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

Nanocarriers

Co-Delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy Weicai Chen, Yuanyuan Yuan, Du Cheng, Jifeng Chen, Lu Wang, and Xintao Shuai*

Drug resistance is the greatest challenge in clinical cancer chemotherapy. Co-delivery of chemotherapeutic drugs and siRNA to tumor cells is a vital means to silence drug resistant genes during the course of cancer chemotherapy for an improved chemotherapeutic effect. This study aims at effective co-delivery of siRNA and anticancer drugs to tumor cells. A ternary block copolymer PEG-PAsp(AED)-PDPA consisting of pH-sensitive poly(2-(diisopropyl amino)ethyl methacrylate) (PDPA), reduction-sensitive poly(N-(2,2′-dithiobis(ethylamine)) aspartamide) PAsp(AED), and poly(ethylene glycol) (PEG) is synthesized and assembled into a core-shell structural micelle which encapsulated doxorubicin (DOX) in its pH-sensitive core and the siRNA-targeting anti-apoptosis BCL-2 gene (BCL-2 siRNA) in a reductionsensitive interlayer. At the optimized size and zeta potential, the nanocarriers loaded with DOX and BCL-2 siRNA may effectively accumulate in the tumor site via blood circulation. Moreover, the dual stimuli-responsive design of micellar carriers allows microenviroment-specific rapid release of both DOX and BCL-2 siRNA inside acidic lysosomes with enriched reducing agent, glutathione (GSH, up to 10 mM). Consequently, the expression of anti-apoptotic BCL-2 protein induced by DOX treatment is significantly down-regulated, which results in synergistically enhanced apoptosis of human ovarian cancer SKOV-3 cells and thus dramatically inhibited tumor growth.

1. Introduction As one of the three main approaches in clinical cancer therapy (parallel to surgery and radiotherapy), chemotherapy W. C. Chen,[+] Y. Y. Yuan,[+] Prof. D. Cheng, L. Wang, Prof. X. T. Shuai PCFM Lab of Ministry of Education School of Chemistry and Chemical Engineering Sun Yat-sen University Guangzhou 510275, China E-mail: [email protected] J. F. Chen, Prof. X. T. 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/smll.201303951 small 2014, DOI: 10.1002/smll.201303951

currently faces an insurmountable challenge in treating almost all cancer types. That is, cancer cells are apt to develop robust drug resistance against chemotherapy, which may lower drug efficacy or even result in treatment failure. Activation of two types of drug resistance genes in cancer cells, the drug efflux pump and non-pump-related anti-apoptotic genes, is the major underlying cause for drug resistance that arises during the course of chemotherapy. Recent studies have shown that anticancer drugs transported by nanocarriers could circumvent the drug efflux pumps including P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP).[1] This advantage further underlines the clinical application potential of anticancer nanomedicines which employ nanocarriers to mediate drug delivery. Unfortunately, up-regulation of anti-apoptotic protein expression may still induce non-pump-related chemoresistance by decreasing cancer-cell sensitivity to chemotherapeutic drugs.[2] Therefore, the strategy to overcome chemoresistance of cancer cells

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relies, to a great extent, on the suppression of anti-apoptotic gene expression even if anticancer drugs are administered by nanocarrier delivery. Owing to its high efficiency in silencing target genes of various diseases,[3,4] small RNA interference (RNAi) has been regarded as a highly promising option for accomplishing the goal. Furthermore, the most prevalent way to use RNAi for this purpose, according to recent literature, is to incorporate small-molecular anticancer drugs and siRNA into one nanomedicinal system.[5] By this means, not only may the nanomedicine bearing the two therapeutic agents circumvent drug efflux pump but the chemosensitivity of tumor cells may also increase significantly since the co-delivered siRNA will silence the target anti-apoptotic genes.[6] In other words, the two major types of drug resistance may be suppressed simultaneously in tumor chemotherapy. Another distinct virtue of co-delivery is that two therapeutic agents will synchronize their in vivo delivery events and thus act on the same cells at the preset drug/siRNA ratio, which it is hoped will achieve a synergistic effect.[7] To date, most nanocarriers for co-delivering siRNA and anticancer drugs have been based on polymeric micelles.[8–10] Indeed, core-shell structural polymeric micelles have demonstrated great potential in delivering drugs to tumor sites through the enhanced permeability and retention (EPR) effect.[11–16] Yet the applications of polymeric micelles have been greatly hindered by several intrinsic drawbacks. In particular, common polymeric micelles assembled from amphiphilic block copolymers such as poly(ethylene glycol)b-polylactide (PEG-b-PLA) generally exhibit a drug-release profile which is not favorable for site-specific drug delivery. Drugs are encapsulated into micelle cores by exploiting the hydrophobic interaction between the polymer chain and drug, and their release from micelles is predominantly controlled by a molecular diffusion mechanism. On one hand, drug molecules located in or near the interphase between core and shell diffuse out of micelles very quickly, a phenomenon generally called the early stage “burst release” that is the underlying cause for off-target drug loss in storage and during the blood circulation. On the other hand, the time taken for drug molecules entrapped at or close to the center of micelle core to release is too long; weeks, or even longer.[6,17,18] Both factors result in low drug availability inside cancer cells, which is not only insufficient for killing cells but also leads to drug resistance. In addition, premature drug leakage in the bloodstream brings about toxicity problems. Therefore, development of polymeric micelles with better drug release profiles has become a hot topic. Recent research has shown the great potential of in vivo microenvironment-sensitive polymeric micelles in improving drug-release properties for effective cancer treatment. For instance, by utilizing the slightly acidic microenvironments of tumor tissues (pH 6.5–6.8), endosomes (pH 5.5–6.5), and lysosomes (pH 4.5–5.5) that are different from that of bloodstream and normal tissues (pH 7.4), site-specific rapid release of anticancer drugs from pH-sensitive micelles can be achieved.[19–23] In addition, the concentration of the reducing agent glutathione (GSH) inside lysosomes is 100–1000 times higher than in the extracellular fluids and bloodstream, which allows the design of reduction-sensitive polymeric micelles for pinpointed intracellular release of various drugs.[24–27] So far,

