Article pubs.acs.org/Biomac
Salt-Induced Stability and Serum-Resistance of Polyglutamate Polyelectrolyte Brushes/Nuclear Factor-κB p65 siRNA Polyplex Enhance the Apoptosis and Efficacy of Doxorubicin Yuefang Zhao,† Yuting Qin,‡ Yang Liang,† Haijuan Zou,† Xiao Peng,§ Huan Huang,† Ming Lu,† and Min Feng*,† †
Department of Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, University Town, Guangzhou, People’s Republic of China 510006 ‡ Laboratory of Molecular Rheumatology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China 200025 § College of Life Sciences, Shenzhen University, Shenzhen, People’s Republic of China 518060 S Supporting Information *
ABSTRACT: Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants Kb under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.
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suffer from relatively low gene transfer efficiency.4,5 Besides the intracellular barriers such as cellular internalization and endosomal escape, the most common problem associated with the low delivery efficiencies of current nonviral polycationbased gene delivery systems is their low colloidal stability in the presence of salt and serum. The low colloidal stability is not a problem in culture when it could be performed under low ionic strength and serum-free conditions, but is a key problem for systemic in vivo delivery. Due to the electrostatic nature of the polycation−gene interaction, the biological fluids containing a high concentration of ions and negatively charged blood components potentially cause the screening of the polyelectrolyte ionic interactions and
INTRODUCTION RNA interference (RNAi) is a powerful sequence-specific gene silencing technology that holds great promise for a significant improvement in cancer therapy. Short interfering RNAs (siRNAs) are one of the most notable applications of RNAi technology and have achieved successful advances in some clinical trials.1,2 The advantage of siRNA-based approaches in cancer therapy is that can stop tumor growth and kill the cancer cells by targeting complementary mRNA strands for degradation, thus specifically inhibiting tumor-associated gene expression while minimizing untoward side effects on normal cells.3 A key component to the outcome of these trials is the effectiveness of their delivery system. Nearly 70% of gene therapy clinical trials involve the use of viral vectors because of their high transfection efficiency. However, their use has been limited due to biosafety concerns. Nonviral polycation-based vectors as the safe alternatives to viral vectors also continue to © 2013 American Chemical Society
Received: February 3, 2013 Revised: April 10, 2013 Published: April 25, 2013 1777
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activation.23 Thus, inhibition of NF-κB activity represents a potential therapeutic strategy for cancer treatment and increases sensitivity of cancer cells to chemotherapeutic drugs. Doxorubicin is one of the most effective chemotherapeutic drugs in the treatment of many types of cancers including bone tumors, ovarian, breast, uterine, and cervical cancers.24 Doxorubicin causes DNA damage and kills cancer cells mainly by apoptosis.25 Several studies indicate that inhibition of apoptosis is associated with doxorubicin resistance which becomes a major obstacle for successful cancer treatment.26,27 Activation of NF-κB-regulated antiapoptotic gene expression is one important cause of resistance to doxorubicin. Thus, suppression of NF-κB activation is expected to increase the effectiveness of doxorubicin.28 In the present study we aimed to develop the biodegradable polyelectrolyte brushes and polyglutamate derivatives (PGS) as carriers for NF-κB p65 siRNA delivery. The spermine presenting one or two more hydrophobic alkyl groups than repeat units of polyethylenimine (PEI) was introduced into poly(L-glutamic acid) as dense pendant chains to form the PGS polyelectrolyte brushes. We anticipated the combined use of polyelectrolyte brushes and hydrophobic modification of polycation would achieve the high stability in physiological saline and serum resistance of siRNA/PGS delivery ststem. Colloidal stability of PGS polyelectrolyte brushes/siRNA polyplex was evaluated in terms of particle size in physiological saline and cellular uptake efficiency in serum containing medium. The interplay between PGS and serum proteins was further investigated using isothermal titration calorimetry. Whether PGS polyelectrolyte brushes delivering NF-κB p65 siRNA could efficiently down-regulate the expression of NF-κB p65 protein and enhance apoptosis in HeLa cells were specifically evaluated. Furthermore, whether the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex could increase the effectiveness of doxorubicin against cancer cells was also evaluated.
