Efficient siRNA Targeted Delivery into Cancer Cells by Gastrin

For GRP and GnRH receptor analysis, the cells were stained for 30 min at 4 °C ...... Flatekval , G. F. and Sioud , M. (2009) Modulation of dendritic ...
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Efficient siRNA Targeted Delivery into Cancer Cells by Gastrin-Releasing Peptides Mouldy Sioud* and Anne Mobergslien Department of Immunology, Molecular Medicine Group, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0310, Oslo, Norway ABSTRACT: Small interfering RNAs (siRNAs) have displayed considerable promise for the treatment of cancer. However, their delivery to the desired cell population remains a challenging task. Here we have covalently conjugated a siRNA against survivin to gastrin-releasing peptides (GRPs) to direct siRNA molecules to cancer cells that express the GRP receptor. The cellular uptake of the peptide−siRNA conjugates was tested in breast MDA-MB 231 cancer cells, which express the GRP receptor. Fluorescein-tagged GRP−siRNA conjugates were taken up by cancer cells but not normal mammary epithelial cells or human blood monocytes. By 120 min of incubation, most of the cells have taken up the conjugates. Excess free peptide inhibited uptake, implying dependence of uptake on GRP receptor. Moreover, bitargeting of siRNA molecules by GR and luteinizing hormone-releasing peptides accelerated the uptake kinetics by MDA-MB 231 cells when compared to monotargeted siRNAs. Peptide−siRNA conjugates, but not free siRNAs, inhibited the expression of survivin, an endogenous gene involved in cancer cell survival. None of the peptide−siRNA conjugates induced the expression of inflammatory cytokines or interferon α in human blood leukocytes. Overall, the data demonstrate the feasibility of GRP receptor-mediated targeted delivery of siRNAs to cancer cells, an important step for RNA interference therapy in humans.

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tumor vasculature expressing αvβ3 integrin was shown to deliver VEGF siRNAs to tumor cells.23 A peptide mimetic of the insulin-like growth factor 1 (IGF-1) was conjugated to siRNAs, and about 60% of the target gene was inhibited by the peptide− siRNA conjugates.14 Furthermore, a 29-amino acid peptide derived from rabies virus glycoprotein facilitated the delivery of siRNAs to the brain through the binding to acetylcholine receptor expressed by neuronal cells.15 Given their exogenous origin, microorganism-derived peptides and peptides selected from random peptide libraries or artificially designed may activate the immune system and induce some side effects.24 By contrast, naturally occurring peptides are expected to be nonimmunogenic and more stable in extracellular microenvironment where they are expected to function. Immunogenicity and stability are likely to be more of an issue for any artificial delivery formats for siRNAs. A number of hormone receptor binding peptides have been successfully explored for magnetic resonance imaging of tumors.25 More importantly, the receptors of these naturally circulating peptides are overexpressed in a large variety of human cancers but not normal human tissues, thus permitting an in vivo targeting of tumor cells. For example, the BB2 receptor for the human gastrinreleasing peptide (GRP) is overexpressed on the cell surfaces of several malignant tissues, particularly in the cases of colon cancer, breast cancer, lung cancer, and prostate cancer.26−28 Another receptor, which also represents an attractive targeting molecule, is the gonadotropin releasing hormone receptor (GnRHR), which is

INTRODUCTION RNA interference (RNAi) has recently emerged not only as a powerful tool to study gene function but also as a therapeutic strategy to silence genes involved in the initiation and/or perpetuation of disease.1−3 Although much has been achieved about the mechanisms of RNAi, there are a number of challenges that applications of this gene-silencing technology need to overcome, including off-target effects and delivery.3,4 To date, siRNA delivery has been achieved by a number of strategies including lipid-based agents, nanoparticles, magnetofection, and electroporation.1,5 As the most used delivery agents enter all cell types, specificity must be built into the delivery agents or directly attached to the siRNA molecules. With respect to peptides, a wide range of cell penetrating peptides (CPPs) have been identified and used as a vehicle for intracellular delivery of siRNAs. Well-known examples of CPPs are TAT-transactivator protein from human immunodeficiency virus type 1, penetratin from the homeodomain protein of Antennapaedia, transportan, a hybrid amino acid sequence, and MGP peptide.6−9 Although the current CPP siRNA-delivery approaches have gained some merits, they generally target the entire cell population.10 To achieve specific delivery to target cells, a variety of siRNA targeting strategies have been used, including antibodies, CpG oligonucleotides, RNA aptamers, peptides, and chemical modifications.11−17 Peptides that target specifically one given cell type have been identified using different techniques such as peptide libraries.18−20 For example, we have identified a short peptide (LTVSPWY) capable of delivering antisense oligonucleotides to breast carcinoma cell lines.20 Recently, this peptide was able to deliver therapeutics and imaging agents to cancer cells in vitro and in vivo.21,22 Similarly, a RGD peptide targeting © 2012 American Chemical Society