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Figure 1. Illustrative preparation of nanocomplex and the dually sensitized release of DOX and siRNA inside cell.

the strategy has not been applied in the co-delivery of siRNA and small-molecular anticancer drugs. Herein, we developed a novel dual-stimuli-sensitive micelle which comprises a PEG corona to stabilize the nanoparticles, a reduction-sensitive interlayer to complex siRNA, and a pH-sensitive core to encapsulate a small molecular anticancer drug. The unique structural design was expected to endow the micelle with drug-release behavior in response to the in vivo microenvironment. In other words, the micelle should be stable in an environment with neutral pH and low GSH concentration (e.g. bloodstream) but disassemble to rapidly release siRNA and anticancer drug inside lysosomes with enriched GSH and low pH (4.5–5.5; Figure 1). As a proof of concept study, doxorubicin (DOX) and siRNA targeting the anti-apoptotic BCL-2 gene (BCL-2 siRNA) were selected as the anticancer drug/siRNA pair for a co-delivery study in human ovarian SKOV-3 cancer since DOX treatment induces BCL-2 up-regulation in this tumor cell.[28] BCL-2 protein is a primary anti-apoptotic protein which acts against cell apoptosis by suppressing mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome c (cyt c), the triggers for the caspase cascade in cell apoptosis.[29] Both in vitro and in vivo studies were conducted to explore the application potential of this dual-stimuli-sensitive delivery system.

2. Results and Discussion 2.1. Polymer Synthesis and Drug/siRNA Encapsulation Reduction- and pH-sensitive triblock copolymer was synthesized by multiple steps as outlined in Figure S1 (Supporting Information) and characterized by using 1H NMR, UV-vis, gel permeation chromatography (GPC), and Raman spectral analyses (Figures S2–S5). Poly(2-(diisopropyl amino) ethyl methacrylate) (PDPA) was selected as the pH-sensitive building block due to its sharp responsiveness following a pH


small 2014, DOI: 10.1002/smll.201303951

Co-Delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy

Figure 2. TEM images of A) blank micelle at pH 7.4 (B-PEAD), B) nanocomplex loaded with siRNA and DOX (D-PEAD/BCL-2, N/P = 15), C) D-PEAD/BCL-2 at pH 5.0, D) D-PEAD/BCL-2 at pH 5.0 in the presence of GSH (10 mM).

decrease from 7.4 to 5.0 and high hydrophobicity at neutral pH compared to poly(amino acid)-based pH-sensitive polymers such as PAsp(DIP).[24,30] DOX-free and -loaded micelles, denoted as B-PEAD and D-PEAD respectively, were prepared by using a solventevaporation method based on the pH- and reduction-sensitive copolymer PEG-PAsp(AED)-PDPA. Since the PDPA block is insoluble in water at neutral pH, it assembled into the pH-sensitive micelle core which encapsulated the hydrophobic DOX (deprotonated form). PEG formed a watersoluble corona while the reduction-sensitive PAsp(AED) formed the cationic interlayer of the micelle. Transmission electron microscopy (TEM) observation showed that the B-PEAD micelle was of uniform and relatively small size of around 40 nm (Figure 2A), and siRNA complexation had little effect on the micelle size or morphology (Figure 2B). After loading 10.2% DOX, the size of cationic micelle slightly increased to 60.3 from 44.9 nm, as measured by dynamic light scattering (DLS; Table 1). Moreover, the zeta potential of D-PEAD is very close to that of B-PEAD (21.7 vs. 24.5 mV), which indicates that DOX encapsulation has little effect on the siRNA-complexing capacity of the cationic micelle. The nanocomplex turned into a highly expanded nanocage (about 400 nm) at pH 5.0 in the absence of GSH (Figure 2C). Table 1. Size changes of the nanoassembly in different conditions measured by using DLS.

Size [nm] a)N/P

B-PEAD

D-PEAD/BCL-2a)

D-PEAD/BCL-2a)

D-PEAD/BCL-2a)

pH 7.4

pH 7.4

pH 5.0

pH 5.0 + GSHb)

44.9 ± 3.2

63.7 ± 5.1

455 ± 34

NDc)

= 15; b)GSH concentration = 10 mM; c)none detected.

