reluctant to penetrate even the leaky tumor blood vessels. Finally, the question arises as to whether the RP can silence the expression of target gene more potently than the PP since it appeared to accumulate in tumor site more effectively. Therefore, in vivo gene silencing in tumor site was performed by delivering luciferase siRNA to firefly luciferase-expressing A549 xenografts in nude mice upon tail-vein injection. The change of luciferase activity inside each group reflects the gene silencing ability of the polyplex. Therefore, the bioluminescence intensities relative to the basic level was analyzed within a group. As shown in Figure 5a and Figure S13b in the Supporting Information, in vivo fluorescence imaging detected constantly increased luciferase expression in tumor site of mice receiving PBS

6


and SCR (control groups). Compared with basic gene expression, no appreciable luciferase gene knockdown was shown in mice receiving the NP, which is in line with the aforementioned results that the NP could neither effectively accumulate in tumor site nor enter the A549 cells to virtually enable RNA interference. However, the animals clearly showed gradual decrease of luciferase fluorescence intensity after receiving the RP and PP, indicating luciferase gene silencing by the delivered luciferase siRNA. In comparison with the PP, the RP appeared to be even more effective in down-regulating luciferase expression. Immunohistochemical studies were highly supportive of the above in vivo fluorescence imaging results (Figure 5b). When the RP was administered, the level of luciferase protein was most significantly lowered in tumor tissue. By contrast, medium level of luciferase protein was shown in the tumor tissue from mice receiving the PP. Moreover, the NP group polyplex demonstrated no appreciable target gene silencing effect in vivo, as compared with the PBS control group. In conclusion, a novel in vivo microenvironment pH and reduction dual-sensitive polyplex was successfully developed. The polyplex is stable and long-circulating in the bloodstream (neutral pH and non-reductive), which assists tumor accumulation via EPR effect. After reaching the acidic tumor tissue, the polyplex can be effectively internalized into tumor cells upon pH-induced surface charge reversal from “negative” to “positive”. The endocytosed polyplex can release siRNA into cytosol by untying the reducible interlayer package and triggering PEI proton buffering in response to intracellular enriched reducing agents (e.g., GSH) and pH gradients. Due to these distinct features, highly effective siRNA delivery leading to target gene silencing was achieved in animal test at the N/P of 3, which is significantly lower than that required for common cationic vectors to achieve an ideal nucleic acid transfection in vivo (usually above N/P 10 in order to have high surface PEG density for antibiofouling). In addition, the pH-inducible surface charge reversal strategy enables tumor specific gene silencing with siRNA since the polyplex cannot reverse its surface charge




COMMUNICATION Figure 5. a) In vivo luciferase activity in firefly luciferase-expressing A549 tumors of nude mice after receiving different polyplexes bearing luciferase siRNA (Dose: 2 mg siRNA/kg body weight). b) Ex vivo histological and immunohistochemical analyses of A549 tumor tissue sections at the postinjection time of 72 h. Nuclei were stained blue while extracellular matrix and cytoplasm were stained red in H&E staining. In the immunohistochemical assays, brown and blue stains indicated luciferase protein and nuclei, respectively. The scale bars represent 100 µm.

to “positive” for effective endocytosis in normal tissues with neutral pH. Cell and animal studies revealed the great potential of the dual-sensitive and surface charge-reversible polyplex as highly potent siRNA nanomedicine.

Acknowledgements This research was supported by the National Natural Science Foundation of China (51225305, 21174166), the Natural Science Foundation (S2012020011070) of Guangdong Province, the Guangdong Innovative and Entrepreneurial Research Team Program (2013S086). Received: August 25, 2014 Revised: September 24, 2014 Published online:

Experimental Section Polyplex Preparation: Details for the copolymer synthesis and characterization are shown in the supporting information. To prepare the interlayer-crosslinked polyplexes, the triblock copolymer PEGPAsp(MEA)-PEI and siRNA were separately dissolved in PBS of pH 5.0 at predetermined amounts according to N/P ratios. The two solutions were mixed by vigorous pipetting. After the mixture was kept at room temperature for 30 min to allow polyplex formation, it was then stirred under bubbling of an oxygen flow for 1 h to crosslink the PAsp(MEA) interlayer of polyplex via disulfide formation (Figure 1a). The solution was adjusted to pH 7.4 or pH 6.8 for other experiments. The interlayernon-crosslinked polyplexes were prepared at the same conditions except that the bubbling of an oxygen flow was not applied. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.


[1] S. S. Dunn, S. Tian, S. Blake, J. Wang, A. L. Galloway, A. Murphy, P. D. Pohlhaus, J. P. Rolland, M. E. Napier, J. M. DeSimone, J. Am. Chem. Soc. 2012, 134, 7423. [2] B. Ballarin-Gonzalez, K. A. Howard, Adv. Drug Delivery Rev. 2012, 64, 1717. [3] S. Yang, Y. Chen, R. Ahmadie, E. A. Ho, J. Controlled Release 2013, 167, 29. [4] E. Mastrobattista, W. E. Hennink, Nat. Mater. 2012, 11, 10. [5] M. Breunig, C. Hozsa, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferic, J. Controlled Release 2008, 130, 57. [6] W. Zauner, M. Ogris, E. Wagner, Adv. Drug Delivery Rev. 1998, 30, 97. [7] M. Morille, C. Passirani, A. Vonarbourg, A. Clavreul, J. P. Benoit, Biomaterials 2008, 29, 3477. [8] M. X. Tang, C. T. Redemann, F. C. Szoka Jr., Bioconjugate Chem. 1996, 7, 703.