the destruction of the nanostructure of polycation-gene polyplex.6 In our early work, we observed the obvious aggregations of DNA/PEI 25 KDa polyplex (more than 1 μm of size) induced by serum.7 The low colloidal stability of DNA/ PEI 25 KDa polyplex decreased their cellular uptake, and consequently, their gene transfection was compromised in the presence of serum. Therefore, the sufficient colloidal stability of polycation−gene polyplex under physiological conditions is an important prerequisite for achieving high delivery efficiency of polycation−gene polyplex by systemic administration. The most commonly used methods to overcome the aggregation and improve colloidal stability involved the use of hydrophilic groups grafted onto the polycation.8 Poly (ethylene glycol) (PEG) was widely used because PEGylated polycation exhibited the reduction of nonspecific binding of serum proteins as well as decreased cytotoxicity.9 However, the steric interactions of PEGylated polycation caused the polyplex to dissociate faster than non-PEGylated polycation in vivo.10 In vivo imaging assays further showed that PEGylated polycation− gene polyplex dissociated during liver passage and so was not suitable for systemic applications.11 Thus, other strategies are needed to improve the colloidal stability of polycation−gene polyplex in the presence of salt and serum. Polyelectrolyte brushes consisting of densely grafted charged side chains on linear backbone chain, planar and spherical systems constitute a new class of material which has recently received considerable interests. Their high osmotic pressure within the brushes and ultralow interface friction bring about new physical properties and applications in biomedicine compared to conventional linear or branched polyelectrolytes.12−14 The polyelectrolyte brushes trap the counterions to form a layer with a high interior salt concentration, and a concomitantly high osmotic pressure within the polyelectrolyte layer, so-called “osmotic brush” regime.15 The osmotic repulsion between polyelectrolyte brushes can enhance the colloidal stability. Furthermore, it was found that the polyelectrolyte brushes became proteinadsorption resistant when the ionic strength of NaCl in medium was raised to a few 100 mM.16 These physical properties of polyelectrolyte brushes have an advantage in stabilizing polyelectrolyte complex against aggregation induced by salt and oppositely charged proteins. Moreover, that hydrophobic modification of polycation led to increasing the colloidal stability of polycation−gene polyplex has been recently reported.17,18 They also tended to increase biocompatibility, cellular uptake, and transfection efficiency.19,20 Nuclear factor-κB (NF-κB) is a transcription factor that regulates the expression of various genes involved in the immune and inflammatory response and also regulates the tumor development. Preclinical and clinical data support that NF-κB has been associated with many aspects of tumor formation and progression, including inhibition of apoptosis, by increasing the expression of antiapoptotic and survival factors.21 There is also increasing evidence that NF-κB plays an important role in inducible drug resistance to chemotherapy.22 The best characterized form of NF-κB is the p50/p65 heterodimer composed of p65 (65 kDa) and p50 (50 kDa) subunits, which is an antiapoptotic gene regulator. The p65 subunit provides the gene regulatory function. In resting cells, the p50/p65 heterodimer is in inactive state by binding to an inhibitor molecule IκBs in the cytoplasm. In contrast, the p50/ p65 heterodimer is activated by rapidly released from complexes with IκBs (within 20 min) upon cellular stimulation in various human cancer cells, resulting in antiapoptotic gene
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EXPERIMENTAL SECTION
Materials. Glutamic acid and N,N-dimethylformamide (DMF) were obtained from Sinopharm Chemical Reagent Co.. Spermine was purchased from Aladdin Chemistry Co. Ltd. Poly-γ-benzyl-L-glutamate (PBLG) was synthesized by using the molar ratio of monomer to initiator of 100:1 in our laboratory and the detailed synthesis can be found in refs 29 and 30. The HeLa cell line was obtained from the American Type Culture Collection (ATCC). RPMI-1640 medium, fetal bovine serum (FBS), trypsin and penicillin-Streptomycin were purchased from Gibco. Bovine serum albumin (BSA) was purchased from Qiyun Co. Branched polyethylenimine, with an average molecular weight of 25 kDa (PEI 25KDa), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich. Lipofectamine 2000 was purchased from Invitrogen Co. The siRNA targeting nuclear factor-κB p65 mRNA (siP65, antisense strand, 5′-GGACAUAUGAGACCUUCAAdTdT-3′), Cy5-labeled siRNA and negative control siRNA were purchased from Ribobio Co. RIPA lysis buffer and FITC labeled goat antirabbit IgG were purchased from Beyotime Co. BCA protein assay was obtained from Thermo Co. Rabbit anti-p65 monoclonal antibody and rabbit anti-β-actin monoclonal antibody were purchased from Cell Signaling Technology Inc. Goat antirabbit IgG were obtained from Merk.Co. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Sigma-Aldrich. Annexin VFITC apoptosis detection kit was purchased from BD Biosciences. All the other reagents were of analytical grade. Preparation of Self-Assemble PGS Polyelectrolyte Brushes/ siRNA Polyplex. The PGS polyelectrolyte brushes composed of poly(L-glutamic acid) with grafting high density of spermine was 1778
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Western Blot Analysis of NF-κB p65 Protein. For Western blot analysis, HeLa cells were lysed with RIPA buffer for at least 20 min on ice. The cells were collected in tubes and centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was transferred and the total protein content was measured using BCA protein assay. All samples were prepared with 30 mg of total protein and then loaded in each lane of a 10% sodium dodecyl sulfate polyacrylamide gel. The electrophoresis was performed at 100 V for 2 h, and subsequently proteins were electro-transferred on a PVDF membrane for 1.5 h at 100 V with a Bio-Rad blotter. Nonspecific binding to the membrane was blocked using 5% nonfat milk powder in Tris-HCl buffer which containing 1% Tween 20 at pH7.4 (TBST) for 1 h at room temperature. The membrane was incubated with two primary antibodies, rabbit anti-p65 monoclonal antibody at a dilution of 1:2000 and rabbit anti-β-actin monoclonal antibody at a dilution of 1:1000, followed by incubation with goat antirabbit IgG peroxidase conjugate secondary antibodies at a dilution of 1:6000 in TBST containing 5% nonfat milk powder. Protein bands were detected by the enhanced chemiluminescence method. Immunofluorescent Staining Study. For the immunofluorescent staining study, the above treated cells were fixed with 4% paraformaldehyde and penetrated with 1% Triton X-100 both for 15 min at room temperature. After blocking with goat serum for 30 min at 37 °C, the cells were stained with rabbit anti-p65 monoclonal antibody (1:100) and then Alexa Fluoror 488 -conjugated goat antirabbit IgG (1:1000). The cell nuclei were counterstained with DAPI. The cellular fluorescence was examined under the Zeiss fluorescence microscope. Analysis of Cellular Apoptosis. HeLa cells were seeded 24 h prior to experiments into 12-well plates at a density of 2.5 × 104 cells per well in 1.0 mL of culture medium and allowed to grow until 50% confluence. The cells were treated with the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex containing 100 nM NF-κB p65 siRNA at the N/P ratio of 20. After transfection for 4 h at 37 °C in 90% humidified atmosphere and 5% CO2, the cells were rinsed with warm PBS and supplied with 2.0 mL culture medium and further incubated for 48 h to effectively down-regulate the activity of NF-κB p65. Then 0.8 μg/mL doxorubicin was added to the cultured cells. After 24 h of incubation, the cells were trypsinized and collected by centrifugation. Apoptotic cells were detected by flow cytometry after staining with Annexin V and Propidium Iodide (PI) using the Annexin V-FITC apoptosis detection kit. Lipofectamine 2000 and PEI 25KDa carrying siRNA transfected HeLa cells were used as positive controls. Annexin V-FITC binding was analyzed by FACS-Calibur Instrument (Becton−Dickinson; Ex = 488 nm, Em = 530 nm) using FITC signal detector FL1, and PI staining was analyzed by the phycoerythrin emission signal detector FL3. Data was acquired in linear mode and visualized in linear mode, analyzed with CellQuest software (Becton− Dickinson). Statistical Analysis. Data were expressed as the mean ± the standard error of the mean. Statistical comparisons were performed using one-way analysis of variance (ANOVA). P values smaller than 0.05 and 0.01 were statistically significant and highly statistically significant, respectively.