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Complexation of B-PEAD or D-PEAD with BCL-2 siRNA to form nanocomplexes B-PEAD/BCL-2 or D-PEAD/BCL-2 was evaluated by using agarose gel electrophoresis. At high N/P ratios (molar ratio of polymer nitrogen to siRNA phosphorus), retardance of siRNA motion became more obvious (Figure S6). When the N/P ratio reached 4, siRNA completely lost mobility in the electric field. D-PEAD and B-PEAD showed no difference in complexing siRNA, consistent with their almost identical zeta potentials. Nanoparticles with high zeta potential attach more easily to negatively charged cell membranes but also lead to high cytotoxicity. In addition, small nanoparticle size favors easy cell uptake by endocytosis. To find out the appropriate zeta potential and size, siRNA nanocomplexes (D-PEAD/BCL-2) prepared at various conditions were analyzed by using DLS. As shown in Figure S7, as the N/P ratios increased from 5 to 30, the zeta potential increased to +15.4 from +1.2 mV, whereas the particle size decreased to 60.5 from 79.4 nm upon more sufficient siRNA complexation. At N/P = 15, the nanocomplex showed a relatively weak positive charge (+5.3 mV) and a small particle size (64 nm). Consequently, the nanocomplex of N/P = 15 was selected for the biological studies. The serum stability of nanocomplex was checked in simulated physiological conditions as reported previously.[31] No obvious change in particle size was detected in a prolonged experimental time (72 h) when 10% fetal bovine serum (FBS) was present in the solution (Figure S8).

2.2. Reduction and pH-Sensitive Drug Release The reduction and pH dually sensitive structure of the micelle was designed to achieve site-specific release of DOX and siRNA inside lysosomal compartments with a lower pH value but higher GSH concentration than the bloodstream (pH = 5.0 vs. 7.4; GSH=10 mm vs. 5 µm). The PDPA block contains tertiary amino groups that have a pKa value of 6.3.[32] The block is hydrophobic at neutral pH values and thus can form a stable micelle core to encapsulate hydrophobic DOX (deprotonated form). siRNA molecules were complexed in the interlayer of micelle by means of electrostatic interaction with amino groups which were linked to the midblock by a reducible disulfide bond. It was expected that the PDPA core would be solubilized upon tertiary amine protonation and the pendant mercaptoethylamine group would be cleaved from the midblock by GSH inside lysosomes, which would result in fast release of DOX and siRNA, respectively. To verify the dually sensitive drug-release behavior of the nanocomplex loaded with DOX and siRNA, in vitro release studies were conducted in lysosome-mimicking environment by measuring the fluorescence intensities of the micelle solution. As shown in Figure 3A, DOX fluorescence intensity was very low at pH 7.4 due to the fluorescence-quenching that results from the aggregation of DOX inside the micelle core. When the solution pH dropped to 5.0, DOX fluorescence intensity increased quickly over time, which indicates fluorescent dequenching owing to DOX release from the micelle. Moreover, dual stimuli resulted in even faster DOX release. That is, DOX release at pH 5.0 was further accelerated in


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(Figure 3B). Consistent results were obtained when quantitatively determining DOX release from nanocomplexes in response to pH change and reduction (Figure 3C). The release rate was very slow at neutral pH without adding GSH. In comparison, addition of GSH (10 mm) to the solution resulted in an obviously increased DOX release rate which was fairly close to that of the siRNA-free micelle. Moreover, DOX release appeared even faster at pH 5.0, and dual stimuli (pH 5.0 and 10 mm GSH) led to the quickest DOX release. Fast DOX release at pH 5.0 was due to the solubilization of the micelle PDPA core. In addition, the cationic interlayer, after complexing siRNA, acted as a reduction-sensitive on-off to co-manipulate DOX release from nanocomplex. At neutral pH without adding GSH to the solution, the siRNAcomplexed interlayer of nanocomplex had a compact structure that effectively hampered DOX diffusion across it (i.e. release “off”).[19] When siRNA was released in response to GSH reduction at pH 7.4, the interlayer was dissolved to allow free diffusion of DOX molecules across it (i.e., release “on”), just like the cationic micelle (D-PEAD) without complexed siRNA. Most importantly, in the presence of GSH (10 mm) at pH 5.0, not only was siRNA rapidly released but the nanocomplex also disassembled to enable the quickest DOX release. The dual-stimuli-sensitive drug-release behaviors of the nanocomplex are in line with its structural transitions observed in the TEM measurements. Finally, we measured the profiles of drug and siRNA release at pH 6.5 and 5 µm GSH (Figure S9 and Figure S10). The release rates of drug and siRNA remained almost unchanged when the solution pH decreased to pH 6.5 from 7.4 or 5 µm GSH was present in the release media, which implies that the weak acidity of tumor tissue (pH ca. 6.5) and very low level of GSH (5 µm) in bloodstream will not obviously affect the stability and payload-release profile of the nanocomplex.