Received: February 3, 2012 Revised: March 22, 2012 Published: March 25, 2012 1040

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RNA stability, the sense and antisense strands were annealed before chemical modification and purification. In some experiments, siRNA coupling to the GRP1 peptide was done as described by Davidson et al.33 The final concentration of the conjugates was adjusted at 0.5 μg/μL. The samples were stored in aliquots at 20 °C until use. Peptide and siRNA Conjugation to Streptavidin. Equimolar amounts (10 nmol) of the 5′-biotin siRNA with 3′ fluorescein, biotin-GRP1 peptide, and biotin-GnRH peptide were mixed with 8 nmol of streptavidin (Promega) in PBS buffer. The mixture was incubated at room temperature for 30 min and was then stored at 4 °C in the dark until use. As controls, streptavidin was incubated with siRNA combined with either GRP1 or GnRH peptide. Peptide−streptavidin−siRNA conjugates were added directly to cells at a final concentration of 50 nM. The attachment of the siRNA molecules and peptides to streptavidin protein was verified with PAGE shift assay. Under our experimental conditions, all siRNAs and peptide molecules were bound to the streptavidin protein. Cellular Uptake of the Peptide−siRNA Conjugates. The cells were seeded onto a six-well plate (5 × 105/well) 1 day before the experiment. Subsequently, they were washed and replenished with 2 mL of fresh X-vivo 15 medium (2 mL/well) and then incubated with peptide−siRNA conjugates (50 nM) or free siRNA (50 nM) for 3 h at 37 °C, 5% CO2. At the end of the uptake period, Hoechst 33342 dye was added for 10 min to visualize nuclei. The cells were washed twice with PBS to remove fluorescent siRNAs and then analyzed by epifluorescence microscope (Leica). To confirm internalization, the cells were cultured on Lab-Tek chamber slides (Nalge Nunc International), incubated with the siRNA conjugates, washed, and then analyzed by a Zeiss LSM 510 confocal microscope. Flow Cytometry Analysis. For GRP and GnRH receptor analysis, the cells were stained for 30 min at 4 °C with goat anti-GRPR or anti-GnRHR polyclonal IgG antibodies in phosphate buffered saline (PBS) containing 1% fetal calf serum (staining buffer). Subsequent to washing, the cells were incubated with FITC-conjugated anti-goat IgG antibody for 30 min in staining buffer, washed, and then analyzed on a BD LSR II flow cytometer (BD Biosciences). Cells were also stained with control antibody isotypes. For peptide binding, the cells were incubated with fluorescein-tagged peptides (5 μg/mL) in phosphate-buffered saline (PBS)/1% FCS for 30 min at room temperature. Subsequently, they were washed and analyzed by flow cytometry. The uptake of peptide−siRNA conjugates was also analyzed by flow cytometry. In these experiments the cells were seeded into six-well plates (3 × 105 cells/well) in RPMI medium and cultured for 24 h. Prior to treatment, RPMI medium was replaced by X-vivo 15 medium (2 mL/well) and the cells were incubated with peptide−siRNA conjugates (50 nM) for the desired time at 37 °C in 5% CO2. Subsequent to incubation, the cells were harvested by scraping, washed 3 times with PBS/ 1% FCS, and then analyzed by flow cytometry. Cellular uptake was also performed in RPMI supplemented with 10% FCS. All data were analyzed by FlowJo software. Real-Time Quantitative Reverse-Transcription PCR. Total RNA was isolated from cells using the TRIzol reagent (Invitrogen). GRP and GnRH receptor expression was analyzed in MDA-BM 231 cells by quantitative RT-PCR using SYBR Green. Survivin mRNA expression in controls and siRNA-treated samples was also analyzed by RT-PCR. All reactions were performed in triplicate and normalized to GAPDH mRNA. Relative expression was calculated using the Ct method. The sequences of

strongly expressed by tumors such as ovarian, colon, breast, prostate, and lung cancer.29 The ability of GRP agonists to be internalized is one factor that has led to their use as vehicles for radionuclide imaging and therapy.28,30 In this work, first we tested whether the covalent linkage of GRP to siRNA against survivin would enable the delivery of the siRNA to cancer cells that express the GRP receptor. Second, we bitargeted the siRNA molecules to breast cancer cell line MDA-MB 231 using GRP and GnRH peptide. Third, we investigated gene silencing by peptide−siRNA conjugates.