7

www.advmat.de

COMMUNICATION

www.MaterialsViews.com [9] W. Y. Seow, Y. Y. Yang, Adv. Mater. 2009, 21, 86. [10] N. Kanayama, S. Fukushima, N. Nishiyama, K. Itaka, W. D. Jang, K. Miyata, Y. Yamasaki, U. Chung, K. Kataoka, ChemMedChem 2006, 1, 439. [11] H. Wei, L. R. Volpatti, D. L. Sellers, D. O. Maris, I. W. Andrews, A. S. Hemphill, L. W. Chan, D. S. H. Chu, P. J. Horner, S. H. Pun, Angew. Chem. Int. Ed. 2013, 52, 5377. [12] M. P. Xiong, M. L. Forrest, G. Ton, A. Zhao, N. M. Davies, G. S. Kwon, Biomaterials 2007, 28, 4889. [13] T. Sun, J. Du, L. Yan, H. Mao, J. Wang, Biomaterials 2008, 29, 4348. [14] D. E. Owens lll, N. A. Peppa, Int. J. Pharmaceut. 2006, 307, 93. [15] F. Alexis, E. Pridgen, L. K. Molnar, O. C. Farokhzad, Mol. Pharmaceutics 2008, 5, 505. [16] S. Huang, K. Shao, Y. Kuang, Y. Liu, J. Li, S. An, Y. Guo, H. Ma, X. He, C. Jiang, Biomaterials 2013, 34, 5294. [17] X. Yang, J. Du, S. Dou, C. Mao, H. Long, J. Wang, ACS Nano 2012, 6, 771.

8


[18] Z. Poon, D. Chang, X. Zhao, P. T. Hammond, ACS Nano 2011, 5, 4284. [19] Y. Matsumura, H. Maeda, Cancer Res. 1986, 46, 6387. [20] C. Scholz, E. Wagner, J. Controlled Release 2012, 161, 554. [21] O. Boussif, F. Lezoualch, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. USA 1995, 92, 7297. [22] A. Akinc, M. Thomas, A. M. Klibanov, R. Langer, J. Gene Med. 2005, 7, 657. [23] J. Dai, S. Lin, D. Cheng, S. Zou, X. Shuai, Angew. Chem. Int. Ed. 2011, 50, 9404. [24] R. J. Verheul, S. V. D. Wal, W. E. Hennink, Biomacromolecules 2010, 11, 1965. [25] W. Stephanie, U. K. Beata, D. Lige, H. Sabrina, G. Marius, B. Udo, C. Frank, A. Achim, J. Controlled Release 2006, 112, 257. [26] K. Albert, L. H. Stephen, Nanomed. Nanotechnol. 2011, 7, 210. [27] S. J. Tseng, Y. Zeng, Y. Deng, P. Yang, J. Liu, I. M. Kempson, Chem. Commun. 2013, 49, 2670. [28] P. Vaupel, F. Kallinowski, P. Okunieff, Cancer Res. 1989, 49, 6449.



Multifunctional polymeric micelles for delivery of drugs and siRNA.

Co-delivery of doxorubicin and siRNA with reduction and pH dually sensitive nanocarrier for synergistic cancer therapy.

PH responsive polypeptide based polymeric micelles for anticancer drug delivery.

Smart pH- and reduction-dual-responsive folate-PEG-coated polymeric lipid vesicles for tumor-triggered targeted drug delivery.

Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

Oral delivery of shRNA and siRNA via multifunctional polymeric nanoparticles for synergistic cancer therapy.

pH- and reduction-responsive polymeric lipid vesicles for enhanced tumor cellular internalization and triggered drug release.

pH-Sensitive carboxymethyl chitosan-modified cationic liposomes for sorafenib and siRNA co-delivery.

Ultra-pH-Responsive and Tumor-Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy.

Charge-conversional and pH-sensitive PEGylated polymeric micelles as efficient nanocarriers for drug delivery.

Colon-targeted delivery of budesonide using dual pH- and time-dependent polymeric nanoparticles for colitis therapy.

Low molecular weight chitosan-coated polymeric nanoparticles for sustained and pH-sensitive delivery of paclitaxel.

A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.

Delivery materials for siRNA therapeutics.

A Polymeric Bowl for Multi-Agent Delivery.

Matrix metalloproteinase 2-sensitive multifunctional polymeric micelles for tumor-specific co-delivery of siRNA and hydrophobic drugs.

The pH-Triggered Triblock Nanocarrier Enabled Highly Efficient siRNA Delivery for Cancer Therapy.

Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA.

Polymeric delivery systems for dexamethasone.

Characterization of polyethylene glycol-polyethyleneimine as a vector for alpha-synuclein siRNA delivery to PC12 cells for Parkinson's disease.

In vitro evaluation of pH-sensitive cholesterol-containing stable polymeric micelles for delivery of camptothecin.

Polymeric micelles with citraconic amide as pH-sensitive bond in backbone for anticancer drug delivery.

Self-assembling gelling formulation based on a crystalline-phase liquid as a non-viral vector for siRNA delivery.

Simultaneous delivery of Paclitaxel and Bcl-2 siRNA via pH-Sensitive liposomal nanocarrier for the synergistic treatment of melanoma.