2.3. Cell-Culture Study

Figure 3. A) DOX fluorescence intensity changes of D-PEAD/BCL-2 solution after decreasing pH and adding 10 mM GSH (DOX loading content of nanocomplex: 10.2%; N/P = 15). B) FITC fluorescence intensity changes of B-PEAD/FITC-SCR solution after adding 10 mM GSH (nanocomplex N/P = 15). C) In vitro DOX release from nanocomplex in phosphate-buffered saline (PBS) in different conditions (nanocomplex N/P = 15; GSH concentration 10 mM if added; study performed at 37 °C; data are mean ± standard error of three parallel samples).

the presence of 10 mm GSH. Meanwhile, siRNA complexed in the interlayer showed a clear reduction-sensitive-release behavior. Addition of 10 mm GSH into the micelle solution rapidly increased fluorescein isothiocyanate (FITC) fluorescence intensity, which indicates release of FITClabeled siRNA which led to FITC fluorescence dequenching


All cell studies were performed in human ovarian cancer SKOV-3 cells. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, micelles without loaded DOX showed very low cytotoxicity whether scrambled siRNA (SCR) was complexed or not. More than 70% of cells remained viable even at the highest experimental concentration of polymer (300 µg mL−1; Figure S11). Cell uptake and intracellular distribution of nanocomplex (D-PEAD/FITC-SCR) were evaluated by using flow cytometry and confocal laser scanning microscopy (CLSM). FITC-labeling of scrambled siRNA (SCR) allowed quantitative analysis of its cell uptake with flow cytometry and direct visualization of its intracellular distribution with CLSM, respectively. As shown in Figure S12, flow cytometric analysis clearly showed the effective co-delivery of siRNA and DOX into SKOV-3 cells. Very few cells were DOX- or FITC-positive only. However, DOX and FITC dually positive cells accounted for 68.4% and 82.8% of the population after incubation with nanocomplex for 2 and 4 h, respectively.


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Figure 4. A) CLSM images of SKOV-3 cells incubated with D-PEAD/FITC-SCR (N/P = 15). Blue fluorescence: nuclei stained with DAPI; red fluorescence: DOX; green fluorescence: FITC-SCR; yellow stains: overlapping of red and green fluorescence; pink light: overlapping of red and blue fluorescence. B) Typical in vivo AF750 fluorescence images at different times after tailvein injection (dose: 3 mg AF750 per kg body weight).

CLSM observation clearly showed the intracellular distribution of DOX and siRNA at different cell incubation times. To oversee the intracellular DOX release behavior, cells were incubated in the presence (4 h) of and then the absence (further 2 or 4 h) of D-PEAD/FITC-SCR. For all experimental time points, siRNA was observed mainly in the cytoplasm. By contrast, DOX showed incubation-time-dependent intracellular distribution patterns. That is, DOX fluorescence was observed mainly in the cytoplasm at 4 h but predominantly in nuclei at 6 and 8 h (Figure 4A). At 4 h, the intracellular distribution patterns of FITC-SCR and DOX are almost the same, which implies a co-delivery event as also shown in the flow cytometry assay. Overlapping of the green FITC fluorescence and red DOX fluorescence generated yellow stains in the cytoplasm. At 6 and 8 h, DOX separated from FITC-SCR and entered nuclei, which turned the nuclei pink due the overlap of the red DOX and blue 4′,6-diamidino2-phenylindole (DAPI) fluorescence. Nanomedicines are taken up by cells by endocytosis, and thus will be wrapped inside the endosomal and thereafter lysosomal compartments. According to the previous report, DOX delivered by a nonsensitive micelle was barely released and remained in the lysosomes even after 24 h.[33] The intracellular drug distribution data in the present study indicated that DOX was quickly released from the nanocomplex inside lysosomes and then migrated into nuclei, as a result of the dual sensitivity of the micelle to pH decrease and GSH reduction. These results are consistent with the in vitro drug-release data. Silencing of the anti-apoptotic BCL-2 gene in SKOV-3 cells by a nanocomplex bearing BCL-2 siRNA was evaluated at protein and mRNA levels with Western blotting small 2014, DOI: 10.1002/smll.201303951

Figure 5. A) Efficacy of nanocomplexes in suppressing BCL-2 expression in SKOV-3 cells at mRNA level, quantified by real-time PCR analysis (n = 3). B) The SKOV-3 cell apoptosis assessed by TUNEL staining assay after cell incubation with a) B-PEAD/SCR, b) B-PEAD/BCL-2, c) D-PEAD/ SCR, d) D-PEAD/BCL-2. *P < 0.05, compared with D-PEAD/BCL-2; #P < 0.05, compared with B-PEAD/BCL-2; ΔP < 0.05, compared with D-PEAD/SCR. C) Viability of SKOV-3 cells after incubation with D-PEAD/ SCR and D-PEAD/BCL-2 at various DOX concentrations, as determined by MTT assay (N/P = 15; incubation time: 48 h; dose: 20 nM siRNA per well).

and real-time polymerase chain reaction (PCR) assays. As shown in Figure 5A, the cells incubated with the nanocomplex bearing SCR only (B-PEAD/SCR) showed almost the same BCL-2 mRNA level as the normally cultured cells