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MATERIALS AND METHODS Cells. The breast MDA-MB-231 cancer cell line was purchased from American Type Culture Collection. Cells were cultured in RPMI medium supplemented with 10% FCS or in X-vivo 15 medium. Human mammary epithelial cells (HMECs) were obtained from Clonetics/BioWhittaker (San Diego, CA, U.S.) and cultured in MEGM, Ham’s F12 medium supplemented with 10% FCS. Human peripheral monocytes, dendritic cells, T cells, and B cells were prepared from buffy coats obtained from normal volunteers as described previously.31 Peptides, siRNAs, and Oligonucleotides. Synthetic peptides were obtained from Eurogentec (Seraing, Belgium). The amino acid sequences of GRP1, GRP2, GnRH peptide, and control peptide are CGGNHWAVGHLM, CKMYPRGNHWAVGHLM, CQHWSYGLRP, and ADGGAQGTAC, respectively. A cysteine residue was added to the NH2- or C-terminus to allow conjugation of the peptides to thiol-containing siRNAs, 6-(iodoacetamide)fluorescein (6-IAF), or biotin. Survivin siRNA sense strand with 5′-thiol group and 3′-fluorescein, unmodified siRNA sense and antisense strands were made and purified by Eurogentec (Seraing, Belgium). 5′-biotinylated siRNA duplexes with or without 3′-fluorescein at the 3′-end of the sense strand, and DNA oligonucleotides were made by Eurofins MWG (Ebersberg, Germany). The sequences of siRNA sense strand were the following: survivin, 5′-GAGCCAAGAACAAAAUUGC-3′; control siRNA (scrambled sequence), 5′UAUAGCAAGACAGCAGAAC-3′. Conjugation of the 6-IAF to Peptides. Peptides were dissolved in water at 1 mg/mL. Around 0.5 nmol of each peptide was mixed with 5 nmol of the fluorescein 6-iodoacetamide in PBS buffer, and then the mixture was incubated at room temperature for 2 h with agitation. The labeled peptide conjugates were purified on a gel filtration column, Sephadex G-10, in PBS buffer as described by the manufacturer's instructions (Nuclear Probes, Invitrogen). Aliquots from all fractions were analyzed on 15% native PAGE gels, and fluorescein-conjugated peptides were visualized by UV exposure. Positive fractions were collected and stored at −20 °C until use. Conjugation of GRP1 to siRNAs. The siRNA targeting human survivin with a thiol group at the 5′-end and fluorescein at the 3′-end of the sense strand was dissolved in 10 mM HEPES and 1 mM EDTA, pH, 8.0, at 20 μM. The conjugation of the siRNA to the peptide-containing a thiol group was mainly performed as described by Muratovska and Eccles.32 Briefly, equimolar concentrations of siRNA, peptide, and the thiol oxidant diamide (20 nmol) were mixed and incubated for 1 h at 40 °C. Subsequently, the mixture was run on 15% PAGE and the band corresponding to the peptide−siRNA conjugates was cut out, crushed into a fine slurry, and then incubated in RNase free water at 37 °C for 6 h with agitation. Subsequent to centrifugation at 12000g for 10 min, supernatants were collected and concentrated to 200 μL using a Centricon-3 device (Ambion). To increase 1041

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proteins were prepared as previously described.31 Equal amount of proteins (50 μg) were resolved by denaturing electrophoresis in 0.1% sodium dodecyl sulfate (SDS) on 12% PAGE and electrotransferred to nitrocellulose membrane. After blocking in 5% milk in TBST (0.1% Tween) for 2 h, membranes were probed with primary antibodies against survivin (Santa Cruz Biotechnology) and HRP-conjugated secondary antibodies. Immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) system (GE Healthcare). In order to control for protein loading, membranes were stripped and then incubated with anti-β-actin polyclonal antibodies. Gene Silencing. One day prior to transfection, the breast MDA-MB 231 cancer cells were seeded in a six-well plate at a density of 5 × 105 cells per well in RPMI supplemented with 10% FCS. Subsequently, the medium was replaced by X-vivo 15 medium (2 mL/well) containing 50 nM either peptide−siRNA conjugates or unconjugated siRNAs. Cells were harvested 24 h after the addition of the test molecules and monitored for survivin gene expression by real-time PCR and Western blots. In some experiments, the conjugates were directly added to cells cultured in RPMI medium supplemented with 10% FCS. Cytokine ELISA. Peripheral blood mononuclear cells were prepared from buffy coats from healthy donors. Cells were plated in a 96-well plate (2 × 105/well/200 μL) and than incubated with either free siRNA or siRNA−peptide conjugates. DOTAP-formulated siRNA was used as positive control. Culture supernatants were collected at 24 h after treatment and assayed for TNF-α and IFN-α by ELISA according to the manufacturer’s protocol (R&D Systems). All molecules were tested at 100 nM in triplicate.