synthesized via aminolysis of the benzyl protecting groups in poly-γbenzyl-L-glutamate (PBLG) with spermine as described in Supporting Information, Figure S1. The PGS polyelectrolyte brushes were selfassembly with siRNA to form polyelectrolyte complexes (PGS polyelectrolyte brushes/siRNA polyplex). Briefly, 100 nM of siRNA was mixed with various amounts of PGS in PBS buffer based on the N/P ratio (the molar ratio of amino groups of PGS to phosphate groups of siRNA) by gentle vortexing. The resulting mixture was incubated for 30 min at room temperature. Particle Size of PGS Polyelectrolyte Brushes/siRNA Polyplex in Physiological Saline. Stability of siRNA polyplex was evaluated in terms of the particle size of PGS polyelectrolyte brushes/siRNA polyplex at the N/P ratio of 20 both in deionized water and in physiological saline (containing 150 mM NaCl). The siRNA/PEI 25KDa polyplex at the N/P ratio of 10 was as a control. The particle size and size distribution of various siRNA polyplex samples were measured by using a photon correlation spectroscopy (PCS) on a Malvern Zetasizer NS90 (Malvern Instruments, U.K.). Each data point is comprised of at least three independent experiments. siRNA Cellular Uptake. The ability of PGS to transfer siRNA into HeLa cells, one of the most commonly used cancer cell lines, was monitored by fluorescence-activated cell sorting (FACS). Briefly, HeLa cells were seeded 24 h prior to experiments into 12-well plates at a density of 2.5 × 104 cells per well in 1.0 mL of culture medium. A total of 100 nM Cy5-labeled siRNA was formulated in the PGS polyplex at varying N/P ratios. After 4 h of incubation with the Cy5labeled siRNA/PGS polyplex in serum free or serum containing medium, the cells were rinsed with cold PBS. Subsequently, cells were treated with trypsin for 2 min, collected by centrifugation, suspended in 0.5 mL of PBS and kept on ice until analysis. The cellular uptake of Cy5-labeled siRNA was detected by FACS-Calibur Instrument (Becton−Dickinson) equipped with an argon laser (Ex = 650 nm, Em = 680 nm). Data was acquired in linear mode and analyzed with Cell Quest software (Becton−Dickinson). Intracellular Distribution. Confocal laser scanning microscopy (CLSM) was used to assess the intracellular distribution of siRNA delivered by PGS. Cy5-labeled siRNA was complexed with PGS at the N/P ratio of 20:1, PEI 25KDa at the N/P ratio of 10:1, or Lipofectamine 2000 according to the manufacturer’s instructions. Cells seeded on the glass coverslips in a six-well plate were incubated with various Cy5-labeled siRNA complexes in serum-free and serumcontaining media for 4 h. Then the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min. For nucleus labeling, fixed cells were incubated with DAPI for 15 min. Then HeLa cells were washed with PBS and stored at 4 °C. Localization of Cy5-labeled siRNA complexes in HeLa cells was monitored by Zeiss LSM710 CLSM (Carl Zeiss, DE). Red fluorescence from Cy5 was induced by the 650 nm excitation and detected at 680 nm. Blue fluorescence from DAPI was induced by the 350 nm excitation with a UV laser and detected at 430 nm. Isothermal Titration Calorimetry. The affinity of bovine serum albumin (BSA) binding to PGS was determined by isothermal titration calorimetry (ITC) with a VP-ITC Microcalorimeter (MicroCal, Inc.) at 25 °C. PGS (0.25 mM) and BSA (0.0147 mM) were prepared in tris-HCl buffer at pH 7.4 (0. 1 M Tris). To determine the binding affinity, 250 μL of polymer stock solution was added stepwise. Typically, 25 injections of 10 μL volume were made with intervals of 200 s between each addition. The first 10 μL of polymer solution titration in each experiment was subsequently deleted in the data analysis. Titration data were integrated and analyzed using Origin by the MicroCal software provided. The affinity of siRNA binding to PGS was determined by ITC at 25 °C. PGS (0.25 mM) and siRNA (0.0015 mM) were prepared in trisHCl buffer. To determine the binding affinity, 250 μL of polymer solution was added stepwise. Typically, 25 injections of 10 μL volume were made with intervals of 200 s between each addition. The first 10 μL of polymer solution titration in each experiment was subsequently deleted in the data analysis. Titration data were integrated and analyzed using Origin by the MicroCal software provided.