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(control group), which indicates the ineffectiveness of SCR in down-regulating BCL-2 expression. By comparison, the BCL-2 mRNA level doubled when the SKOV-3 cells were incubated with the nanocomplex bearing DOX and SCR (D-PEAD/SCR), which implies significant BCL-2 up-regulation induced by DOX treatment. Similar results were also reported in previous studies; DOX treatment stimulated cellular anti-apoptotic defense via up-regulation of BCL-2 protein.[2] More excitingly, cell treatment with nanocomplexes containing BCL-2 siRNA resulted in a remarkably lower level of BCL-2 mRNA in SKOV-3 cells than the initial level, no matter whether DOX was co-delivered into the cells or not. In particular, compared to the cells incubated with D-PEAD/SCR, the cells incubated with D-PEAD/ BCL-2 showed a BCL-2 mRNA level that was decreased by more than three-quarters. Determination of BCL-2 gene expression at the protein level using Western blotting showed similar results. As shown in Figure S13, compared to the control cells or cells incubated with B-PEAD/SCR, cells incubated with D-PEAD/SCR showed remarkably upregulated BCL-2 expression. Furthermore, cells incubated with D-PEAD/BCL-2 or B-PEAD/BCL-2 showed an obviously lower level of BCL-2 protein than cells incubated with D-PEAD/SCR. The above data underline the significance of silencing BCL-2 gene in the chemotherapy of SKOV-3 cancer, and strongly evidence that the DOX treatmentinducible up-regulation of anti-apoptotic BCL-2 gene could be effectively suppressed by using our reduction and pH dually sensitive nanocarriers to co-deliver BCL-2 siRNA and DOX. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was then conducted to ascertain if there is a synergistic effect of siRNA and DOX in inducing apoptosis of SKOV-3 cells. Nuclei of the apoptotic cells were stained brown due to the existence of fragmented DNA whereas the normal cells were not stained (Figure 5B). The TUNEL-positive cells were counted to obtain the percentage of apoptotic cells. Cells incubated with various nanocomplexes displayed significantly different apoptosis levels (Figure S14). Cells incubated with nanocomplex containing no therapeutic agent (B-PEAD/SCR) only showed 8.0 ± 1.1% apoptosis, which is very close to that of the normally cultured control (2.0 ± 1.0%). Cells incubated with nanocomplexes that contained only BCL-2 siRNA (B-PEAD/BCL-2) or DOX exhibited higher apoptosis around 19.7 ± 1.5% or 43.5 ± 2.2%, which indicates that BCL-2 siRNA or DOX alone may induce apoptosis in SKOV-3 cells. The highest apoptosis level (76.1 ± 3.1%) was in cells incubated with nanocomplex bearing both DOX and BCL-2 siRNA (D-PEAD/BCL-2), which means that co-delivery of BCL-2 siRNA and DOX resulted in a cell apoptosis value which is 13% higher than the sum (63%) caused by the independently delivered DOX and BCL-2 siRNA. These data clearly demonstrate the synergistic effect of the two therapeutic agents in promoting SKOV-3 cell apoptosis, which appears reasonable since real-time PCR and Western blot assays have shown that DOX inducible up-regulation of anti-apoptotic BCL-2 gene can be effectively suppressed by the co-delivered BCL-2 siRNA.


An MTT assay showed that BCL-2 gene silencing with the co-delivered BCL-2 siRNA virtually sensitized the SKOV-3 cells to DOX treatment. As shown in Figure 5C, D-PEAD/ BCL-2 exhibited higher toxicity at all DOX concentrations compared to D-PEAD/SCR. The IC50 value of D-PEAD/ BCL-2 is as low as 0.012 µm DOX, at which D-PEAD/SCR only killed 10% of the cells. The IC50 value of D-PEAD/SCR is 1.0 µm DOX, which is two orders of magnitude higher than that of D-PEAD/BCL-2.

2.4. In Vivo Tumor Accumulation and Distribution of Nanocomplex Our dually sensitive nanomedicine with a relatively small particle size of around 60 nm was expected to cross the tumor blood vessels easily and accumulate in the tumor site through the EPR effect.[11] To verify this potential, in vivo fluorescence imaging was performed to monitor the distribution and tumor accumulation of nanocomplex in nude mice bearing a subcutaneous SKOV-3 xenograft. Since massive background fluorescence of animal can be excitated at the DOX excitation wavelength (600 nm), in vivo imaging was not conducted by determining the DOX fluorescence intensity. Instead, to enable high quality in vivo fluorescence imaging without exciting much background fluorescence, siRNA in the nanocomplex was labeled with a near-infrared fluorescent dye AF750 and then the AF750 fluorescence intensity was monitored in animals that received D-PEAD/AF750-SCR. Figure 4B shows the in vivo distribution of nanocomplex in tumor-bearing mice at various times after tail-vein injection. The AF750 fluorescence intensity in tumor site reached the highest value at 4 h, and was maintained at a high level up to 12 h. At 24 h after injection, the AF750 fluorescence intensity in the tumor site was still much stronger than anywhere except the liver. These results show that the dually sensitive nanomedicine applied via intravenous injection was effectively delivered to the tumor site. Our results are in good agreement with the previous report that nanomedicines with similar sizes (60–70 nm) may accumulate preferentially in the tumor site through the EPR effect and at reticuloendothelial systems (RES) such as the liver.[34] The mice were sacrificed after in vivo imaging, and ex vivo imaging of tumor and major organs was performed to verify the in vivo imaging results. As shown in Figure S15, the nanocomplex mainly accumulated in the tumor tissue and liver just as detected in the in vivo imaging study.