the used primers are the following: GRP receptor, 5′-CTCCCCGTGAACGATGACTGG-3′ (forward) and 5′-ATCTTCATCAGGGCATGGGAG-3′ (reverse); GnRH receptor, 5′-CCAGAGACACAAGGCTTGAAG-3′ (forward) and 5′-TGACAATCAGAGTCTCCAACAG-3′ (reverse); survivin 5′-AGGTTCCTTATCTGTCACAC-3′ (forward) and 5′-TCCCCAATGACTTAGAATGG-3′ (reverse); GAPDH, 5′-CTTCCAAGGAGTAAGACCCC-3′ (forward) and 5′-TGTGAGGAGGGAGATTCAC-3′ (reverse). Western Blot Analysis. Subsequent to treatments, cells were collected by scraping and washed with PBS, and cytoplasmic

Figure 1. MDA-MB cancer cells express the GRP receptor. (A) GRP receptor expression was analyzed by quantitative real time PCR in MDA-MB 231 and human mammary epithelial cells (HMEC). (B) GRP receptor expression (orange lines), compared with isotype control (blue lines) and unstained cells (red lines), was analyzed by flow cytometry in both cell types. Data are representative of at least three independent experiments.

Figure 2. Binding of GRP peptides to human cells. Binding of the carboxyfluorescein-tagged GRP1 and GRP2 peptides (orange lines), or control peptide (blue lines) to MDA-MB 231 (A). The binding to HMEC and blood monocytes is shown in (B). The cells were incubated with the peptides (5 μg/mL) for 30 min at room temperature, washed three times, and then analyzed by flow cytometry. The cells incubated with only the staining buffer are represented by the red lines. Data are representative of three independent experiments. 1042

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Statistical Analysis. Statistical analysis was performed with a Student’s t test. Values with P < 0.05 were considered significant.

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RESULTS Analysis of Peptide Binding by Flow Cytometry. The overexpression of GRP receptor BB2 in various neoplasias relative to normal tissues has been a major factor for driving the development of GRP-based radiopharmaceuticals.34 To assess the utility of this receptor as a targeting strategy for siRNAs, first we have examined its expression in the breast MDA-MB 231 cancer cell line at the mRNA level by RTPCR (Figure 1A) and at the protein level by flow cytometry (Figure 1B and Figure 1C). The receptor is expressed by breast MDA-MB 231 cancer cells but not normal human mammary epithelial cells (HMEC). To investigate the targeting potential of GRP receptor, two synthetic peptides (GRP1, CGGNHWAVGHLM; GRP2, CKMYPRGNHWAVGHLM) were designed, conjugated to 6-iodoacetamidofluorescein (6-IAF), and tested for binding to MDA-MB 231 cells (Figure 2A). A significant binding to both peptides when compared to control peptide was found (P < 0.001). HMEC and blood monocytes did not express the receptor, and they did not bind to GRP1 or GRP2 (Figure 2B and data not shown). Collectively, these results confirm the binding specificity of the GR peptides and indicated that the addition of a cysteine and a fluorescent dye to the NH2 terminus of the peptides does not hamper their binding to the GRP receptor expressed by breast MDA-MB 231 cancer cells. Cellular Uptake of the Peptide−siRNA Conjugates. To determine whether the short peptide (GRP1) could deliver siRNAs to cancer cells, we prepared peptide−siRNA conjugates with fluorescently labeled sense strand and then evaluated their cellular uptake. The peptide was covalently attached to the 5′end of the sense siRNA strand via disulfide linkage. Breast MDA-MB 231 cancer cells were incubated with the conjugates for 3 h at 37 °C in ex vivo 15 medium. Subsequent to incubation, Hoechst 33342 dye was added to the cultures for 10 min to visualize the nuclei in living cells, and then the cells were washed and analyzed with an epifluorescence microscope (Figure 3A). The conjugation of the peptide to survivin siRNA dramatically facilitated the uptake of siRNA by breast MDAMB 231 cells. The same results were obtained with breast T47D cancer cells (data not shown). In contrast, free siRNA molecules were not taken up by the cells when compared to peptide−siRNA conjugates (Figure 3A). We further validated peptide−siRNA delivery to MDA-MB 231 cells by confocal microscopy. Fluorescently labeled siRNA delivered with GRP1 exhibited a punctate pattern characteristic of intracellular delivery and compartmentalization (Figure 3B). To investigate the kinetics of uptake, MDA-MB 231 cells were incubated with GRP1−siRNA conjugates at 37 °C, and then they were harvested at various time points, washed, and analyzed with flow cytometry (Figure 4). GRP1−siRNA conjugates were efficiently taken up by the cells. Indeed, within 120 min of incubation nearly 70% of the cells have taken up the siRNAs. To validate that the uptake is receptor-dependent, we performed competition experiments. As shown in Figure 5A, excess free peptide inhibited uptake (58% vs 22.4%; 58% vs 9.0%, P < 0.001), implying dependence of uptake on the GRP receptor. In contrast to cancer cells, the peptide−siRNA conjugates were not taken up by blood monocytes (Figure 5B) or HMEC that do not express the GRP receptor (Figure 5C).