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RESULTS AND DISCUSSION Colloidal Stability of PGS Polyelectrolyte Brushes/ siRNA Polyplex in Physiological Saline. The numbers of RNAi-based preclinical and clinical trials for treating fatal and cureless disorders have grown dramatically over the past several years.31 One of the primary problems associated with the low delivery efficiency of current polycation-based gene delivery systems is their low colloidal stability under physiological salt and serum conditions. The DNA/polycation polyplex tends to bind nonspecifically to the oppositely charged blood components at physiological pH.32 The surface charge neutralization of DNA-polycation colloidal particles results in the destabilization and aggregations. In our early work, we observed the obvious aggregations of DNA/PEI 25KDa 1779
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Scheme 1. Polyglutamate Derivatives (PGS) Polyelectrolyte Brushesa
a
Spermine presenting one or two more hydrophobic alkyl groups than repeat units of polyethylenimine was densely grafted to poly(Lglutamic acid) backbone.
polyplex (more than 1 μm of size) induced by serum.7 The low colloidal stability of DNA/PEI 25KDa polyplex decreased their cellular uptake and consequently their gene transfection was compromised in the presence of serum. Therefore, the sufficient colloidal stability of polycation−gene polyplex under physiological conditions is an important prerequisite for achieving high delivery efficiency of polycation−gene polyplex by systemic administration.33 It has been reported that the polyelectrolyte brushes could trap their counterions to form a layer with a high internal ion concentration and a concomitantly high osmotic pressure within the polyelectrolyte
Figure 2. Cellular uptake of Cy5-siRNA polyplex. (A) Percentages of Cy5-positive cells and (B) Representative flow cytometric histogram after HeLa cells incubated with Cy5-siRNA polyplex in serum-free and serum-containing medium. (C) Confocal microscopy images of HeLa cells after treatment with Cy5-siRNA complexes. The nuclei were stained blue (DAPI) and the internalized siRNA appeared as red (Cy5).
layer.15 The osmotic repulsion force could enhance the colloidal stability. Moreover, it has been found that hydrophobic modification of polycation could increase the colloidal
Figure 1. (A) Particle size and (B) PDI changes of siRNA/PGS polyelectrolyte brush polyplex at the N/P ratio of 20 were determined in deionized water and 150 mM NaCl after incubation for various periods. (C) Size distributions of siRNA polyplex after incubation for 12 h. The siRNA/PEI 25KDa polyplex was used as a control. 1780
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Figure 3. Typical ITC data recorded for the interplays between polymer and BSA. The area underneath each injection peak (upper panels) was equal to the total heat released for that injection. When this integrated heat was plotted against the moles of polymer added to BSA in the cell, a complete binding isotherm for the interaction was obtained (below panels).
Table 1. Thermodynamic Parameters of the Interplays between Polymer and BSA samples
ΔH (cal·mol−1)
ΔS (cal·mol−1·K−1)
ΔG (cal·mol−1)
Kb (M−1)
PGS PEI 25KDa
6.226E4 ± 1.183E4 −1.218E8 ± 4.009E7
230 −4.08E5
−6.280E3 −3.152E5
4.43E4 ± 1.49E4 4.14E5 ± 1.35E5
Figure 4. ITC raw data and resulting integrals for polymer and siRNA interactions.
Table 2. Thermodynamic Parameters of the Interactions between Polymer and siRNA samples
ΔH (cal·mol−1)
ΔS (cal·mol−1K−1)
ΔG (cal·mol−1)
Kb (M−1)
PGS PEI 25KDa
−5.255E5 ± 4.183E5 −2.235E6 ± 2.056E6
−878 −7.47E3
−2.639E5 −9.012E3
1.99E3 ± 1.51E3 3.93E4 ± 1.71E4
stability of polycation−gene polyplex.17 In the present study, to increase siRNA polyplex stability under physiological conditions, spermine presenting one or two more hydrophobic
alkyl groups than repeat units of PEI was introduced into poly(L-glutamic acid) as densely grafted side chains through an ammonolysis reaction to form PGS polyelectrolyte brushes 1781
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Scheme 2. Schematic Illustration of Salt-Stable and Serum-Resistant PGS Polyelectrolyte Brushes/NF-κB p65 siRNA Polyplex for siRNA-Mediated p65 Gene Silencing
(Scheme 1 and Supporting Information, Figure S1). The weight average molecular weight of PGS polyelectrolyte brushes was 54.6 kDa and the percentage of spermine grafting groups was 70.96% (Supporting Information, Table S1). We anticipated the combined use of polyelectrolyte brushes and hydrophobic modification of polycation would achieve the high stability in physiological saline and serum resistance of siRNA/ PGS delivery system. To estimate stability of PGS polyelectrolyte brushes/siRNA polyplex under simulated physiological conditions, their particle size and size distributions were measured in physiological saline solution (containing 150 mM NaCl). Compared with the size measurement in deionized water, PGS polyelectrolyte brushes/ siRNA polyplex at the N/P ratio of 20 incubating in physiological saline solution exhibited no obvious changes in particle size (approximately 150 nm), as shown in Figure 1A. The results suggested that the PGS polyelectrolyte brushes/ siRNA polyplex had good colloidal stability under the physiological saline condition, which is one of advantages for further biomedical applications in vivo. Furthermore, the size distribution of PGS polyelectrolyte brushes/siRNA polyplex maintained monodispersed with the polydispersion index (PDI) value of 0.2 after incubation in physiological saline for 12 h (Figure 1B,C). In contrast, siRNA/PEI 25KDa polyplex exhibited much larger particles above 1 μm with PDI more than 0.5 in physiological saline over incubation periods of 1 h. PGS Polyelectrolyte Brushes/siRNA Polyplex against Serum-Induced Destabilization. The sufficient colloidal stability of siRNA/PGS polyplex under serum-containing