2.5. Combined Therapeutic Effect of siRNA and DOX In Vivo Since DOX and BCL-2 siRNA co-delivered by the dually sensitive nanocarrier synergistically induced SKOV-3 cell apoptosis in vitro, we were anxious to know whether the synergistic anticancer effect will be achieved in vivo as well. Anticancer treatments were performed when tumors grew up to 50 mm3. Four groups of mice receiving PBS, B-PEAD/ BCL-2, D-PEAD/SCR, and D-PEAD/BCL-2 were tested. The tumor volumes and body weights were measured every


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Figure 6. A) Tumor growth inhibition (n = 20) and B) cumulative survival of nude mice bearing SKOV-3 tumor after tail-vein injection of different formulations. C) The histological characteristics and TUNEL assay of SKOV-3 tumor tissue after treatments. Nuclei are stained blue while extracellular matrix and cytoplasm are stained red in H&E analysis. In the immunohistochemical assay, the brown and blue stains indicate BCL-2 or cleaved caspase-3 protein and nuclei, respectively. In the TUNEL analysis, brown and green stains indicate apoptotic and normal cells, respectively. Scale bars: 50 µm; dose: 1 mg DOX per kg body weight per injection for DOX-containing nanocomplexes at an interval of 3 days.

3 days up to 30 days. The mice treated with PBS exhibited obvious weight loss, decreased activity, and loss of appetite during the experimental process. These symptoms were significantly less in the mice treated with D-PEAD/BCL-2. D-PEAD/BCL-2 treatment resulted in the best tumor growth inhibition (Figure 6A). After 30 days, tumors in mice receiving PBS grew up to 980 ± 60 mm3, and were obviously larger than those in mice that received nanocomplex incorporating siRNA or DOX only (840 ± 60 mm3 for B-PEAD/ BCL-2; 380 ± 55 mm3 for D-PEAD/SCR). Meanwhile, the tumor volume was as small as 110 ± 45 mm3 in mice that received D-PEAD/BCL-2. The time course survival rates of mice receiving various treatments are in line with the results of tumor growth inhibition (Figure 6B). The three treatment groups all showed apparent therapeutic effects, and the mice treated with D-PEAD/BCL-2 showed the longest survival time. No mouse in the PBS control group survived longer than 40 days. However, over 75% of the mice survived longer than 45 days when receiving the D-PEAD/BCL-2 treatment. These results highlight the significance of the combined DOX and siRNA therapy for in vivo cancer treatment. The histological changes of tumors after various treatments were compared. As shown in Figure 6C, the small 2014, DOI: 10.1002/smll.201303951

haematoxylin and eosin (H&E) stained section of tumor tissue from the control group receiving PBS was the most hypercellular and showed obvious nuclear polymorphism. Signs of tumor growth inhibition were clearly observed in tumor tissues for the three therapeutic groups. The D-PEAD/ BCL-2 treatment resulted in the fewest tumor cells and the highest level of tumor necrosis. Immunohistochemical assays were performed to detect BCL-2 expression and caspase-3 activation (i.e., cleaved caspase-3 protein) in tumor tissues of mice after different treatments. Cell nuclei were stained blue and the brown blots indicated the BCL-2 or cleaved caspase-3 protein in the tumor tissue (Figure 6C). Treatment with DOX alone (D-PEAD/SCR) led to the highest level of BCL-2 protein expression. As found in the cell study, expression of the anti-apoptotic BCL-2 protein in tumor tissues was effectively down-regulated by BCL-2 siRNA co-delivered with either B-PEAD/BCL-2 or D-PEAD/BCL-2. Cleaved caspase-3 protein indicative of apoptosis activation was expressed at the highest level, and consequently the TUNEL assay showed the most prominent cancer-cell apoptosis in tumor tissues from mice receiving D-PEAD/BCL-2. These results evidence that the co-delivered DOX and BCL-2 siRNA acted synergistically on SKOV-3 cells to induce apoptosis in vivo, which



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is the underlying cause for the improved therapeutic effect achieved with the dually sensitive nanocomplex (D-PEAD/ BCL-2). Similar synergistic effects of siRNA and DOX were also found in other cancer types when they were co-delivered by other nanocarriers.[6,35]

3. Conclusion A novel triblock copolymer PEG-PAsp(AED)-PDPA with reduction and pH dual sensitivity was successfully synthesized. In aqueous media, the copolymer self-assembles into a three-layered spherical nanocarrier that incorporates anticancer drug DOX and BCL-2 siRNA in its pH-sensitive core and interlayer, respectively. Bioenvironment-triggered fast release of the two therapeutic agents from nanocarriers was achieved. The nanomedicine prepared at the appropriate N/P ratio demonstrated remarkable potency of co-delivering DOX and BCL-2 siRNA into cancer cells and accumulation in the tumor site after intravenous injection. Consequently, the codelivered BCL-2 siRNA effectively down-regulated the DOX treatment-inducible overexpression of anti-apoptotic BCL-2 protein, which sensitized cancer cells to chemotherapy. Both in vitro and in vivo studies showed that the co-delivered DOX and BCL-2 siRNA synergistically induced apoptosis of cancer cells for the optimal therapeutic effect. Our results reveal the great potential of the reduction and pH dually sensitive nanocarrier as an effective nanoplatform for achieving combined chemo- and siRNA therapy in tumor treatment.