Figure 3. Fluorescence microscopy images of MDA-MB 231 cells. (A) The cells were incubated with GRP1−siRNA conjugates (50 nM) or free siRNA (50 nM) for 3 h at 37 °C in X-vivo 15 medium and then processed as described in Materials and Methods. Hoechst 33342 staining, shown in blue, was added to visualize nuclei. Green signals represent 3′-fluorescein-labeled siRNA molecules. Data are representative of at least four independent experiments. (B) Confocal microscopy showing cytoplasmic localization of the peptide−siRNA conjugates. The cells were cultured in chamber slides and then processed as in (A). Green signals represent 3′-fluorescein-labeled siRNA molecules.

Enhancement of siRNA Uptake by the Combined Use of GRP and GnRH Peptides. Although the rate of uptake of the GRP1−siRNA conjugates by breast cancer cell line MDAMB 231 cells is relatively good, we have tested the possibility of accelerating the uptake by simultaneously targeting GRP and GnRH receptors. Theoretically, the recognition of two cell surface receptors is expected to increase targeting specificity and uptake by cells that express both receptors. MDA-MB 231 cells expressed the GnRH receptor at mRNA and protein levels (Figure 6A and Figure 6B). In the next experiment, we tested whether a 10 amino acids (CQHWSYGLRP) GnRH peptide bind to MDA-MB 231 cells. Flow cytometry analysis revealed the binding of the 6-iodoacetamidofluorescein (6-IAF) conjugated GnRH peptide to MDA-MB 231 cells (Figure 6C). We used streptavidin−biotin technology to investigate the delivery of siRNA molecules by the combined use of GRP and GnRH peptides. Equimolar amounts of biotinylated peptides and biotinylated siRNA were incubated with streptavidin protein, and then the complexes were added to MDA-MB 231 cells. The siRNA is biotinylated at the 5′-end, and it has fluorescein at the 3′-end. Streptavidin has the ability to bind up 1043

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Figure 4. Uptake kinetics of the GRP1−siRNA conjugates. The cells were cultured in six-well plates and then incubated at 37 °C with GRP1−siRNA conjugates (50 nM) for 0, 15, 30, 45, 60, or 120 min. Subsequent to incubation, the cells were scraped, washed three times, and then analyzed by flow cytometry. Data are representative of three independent experiments.

Figure 5. Competition assays and GRP1−siRNA conjugates binding to normal cells. (A) The competition experiments were performed by adding free peptide (500 nM or 1 μM) to MDA-MB 231 cells 60 min prior to addition of GRP1−siRNA conjugates (50 nM) and further incubation for 120 min. Subsequently, the cells were processed as in Figure 4. (B, C) Uptake of GRP1−siRNA conjugates by monocytes and HMEC, respectively. The cells were incubated with the peptide−siRNA conjugates (50 nM) at 37 °C for 120 min. Subsequently, they were washed and then analyzed by flow cytometry. 1044