conditions was also a step toward successful application of polyplex for in vivo siRNA delivery. The instability of genepolycation polyplex such as PEI and polylysine polyplex in the presence of serum hinders their further application for in vivo transfection.34 The stability of PGS polyelectrolyte brushes/ siRNA polyplex in simulated biological fluid was further investigated by comparison of their cellular uptake efficiency in the presence and absence of serum. When the PGS polyelectrolyte brushes/siRNA polyplex was incubated with HeLa cells under the same experimental conditions used for gene silencing experiments, there was no statistically significant difference in cellular uptake efficiency between in serum-free medium and in serum-containing medium (Figure 2A,B). However, the cellular uptake efficiency of the Cy5-siRNA/PEI 25KDa polyplex showed highly significant reduction in serumcontaining medium, compared with that in serum-free medium. The highly cellular uptake efficiency in serum-containing medium implied that PGS polyelectrolyte brushes/siRNA polyplex was the stable delivery system in simulated biological fluids. The cellular uptake of PGS polyelectrolyte brushes/siRNA polyplex in serum-free and serum-containing medium was further observed by using CLMS (Figure 2C). It provided supporting evidence whether cellular uptake of PGS/siRNA polyplex is inhibited by serum components in the medium. The fluorescence signals emitted from Cy5-siRNA was red and emission from the cell nucleus stained with DAPI was blue. By 4 h post-transfection, the CLSM images revealed that internalized PGS polyelectrolyte brushes/Cy5-siRNA polyplex 1782
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Figure 5. Down-regulation of NF-κB p65 gene expression in HeLa cells. (A) Immunohistochemical analysis of NF-κB p65 expression. Green signals represented the expression of NF-κB p65 protein; Blue signals presented cell nuclei counterstained with DAPI (scale bar = 10 μm). (B, C) Western blot analysis of NF-κB p65 protein (65 kDa) levels. β-Actin (42 kDa) was used as a reference protein.
in HeLa cells did not showed any obviously reduction in the presence of serum, compared with that in serum-free medium. In contrast, the red fluorescence signals emitted from Cy5siRNA dramatically decreased when HeLa cells treated with either Cy5-siRNA/PEI 25KDa or Cy5-siRNA/Lipofectamine 2000 in the serum-containing medium. The results confirmed that the PGS polyelectrolyte brushes/Cy5-siRNA polyplex was resistant to serum inhibition, resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. Serum containing media usually showed inhibitory effects on cellular uptake of polycation gene delivery, due to that cationic polymer nonspecifically and strongly interacted with negatively charged serum proteins in an ion-dependent manner.35 Isothermal titration calorimetry (ITC) was used to investigate the nonspecifically binding affinities of PGS polyelectrolyte brushes to negatively charged bovine serum albumin (BSA) in the presence of salt (100 mM), as shown in Figure 3. Serum albumin is the most abundant plasma protein in mammals. PEI 25KDa was used as a positive control to compare with PGS polyelectrolyte brushes in terms of the electrostatic interaction with BSA. The results revealed that the PGS polyelectrolyte brushes have a low affinity to BSA, as evidenced by their relatively low values of Gibbs free energy and binding constant Kb (Table 1). Furthermore, the interplay between PGS polyelectrolyte brushes and BSA was not dominated by strongly electrostatic bonding as PEI 25KDa did, but by weakly hydrophobic interactions (Figure 3). The results demonstrated that in the presence of BSA and salt, the steric hindrance effects
on PGS polyelectrolyte brushes prevailed over the electrostatic repulsion resulting in reducing the serum-protein adsorption, which was reported as one of the unique properties of polyelectrolyte brushes.16 On the other hand, serum addition remarkably reduced the cellular uptake of PEI 25KDa polyplex, most likely due to PEI 25KDa binding with BSA by strongly electrostatic bonding with both high values of Gibbs free energy and binding constant Kb. It was noticed that PEI 25KDa binding siRNA tightly led to low knockdown efficiency due to insufficient siRNA release from the polyplexes in cells.36 The interactions between siRNA and PGS polyelectrolyte brushes were measured by ITC thermodynamic analysis. There was a cleared difference in the titration profiles of siRNA with PGS polyelectrolyte brushes and PEI 25KDa polyplexes (Figure 4). The initial exothermic quantity measured by ITC is usually represented the interaction strengths of the titrant and the test substance.37 The interactions between the siRNA and the PGS during the early titration showed about 69.3 kcal/moL exothermic quantities, which was significantly lower than those of siRNA/PEI 25KDa polyplexes (325 kcal/moL). Furthermore, binding constants Kb value of the PGS complexed with siRNA was 19.75 times smaller than that of PEI 25KDa complexed with siRNA (Table 2). The results suggested that the PGS polyelectrolyte brushes complexed with siRNA were relatively weaker than PEI 25KDa that allowed for timely unpacking of siRNA in cells. 1783
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Figure 6. (A) Annexin V/PI staining assay of cell apoptosis induced by various NF-κB p65 siRNA polyplex formulations. The lower left quadrant represented the live cells (annexin V negative, PI negative). The upper left quadrant represented the necrotic cells (annexin V negative, PI positive). The upper right quadrant represented the late apoptotic cells (annexin V positive, PI positive). The lower right quadrant represented the early apoptotic cells (annexin V positive, PI negative). (B) Percentages of different phase of apoptotic cells. Percentage of total apoptotic cells including sum of early and late apoptotic cells.