4. Experimental Section Preparation of DOX-Loaded Cationic Micelles and siRNA Complexes: 30.0 mg PEG-PAsp(AED)-PDPA was dissolved in 5 mL methanol. 7.0 mg DOX·HCl was dissolved in 5 mL methanol and then 10 µL triethylamine was added to deprotonate DOX·HCl. The two solutions were mixed and then added dropwise to PBS at pH 7.4 (30 mL) under ultrasonic agitation using a Type 60 Sonic Dismembrator (Fisher Scientific) at a power level of 50%. After methanol was removed by rotary evaporation, the solution was adjusted to pH 7.4 and then dialyzed (MWCO: 14 000 Da) against water for 48 h to remove free DOX. Afterwards, the solution was filtered with a syringe filter (pore size: 0.45 µm) to eliminate aggregates, concentrated, and washed three times using a MILLIPORE Centrifugal Filter Device (MWCO: 100 000 Da). DOX-free micelle (B-PEAD) was prepared in the same way except that DOX was not used in the procedure. Predetermined amounts of siRNA and DOX-loaded micelle or DOX-free micelle were mixed in 0.9% sodium chloride solution by vigorous pipetting. The mixture was then kept still for 30 min at room temperature to allow nanocomplex formation. The amount of polymeric micelle used to complex siRNA was determined based on the designed N/P ratio. Dual Sensitivity Evaluated by Fluorometric Assay: The pH value of D-PEAD/BCL-2 solution with or without 10 mM GSH was adjusted to 5.0 with 0.1 M HCl. DOX fluorescence emission from 500–700


nm was measured with excitation wavelength of 480 nm on a RF5301PC spectrophotometer (Shimadzu) at different time points. Blank micelle was complexed with FITC-SCR at N/P ratio of 15. The complex solution was treated with 10 mM GSH. FITC fluorescence emission from 495–600 nm was measured with excitation wavelength of 488 nm on a RF5301PC spectrophotometer (Shimadzu) at different time points. In Vitro DOX Release: The siRNA complexes were formed at a fixed N/P ratio of 15. The solutions of siRNA complexes (2 mL each) were mixed with 3 mL PBS (pH 7.4). Release was studied at two pH values (pH 7.4 and 5.0) in PBS. To evaluate the reduction sensitivity, GSH was added to the micelle solution at a concentration of 10 mM. Each sample was transferred into a dialysis bag (MWCO: 14 000 Da). The bag was clamped and then placed into the same buffered solution (30 mL). The release study was performed at 37 °C in an incubation shaker. At selected time intervals, 5 mL of solution outside the dialysis bag was replaced with fresh buffer solution for UV-vis analysis. DOX concentration was measured based on the absorbance intensity of DOX at 482.5 nm. The cumulative amount of released drug was calculated, and the percentages of drug released from micelles were plotted against time. Flow Cytometry Analysis: Incubated cells were trypsinized, washed and resuspended in 0.5 mL of PBS, and finally measured by flow cytometry using a 488-nm laser for excitation. The fluorescence emissions from FITC and DOX were recorded at 525 and 575 nm, respectively. Normally cultured cells without transfection were measured to calibrate the background. CLSM: SKOV-3 cells were seeded in three petri dishes at a density of 1 × 104 cells per dish. Cells in all three dishes were first incubated for 4 h with DOX and FITC-SCR loaded nanocomplex in medium containing 3% FBS. Cells in one dish were subject to CLSM observation. In contrast, cells in the other two dishes were further incubated in nanocomplex-free fresh medium for 2 and 4 h, before the measurement. After the cells were washed three times with PBS and nuclei were stained with DAPI to identify the drug and FITC-SCR locations; they were observed on a FluoView FV1000 microscope (OLYMPUS, Japan). FITC, DOX, and DAPI were excited at 490, 485, and 358 nm, respectively. The emission wavelengths of doxorubicin, FITC, DAPI are 595, 525, and 455 nm, respectively. MTT Assay: SKOV-3 cells were seeded into 96-well plates at a density of 1 × 103 cells per well and cultured for 24 h in 100 µL of RPMI-1640 containing 10% FBS in a humidified atmosphere with 5% CO2. To evaluate the synergistic effects of siRNA and DOX, the cells were incubated for 48 h with DOX-loaded micelles complexing either SCR (D-PEAD/SCR) or BCL-2 siRNA (D-PEAD/BCL-2). 100 µL of growth medium was replaced into each well and then 10 µL of MTT solution (5 mg mL−1 in PBS) was added. After cells were further incubated for 4 h, the supernatant in the wells was discarded and 100 µL of N,N-dimethylsulfoxide was added to dissolve the substrate. After gentle agitation for 5 min, the absorbance at 570 nm was recorded on a Tecan Infinite F200 Multimode plate reader. Real-Time PCR Assay for mRNA Level of BCL-2 Gene: 2 µL of cDNA was applied for quantitative analysis by using PCR Plantinum Quantitative PCR SuperMix-UDG Kit (Invitrogen, USA). To determine the target gene copies, the gene forward primer and reverse primer (both at 900 nM) were mixed with the fluorescence probe