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Figure 6. GnRH receptor expression and uptake kinetics. (A) The expression of Gn-RH receptor in cancer (MDA-MB 231) and normal (HMEC) cells was analyzed by RT-PCR. (B) Gn-RH receptor expression (green line), compared with isotype control (blue lines) and unstained cells (red lines), was analyzed by flow cytometry using specific antibodies. (C) Binding of the carboxyfluorescein-tagged GnRH peptide (green line) or of control peptide (blue line) to MDA-MB 231 cells. The cells were incubated with the peptides (5 μg/mL) for 30 min at room temperature, washed, and then analyzed by flow cytometry. The red line represents unstained cells. (D) Uptake kinetics of steptavidin−GRP-Gn-RHP−siRNA conjugates. The cells were cultured in six-well plates and then incubated at 37 °C with streptavidin−GRP-Gn-RHP−siRNA conjugates (50 nM) for various time points. Subsequent to incubation, the cells were scraped, washed three times, and then analyzed by flow cytometry. The cells were also incubated with either streptavidin−GRP1−siRNA (E) or streptavidin−Gn-RHP−siRNA (F) conjugates and processed as in (D). Results are representative of three independent experiments.

been associated with poor prognosis.35 In initial studies, several siRNAs targeting survivin were designed, tested and the most active siRNA was conjugated to GRP1. The cells were incubated for 24 h with either GRP1−siRNA formulations or free siRNA. The disulfide linkage was used for the conjugation of the GRP1 to the 5′-terminus of the sense strand of siRNA. This linkage was chosen to allow the uncoupling of the siRNA from the peptide by reduction in the cytoplasm. We also chose to have the 3′-end of the sense unconjugated. As shown in Figure 7A, treatment of the cells with GRP1−siRNA conjugates resulted in significant gene silencing (15 ± 5% survivin expression relative to untreated cell, P < 0.001). Unconjugated siRNA did not reduce survivin mRNA (94 ± 3% relative to untreated control) nor did the scrambled siRNA conjugated to GRP1 (92 ± 5%, relative to the untreated control). Western

to four biotin molecules, and therefore, each streptavidin molecule is expected to bind to at least two different peptides and one siRNA molecule. As shown in Figure 6D, bitargeting led to accelerated uptake of the siRNA conjugates by MDA-MB 231 cancer cells when compared to cells incubated with either streptavidin−GRP1−siRNA or streptavidin−Gn-RHP−siRNA conjugates (Figure 6E and Figure 6F, respectively). Indeed, within 60 min, nearly 56% of the cells have taken up the bitargeted siRNA, while around 31% of the cells have taken up the monotargeted siRNA molecules (P < 0.05). Endogenous Gene Knockdown Using Peptide−siRNA Conjugates. Having confirmed the delivery of siRNAs using short peptides, subsequently we investigated the targeting of survivin, an endogenous gene, by measuring mRNA and protein levels. Increased survivin expression in cancer cells has 1045

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nonspecific immune activation by siRNAs could contribute to antitumoral effects of siRNA but can also create unwanted toxicities.37 Therefore, we have assessed the induction of TNFα and IFN-α by PBMC in response to GRP1−siRNA conjugates. We used DOTAP-delivered siRNA as positive control.36 In contrast to DOTAP-delivered siRNA, we did not observe TNF-α or IFN-α production following treatment with GRP1− siRNA conjugates (Figure 9).

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DISCUSSION RNAi has been applied as tool to gain insights into gene functions or to understand cellular mechanisms in the context of infection or cancer.2 Also, it has been considered as a promising strategy for the treatment of human diseases.3 Yet in order to exploit the full therapeutic potential of this technology, it is necessary to overcome certain constraints related to delivery, targeting, and activation of innate immunity. Here, we covalently linked a siRNA against survivin to naturally occurring peptides whose receptors are strongly expressed on the majority of tumors, including, breast, colon, ovarian, and prostate cancers to achieve cell-type-specific delivery of the siRNA molecules. The siRNA−peptide conjugates were effectively taken up by cancer cells but not normal cells. In contrast to free siRNAs, the siRNA− peptide conjugates inhibited gene expression. Moreover, none of the conjugates induced the expression of inflammatory cytokines and IFN-α in human blood leukocytes. In general specific interaction between a specific ligand and its cell surface receptor normally enhances the cellular uptake by the aid of a mechanism called receptor-mediated endocytosis. Cell-specific ligands, including aptamers, antibodies, and vitamins, have been used to confer cell-specificity of siRNAs.3,4 The antibody-targeted delivery has attracted much attention owing to the high selectiveness toward cell surface receptors.38 In this respect, a monoclonal antibody targeting the transferrin receptor was directly conjugated to siRNAs via a biotin−streptavidin linkage, and intravenous administration of these formulations blocked the expression of a reporter gene in a rat model bearing intracranially transplanted brain tumor.39 Similarly, single-chain Fv antibody−siRNA conjugates targeting either gp120-expressing cells or the T cell-specific CD7 receptor delivered anti- HIV-1 siRNAs to cells in vitro and in vivo.11 In contrast to antibodies, small peptides are expected to be less immunogenic, cost-effective, and easy to conjugate to small molecules such as siRNAs. Because of their small size, they are also expected to have more tumor penetrance than antibodies. It is interesting to note that GRP and GnRH peptides also have high affinity for their receptors expressed on the cell surface of cancer cells.26,29 Such high affinity would allow the use of low concentrations of peptide−siRNA conjugates, which likely reduces potential nonspecific side effects while being costeffective. Moreover, the clinical usefulness of certain GRP radiopharmaceutical analogues as cancer specific imaging agents was demonstrated in patients with either prostate or breast cancer.40 Overall, the published data indicate that GRP analogues can localize in tumors with high specificity. Again, these promising results in patients may facilitate the therapeutic testing of GRP1−siRNA conjugates in humans. In order to preserve the silencing potency of siRNAs, we have used a disulfide linkage between the peptide and siRNA molecules. This strategy is expected to facilitate the intracellular delivery of siRNAs by the action of the GRP peptides and to release intact siRNA by reductive cleavage of the disulfide