Down-Regulation of NF-κB p65 Protein by PGS Polyelectrolyte Brushes/siRNA Polyplex. Up to now, low delivery efficiency by nonviral carriers is a major barrier to achieving an effective siRNA-based therapeutic.38 Based on the above optimization results, the high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes complexed with NF-κB p65 siRNA at the N/P ratio of 20 was used to investigate the NF-κB p65 gene silencing in HeLa cells, as shown in Scheme 2. Immunohistochemical and Western blot analysis were performed at 48 h post-transfection to evaluate the expression levels of NF-κB p65 protein. Immunohistochemical analysis showed that the expressed NFκB p65 protein was green fluorescence layer localized in the cytoplasm of HeLa cells (Figure 5A). The images revealed that the untreated HeLa cells expressed high levels of NF-κB p65 protein in the cytoplasm. The HeLa cells under treatment of either siRNA negative control/PGS polyelectrolyte brush polyplex or naked NF-κB p65 siRNA did not showed any clear down-regulation of NF-κB p65 protein expression. However, the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex transfection induced remarkably down-regulated NFκB p65 protein in HeLa cells. The effects of siRNA mediated by PGS polyelectrolyte brushes, PEI 25KDa, and Lipofectamine 2000 on inhibiting NF-κB p65 expression was also confirmed at protein levels by Western blotting (Figure 5B,C). The treatment of PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex exhibited significantly lower NF-κB p65 protein levels compared with HeLa cells treated with PEI 25KDa (P < 0.01) and was comparably attenuated as Lipofectamine 2000 did.
Induction of Apoptosis in HeLa Cells by PGS Polyelectrolyte Brushes/NF-κB p65 siRNA Polyplex. It has been widely reported that NF-κB p65 is overexpressed in several cancer and its activation is associated with inducible drug resistance to chemotherapy.21,22 Thus, inhibition of NFκB activity represents a potential therapeutic strategy for cancer treatment and increases sensitivity of cancer cells to chemotherapeutic drugs. The serious side effects of nonspecific inhibition of NF-κB have not been widely reported due to NFκB being in inactive state in resting cells. The potential side effects of nonspecific inhibition of NF-κB were decreasing immune responses. However, the nonviral delivery system mediated siRNA therapy is considered as transient, allowing recovery of immune functions after cessation of the treatment.39 Our data showed that PGS polyelectrolyte brushes/ NF-κB p65 siRNA polyplex transfection could strongly induce down-regulation of NF-κB p65 protein expression levels in HeLa cells. Consequently, we further examined whether the down-regulation of NF-κB p65 protein expression mediated by PGS polyelectrolyte brush polyplex could induce apoptosis of cancer cells. FITC-Annexin V/propidium iodide (PI) double staining was used to identify apoptotic cells, as shown in Figure 6. In comparison with untreated cells, PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex significantly induced apoptosis in HeLa cells. They led to a 2.5- and 1.5-fold increase in the early stage apoptosis induced by PEI 25KDa and Lipofectamine 2000 systems, respectively (lower right quadrant). The PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex treated cells also showed significantly higher levels of 1784
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doxorubicin there was a greater decrease in the number of viable cells compared with cells treated with doxorubicin alone, while doxorubicin demonstrated a dose-dependent decrease in cell proliferation in both groups. Moreover, by the combining use of the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex with doxorubicin, equivalent 50% cytotoxic effects of doxorubicin alone at 1.2 μg/mL could be achieved using 50% less of doxorubicin. The enhanced sensitivity of HeLa cells to doxorubicin was also obtained when the effects of doxorubicin was analyzed on apoptosis using fluorescence-activated cell sorting, as shown in Figure 7B. The total apoptosis (lower right and upper right quadrant) of doxorubicin with pretransfection of the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex was 170% of that treated with same dose of doxorubicin alone. In addition, that the HeLa cells transfected by the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex was exposed to doxorubicin showed significantly increased necrosis. Doxorubicin was reported to cause necrosis when used in high concentrations because doxorubicin triggers membrane peroxidation and disruption.40 These results confirmed that pretreatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex could increase the sensitivity of HeLa cells to doxorubicin, which was consistent with the results of cytotoxicity. It has been reported that cationic polymer using in gene delivery tends to induce apoptosis and cytotoxicity.41,42 Therefore, whether the PGS polyelectrolyte brush carrier enhanced the effectiveness of doxorubicin was measured. The results indicated that doxorubicin plus PGS polyelectrolyte brushes complexed with negative control siRNA at the same concentration used in transfection experiments had no positive effects on apoptosis in HeLa cells, in comparison with the control and treatment with doxorubicin alone groups, respectively. It demonstrated that the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex could enhance the apoptotic effects of doxorubicin. These results also suggested that the enhanced cytotoxic and apoptotic effects of doxorubicin were clearly benefit from NF-κB p65 inhibition mediated by PGS polyelectrolyte brushes/siRNA polyplex. This synergistic effect is clinically important because it can decrease the dosages of doxorubicin and potentially overcome the drug resistance in cancer chemotherapy.