small 2014, DOI: 10.1002/smll.201303951


(250 nM) by using Master Mix and then subjected to the real-time PCR assay. The mRNA level of β-actin gene was also measured as an internal standard. The primer and probe sequences are listed in the Supporting Information. The real-time PCR program was run on a StepOne Plus Realtime PCR System (ABI, USA) first at the thermal cycling conditions of 95 °C for 5 min; then 40 cycles of 95 °C for 30 s, 58 °C for 15 s, and 68 °C for 15 s. All experiments were conducted in triplicate. Western Blotting Analysis: The total proteins were extracted from the cells and the protein content was determined with a bicinchoninic acid protein assay kit (Invitrogen, Carlsbad, CA, USA). 40 µg of protein were separated through 15% SDS-PAGE (Bio-rad, Hercules, CA) and then transferred to a nitrocellulose membrane. The membranes were subsequently incubated with rabbit antibody against BCL-2 (1:500 dilution in PBS/Tween; Cell Signaling Technology, Danvers, USA). β-Actin was used as an internal standard to normalize protein expression, and the protein-containing membranes were simultaneously incubated with β-actin (C4) monoclonal antibodies (1:2000 in PBS/Tween; Santa Cruz biotechnology Inc, Santa Cruz, CA). After being washed three times with TBST and incubated with HRP-conjugated goat anti-rabbit or antimouse antibodies IgG (1:8000 dilution; Cell Signaling Technology, Danvers, USA) at 37 °C for 1 h, the protein–antibody complexes were analyzed by using chemoluminescence (ECL Plus, Amersham Biosciences, USA). TUNEL Assay: 100 µL of proteinase K (20 µg mL−1) in Tris buffered saline (TBS, pH 8.0) was added to incubate the cells for 20 min at room temperature. The cells were washed with TBS, and then 3% H2O2 aqueous solution was added to inactivate endogenous peroxidase at room temperature. The cells were washed with PBS, further treated with terminal deoxynucleotidyl transferase (TdT Enzyme) at 37 °C for 90 min, and washed again with PBS. After the exposed 3′-OH ends of the breakage DNA fragment in apoptotic cells were labeled with biotin-labeled deoxynucleotide, the cells were incubated at room temperature for 30 min with streptavidin–horseradish peroxidase conjugate. The diaminobenzidine (DAB) reacted with the labeled sample to generate an insoluble brown DAB reaction product signal which shaded blue-green to greenish tan to indicate non-apoptotic cells. Animal Model: All surgical interventions and post-operative animal care were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University. Female nude mice Nu/Nu were ordered from Vital River Laboratories (Beijing, China). SKOV-3 cells were implanted into the 6–7 week old nude mice. The mice were anesthetized with chloral hydrate (20 mg kg−1) and placed in a stereotactic frame. Cells (1 × 106) in 200 µL of serumfree RPMI-1640 were subcutaneously injected in the right back, and tumor growth was monitored by caliper measurements of tumor volume. In Vivo and Ex Vivo Fluorescence Imaging: Nanocomplex of DOX-loaded micelle and AF750-SCR (N/P = 15) was injected into mice bearing SKOV-3 tumor via the lateral tail vein (dose: 5 mg DOX kg−1 body mass). The nude mice under anaesthesia were imaged using in vivo fluorescence imaging system (Carestream In-Vivo Imaging System FX PRO, USA) at selected time points. The mice were sacrificed 24 h after injection and the organs of interest were excised for ex vivo imaging. The 530 and 720 nm excitation filters were used and then fluorescence emissions from DOX and AF750 were recorded at 600 and 790 nm, respectively. small 2014, DOI: 10.1002/smll.201303951

Tumor Growth Inhibition: Mice bearing the SKOV-3 tumor were randomly divided into four groups (n = 20) for treatment with PBS, B-PEAD/BCL-2, D-PEAD/SCR, or D-PEAD/BCL-2. When the tumor volume reached approximately 50 mm3, treatments were performed. The nanocomplex in 200 µL of NaCl solution or PBS of the same volume was injected via lateral tail vein at an interval of 3 days for total 30 days at a constant dose of 1 mg DOX Kg−1 body weight. Tumor growth was monitored and tumor volume was calculated by the following equation: Volume = 0.5 × l × w2, where w and l are width and length of the tumor, respectively. Survival rate of mice was analyzed by a log-rank test based on the Kaplan-Meier survival analysis using MedCalc statistical software. Histology and Immunohistochemistry: The mice were sacrificed. Subsequently, the tumors were collected and fixed for 24 h in 10% PBS buffered formalin. Tissue sections (5 µm) were then stained with H&E after deparaffinization (at least five paraffin sections from each animal). Immunohistochemistry was performed as follows: tumor sections were deparaffinized with xylene and alcohol, washed with PBS, and incubated in 10 mM citrate buffer (pH 7.4) for 15 min at 90 °C. The sections were then treated with 0.3% hydrogen peroxide in methanol for 30 min at 4 °C. After blocking with 10% normal horse serum, 2% bovine serum albumin, and 0.5% Triton X-100 for 1 h, the tissues were incubated with rabbit polyclonal primary antibodies for cleaved caspase-3 or BCL-2 (1:500 dilution in PBS/Tween; Cell Signaling Technology, Danvers, USA) for 1 h at 37 °C, washed with PBS, and further incubated for 1 h with horseradish-conjugated donkey antirabbit IgG secondary antibodies (DAKO Corporation, Carpinteria, CA). The immunoreactivity on the tissue sections was visualized by using the peroxidase substrate diaminobenzidine. Statistical Analysis: Statistical analysis of data was performed with the one-factor analysis of variance (SPSS software). The results were expressed as mean ± SE, and P < 0.05 was considered to be statistically significant. All statistical tests were two-sided.

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

Acknowledgements The study was supported by the National Natural Science Foundation of China (51225305, 21174166, 51373203), the Ph.D. Programs Foundation of Ministry of Education of China (20100171110011), the S&T Programs (2010B031500011), and Natural Science Foundation (S2012020011070) of Guangdong Province.

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Received: December 31, 2013 Revised: February 15, 2014 Published online:

small 2014, DOI: 10.1002/smll.201303951

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