Figure 7. Inhibition of survivin gene expression by the GRP1−siRNA conjugates. Survivin siRNA was directly conjugated to the GRP1 via disulfide linkage. The GRP1−siRNA conjugates (GRP1−siRNA) or unconjugated siRNAs (50 nM) were added to MDA-MB 231 cells cultured in X-vivo 15 medium. As a control for gene silencing, a scrambled siRNA conjugated to GRP1 was included (GRP1-Sc− siRNA). Subsequent to 24 h of incubation time, total RNA and cytoplasmic protein extracts were prepared and then the expression of the survivin was analyzed by RT-PCR (A) and Western blots (B). Control = untreated cells. Data are representative of at least three independent experiments.

blot analysis was also used to investigate the relative knockdown of survivin expression (Figure 7B). GRP1−siRNA conjugates significantly reduced survivin protein level in breast MDA-MD 231 cancer cells when compared to control molecules (18 ± 5%, relative to untreatred cells, P < 0.001). These results clearly demonstrate that GRP1 is an efficient siRNA escort agent and indicate that siRNA molecules have reached the cytoplasm for gene silencing, which is consistent with the confocal microscopy data (Figure 3B). Having demonstrated that the conjugates are active in human cells cultured in X-vivo 15 medium, in the next experiments we have tested the uptake in RPMI medium supplemented with 10% FCS. The cells were incubated for 3 h with GRP1−siRNAs conjugates at various concentrations and then analyzed by flow cytometry. As shown in Figure 8A, the peptide−siRNA conjugates were taken up by MDA-MB 231 cells. When tested at 100 nM, the level of survivin mRNA in GRP1−siRNA-treated cells was significantly reduced (46 ± 6%, relative to cell treated with GRP1-scrambled siRNA conjugates, P < 0.05) (Figure 8B). Consistent with mRNA level, treatment of cells with GPR1− siRNA conjugates yielded a significant decrease in survivin protein level (42% relative to cells treated with GRP1-scrambled siRNA conjugates, P < 0.05) (Figure 8 and Figure 8D). Collectively, these results indicate that gene silencing in mammalian cells can be achieved with GRP−siRNA conjugates in the presence of serum. Peptide−siRNA Formulations Did Not Trigger Cytokine Production by Blood Leukocytes. We have previously shown that both chemically made single-stranded and doublestranded siRNAs can activate innate immunity leading to the production of cytokines such as tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), and interferon-α (IFN-α).36 Such 1046

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Figure 8. Uptake and silencing potency of GRP−siRNA conjugates in the presence of 10% FCS. (A) Various concentrations of GRP1−siRNA conjugates were added to MDA-MB 231 cells cultured in RPMI medium supplemented with 10% FCS. Subsequent to 3 h of incubation time, the cells were washed three times and then analyzed by flow cytometry. The silencing potency of the peptide−siRNA conjugates (100 nM) was also tested in RPMI medium supplemented with 10% FCS. Subsequent to 24 h of incubation time, total RNA and cytoplasmic protein extracts were prepared and the expression of the survivin was analyzed by RT-PCR (B) and Western blots (C). (D) Densitometry analysis of survivin protein level and normalization with β-actin level (mean ± SD of three experiments). Control = untreated cells. All data are representative of at least three experiments.