Figure 7. (A) Cytotoxicity of HeLa cells and (B) apoptosis analysis of HeLa cells treated with 0.8 μg/mL of doxorubicin after transfection with or without PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex. Cells were stained with Annexin V-FITC and propidium iodide (**P < 0.01, *P < 0.05).
total apoptosis including the early stage apoptosis and late-stage apoptosis (lower right and upper right quadrant) than those of PEI 25KDa and Lipofectamine 2000 systems. In addition, cell necrosis induced by siRNA/PEI 25KDa polyplex was the highest levels among the three siRNA delivery systems (upper left quadrant), most likely due to the well-known adverse side effects, especially high cytotoxicity of PEI 25KDa. Enhancement of Cytotoxic and Apoptotic Effects of Doxorubicin Combined with PGS Polyelectrolyte Brushes/NF-κB p65 siRNA Polyplex. Doxorubicin is one of the most effective chemotherapeutic agents in the treatment of many types of cancers including bone tumors, ovarian, breast, uterine and cervical cancers.24 Several studies indicate that doxorubicin induces activity of NF-κB resulting in drug resistance in cancer chemotherapy.26 We then explored whether the targeted inhibition of NF-κB p65 mediated by PGS polyelectrolyte brushes/siRNA polyplex could increase doxorubicin effectiveness in cancer cells. First, we evaluated the effects of combined treatment with doxorubicin and PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex on HeLa cell viability (Figure 7A). The cytotoxicity study was conducted using two groups, HeLa cells exposed to doxorubicin with or without NF-κB p65 siRNA polyplex pretreatment. When HeLa cells were per-transfected with the PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex prior to the addition of
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CONCLUSIONS A major challenge of polymer-mediated siRNA delivery in the clinical setting is achieving good stability under physiological conditions and efficient intracellular delivery. In the present study we developed a biodegradable polyglutamate derivative PGS polyelectrolyte brushes composed of poly(L-glutamic acid) densely grafted spermine for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes formed highly stable PGS/siRNA polyplex (about 150 nm) with good dispersity in physiological saline. These size-controlled siRNA/PGS polyelectrolyte brush polyplex did not show any obvious decreases in cellular uptake efficiency in the presence of serum, whereas a marked reduction in cellular uptake of PEI 25KDa polyplex was observed. ITC provided the interesting evidence that a weak hydrophobic interaction was the major driving force within the interplay between PGS polyelectrolyte brushes and serum proteins in the presence of salt (0.1 M), but not in a strongly ion-dependent manner as PEI 25KDa did. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved more efficient gene silencing of p65 gene blocked NF-κB hyperactivation than PEI 25KDa and comparable to Lipofectamine 2000. The early stage 1785
dx.doi.org/10.1021/bm400177q | Biomacromolecules 2013, 14, 1777−1786
Biomacromolecules
Article
(15) Toomey, R.; Tirrell, M. Annu. Rev. Phys. Chem. 2008, 59, 493− 517. (16) Czeslik, C.; Jackler, G.; Hazlett, T.; Gratton, E.; Steitz, R.; Wittemann, A.; Ballauff, M. Phys. Chem. Chem. Phys. 2004, 6, 5557− 5563. (17) Philipp, A.; Zhao, X.; Tarcha, P.; Wagner, E.; Zintchenko, A. Bioconjugate Chem. 2009, 20, 2055−2061. (18) Jones, S. P.; Gabrielson, N. P.; Wong, C. H.; Chow, H. F.; Pack, D. W.; Posocco, P.; Fermeglia, M.; Pricl, S.; Smith, D. K. Mol. Pharmaceutics 2011, 8, 416−429. (19) Sunshine, J. C.; Akanda, M. I.; Li, D.; Kozielski, K. L.; Green, J. J. Biomacromolecules 2011, 12, 3592−3600. (20) Santos, J. L.; Oliveira, H.; Pandita, D.; Rodrigues, J.; Pêgo, A. P.; Granja, P. L.; Tomás, H. J. Controlled Release 2010, 144, 55−64. (21) Tapia, M. A.; González-Navarrete, I.; Dalmases, A.; Bosch, M.; Rodriguez-Fanjul, V.; Rolfe, M.; Ross, J. S.; Mezquita, J.; Mezquita, C.; Bachs, O.; Gascón, P.; Rojo, F.; Perona, R.; Rovira, A.; Albanell. J. Cell Cycle 2007, 6, 2284−2292. (22) Montagut, C.; Tusquets, I.; Ferrer, B.; Corominas, J. M.; Bellosillo, B.; Campas, C.; Suarez, M.; Fabregat, X.; Campo, E.; Gascon, P.; Serrano, S.; Fernandez, P. L.; Rovira, A.; Albanell. J. Endocr. Relat. Cancer 2006, 13, 607−616. (23) Sun, Z.; Andersson, R. Shock 2002, 18, 99−106. (24) Bien, S.; Ritter, C. A.; Gratz, M.; Sperker, B.; Sonnemann, J.; Beck, J. F.; Kroemer, H. K. Mol. Pharmacol. 2004, 65, 1092−1102. (25) Lee, T. K.; Lau, T. C.; Ng, I. O. Cancer Chemother. Pharmacol. 2002, 49, 78−86. (26) Gangadharan, C.; Thoh, M.; Manna, S. K. J. Cell Biochem. 2009, 107, 203−213. (27) Dunkern, T. R.; Wedemeyer, I.; Baumgärtner, M.; Fritz, G.; Kaina, B. DNA Repair 2003, 2, 49−60. (28) Bednarski, B. K.; Baldwin, A. S.; Kim, H. J. PLoS One 2009, 4, e6992. (29) Pan, S. R.; Wang, Q. M.; Yi, W. J. Biomater. Appl. 2007, 22, 181−192. (30) Wen, Y.; Pan, S.; Luo, X.; Zhang, X.; Zhang, W.; Feng, M. Bioconjugate Chem. 2009, 20, 322−332. (31) Davidson, B. L.; McCray, P. B., Jr. Nat. Rev. Genet. 2011, 12, 329−340. (32) Kircheis, R.; Wightman, L.; Schreiber, A.; Robitza, B.; Rössler, V.; Kursa, M.; Wagner, E. Gene Ther. 2001, 8, 28−40. (33) Oupicky, D.; Ogris, M.; Howard, K. A.; Dash, P. R.; Ulbrich, K.; Seymour, L. W. Mol. Ther. 2002, 5, 463−472. (34) Guo, W.; Lee, R. J. J. Controlled Release 2001, 77, 131−138. (35) Haberland, A.; Knaus, T.; Zaitsev, S. V.; Buchberger, B.; Lun, A.; Haller, H.; Böttger, M. Pharm. Res. 2000, 17, 229−235. (36) Nguyen, J.; Reul, R.; Roesler, S.; Dayyoub, E.; Schmehl, T.; Gessler, T.; Seeger, W.; Kissel, T. H. Pharm. Res. 2010, 27, 2670− 2682. (37) Cedervall, T.; Lynch, L.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050−2055. (38) Bora, R. S.; Gupta, D.; Mukkur, T. K.; Saini, K. S. Mol. Med. Report 2012, 6, 9−15. (39) Frelin, C.; Imbert, V.; Griessinger, E.; Peyron, A. C.; Rochet, N.; Philip, P.; Dageville, C.; Sirvent, A.; Hummelsberger, M.; Bérard, E.; Dreano, M.; Sirvent, N.; Peyron, J. F. Blood 2005, 105, 804−811. (40) Hassan, F.; Morikawa, A.; Islam, S.; Tumurkhuu, G.; Dagvadorj, J.; Koide, N.; Naiki, Y.; Mori, I.; Yoshida, T.; Yokochi, T. Clin. Exp. Immunol. 2008, 151, 334−340. (41) Thomas, T. P.; Majoros, I.; Kotlyar, A.; Mullen, D.; Holl, M. M.; Baker, J. R., Jr. Biomacromolecules 2009, 10, 3207−3214. (42) Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1200−1205.
apoptotic rate of HeLa cells induced by PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex was significantly higher than those induced by PEI 25KDa. The combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin led to significantly enhanced cytotoxicity and apoptosis in HeLa cells. These size-controlled PGS polyelectrolyte brushes/siRNA polyplex in the presence of saline and serum have potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.
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ASSOCIATED CONTENT
S Supporting Information *
Summary of synthesis of poly(L-glutamic acid) densely grafted spermine (PGS), 1H NMR spectra of spermine, poly-γ-benzylL-glutamate (PBLG) and PGS polyelectrolyte brushes, gel permeation chromatography (GPC) analysis of the average molecular weight of PGS, characterization of siRNA/PGS polyelectrolyte brush polyplex, and cell viability of PGS polyelectrolyte brushes. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +8620 39943119. Fax: +8620 39943119. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (Project No. 30600789) and Guangdong Natural Science Fund (Project No. S2012010008453) for their financial support of this research.
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REFERENCES
(1) Fougerolles, A.; Vornlocher, H. P.; Maraganore, J.; Lieberman, J. Nat. Rev. Drug Discovery 2007, 6, 443−453. (2) Burnett, J. C.; Rossi, J. J.; Tiemann, K. Biotechnol. J. 2011, 6, 1130−1146. (3) Devi, G. R. Cancer Gene Ther. 2006, 13, 819−829. (4) Patil, S. D.; Rhodes, D. G.; Burgess, D. J. AAPS J. 2005, 7, E61− 77. (5) Huang, L.; Liu, Y. Annu. Rev. Biomed. Eng. 2011, 13, 507−530. (6) Santander-Ortega, M. J.; Fuente, M.; Lozano, M. V.; Bekheet, M. E.; Progatzky, F.; Elouzi, A.; Uchegbu, I. F.; Schätzlein, A. G. Soft Matter 2012, 8, 12080−12092. (7) Wang, C.; Luo, X.; Feng, M.; Zhao, Y.; Zeng, X.; Pan, S.; Wu, C.; Peng, H. Acta Biomater. 2012, 8, 3014−3026. (8) Steele, T. W.; Zhao, X.; Tarcha, P.; Kissel, T. Eur. J. Pharm. Biopharm. 2012, 80, 14−24. (9) Zhong, Z.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christensen, L. V.; Yockman, J. W.; Kim, Y. H.; Kim, S. W. Biomacromolecules 2005, 6, 3440−3448. (10) Fischer, D.; Osburg, B.; Petersen, H.; Kissel, T.; Bickel, U. Drug Metab. Dispos. 2004, 32, 983−992. (11) Merkel, O. M.; Librizzi, D.; Pfestroff, A.; Schurrat, T.; Buyens, K.; Sanders, N. N.; De Smedt, S. S.; Béhé, M.; Kissel, T. J. Controlled Release 2009, 138, 148−159. (12) Raviv, U.; Giasson, S.; Kampf, N.; Gohy, J.; Jérôme, R.; Klein, J. Nature 2003, 425, 163−165. (13) Lau, K. H.; Ren, C.; Sileika, T. S.; Park, S. H.; Szleifer, I.; Messersmith, P. B. Langmuir 2012, 28, 16099−16107. (14) Henzler, K.; Haupt, B.; Lauterbach, K.; Wittemann, A.; Borisov, O.; Ballauff, M. J. Am. Chem. Soc. 2010, 132, 3159−3163. 1786
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