Serum effect on the activity of peptide−siRNA conjugates is an important issue when considering potential in vivo applications. The GRP−siRNA conjugates were active in medium supplemented with 10% FCS. At 100 nM, a significant gene silencing (58% inhibition of protein expression, P < 0.05) effect was obtained with GRP1−siRNA conjugates. Previous studies have shown that peptide−siRNA conjugates can be active in the presence of serum. In a recent study, the authors have treated M21 + GL3 cells RGD−siRNA conjugates for 4 h in serum free medium and then added serum to a final concentration of 2%.41 Under these conditions, a 40% reduction in luciferase activity at 50 nM was obtained with one siRNA. A second siRNA showed 70% inhibition at 25 nM; however, there was no difference in potency between 25 and 100 nM concentrations. In another study, the breast MCF-7 cancer cells growing in DMEM/ F12 medium supplemented with 5% FCS was treated with a nuclease resistant siRNA conjugated to a peptide analogue of insulin-like growth factor. A 57% inhibition of the insulin receptor substrate-1 protein was obtained at 200 nM.14 Notably, most of the published data on peptide delivery of siRNAs have been carried in serum free medium for 4−6 h, and then serum was added to cell culture. One reason for this has been that the siRNA molecules and/or peptides are not so stable in serum. Neither the suvivin siRNA nor the GRP peptides used in this study are modified; therefore, there is room for improvement. By use of stabilized siRNA and/or peptides in combination with optimized conjugation protocols, the GRP− siRNA conjugates are likely to work at lower concentrations in

Figure 9. Immunostimulatory potential of GRP−siRNA conjugates. Peripheral blood mononuclear cells were incubated with either GRP1−siRNA conjugates or DOTAP-formulated siRNAs (100 nM) for 18 h. Subsequently, culture supernatants were harvested and cytokine contents were determined by ELISA. Data are representative of at least three independent experiments.

linkage between the peptide and the siRNAs in the reductive cytoplasmic environment. Gene silencing was achieved at lower siRNA concentrations. Indeed, we obtained around 80% inhibition of survivin gene at 50 nM in X-vivo 15 medium. Other studies have reported high concentration in vitro.14,12,41,42 1047

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medium containing serum. With regard to siRNA stability, a recent study showed that a group of siRNAs are extremely stable in their native form in 10% serum and in vivo.43 Although it exceeded our expectations, the uptake of GRP− siRNA conjugates by breast cancer MDA-MB 231 cells was somehow slow when compared to other targeting strategies.12 The kinetic of uptake seems to be receptor-dependent. Indeed, increasing the peptide−siRNA concentration from 50 to 100 nM did not significantly accelerate the kinetics of uptake in X-vivo 15 medium (data not shown). To enhance cell uptake and/or specificity, one might use bispecific or trispecific branched peptides targeted to different receptors expressed by the same cell type. Enhancement of uptake was achieved by conjugation of siRNA molecules to GRP and GnRH peptide. Therefore, bitargeting of siRNAs to tumor cells is possible and should be further explored. Another problem encountered during siRNA delivery is the activation of innate immunity through the binding of siRNA molecules to TLR7 and TLR8, expressed by immune cells, particularly monocytes and dendritic cells.36,44 These early findings highlight the necessity of testing the immunostimulatory potential of each therapeutic siRNA. We found no significant binding of the peptide−siRNA conjugates to human blood cells such as monocytes, dendritic cells, and T cells (Figure 5B and data not shown). Moreover, no TNF-α or IFNα was produced after overnight incubation of blood leukocytes with peptide−siRNA conjugates. Depending on the intended therapy, immunostimulation could be a wanted or unwanted effect. For treatments like cancer therapy and infection medicine, the immunostimulatory effect could be wanted and beneficial, and in that respect siRNAs can intentionally be designed to induce such an adjuvant effect.45 In summary, the current study shows that naturally occurring peptides are suitable for carrying siRNAs to breast cancer cells. There are other types of cancer that show overexpression of GRP receptor and therefore may also benefit from the use of this targeting strategy. Although further work is needed, the conjugation of two synergistic peptides on the same assembling agents such as streptavidin and nanoparticles will strengthen the targeting specificity and facilitate the uptake in vitro and in vivo.46 In addition to its therapeutic applications, the targeting method described here could potentially be adapted for basic research and target screening in cancer cells that express GRP and/or GnRH receptors.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +47 22 78 14 14. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge support from Helse Sør Øst, the Norwegian Cancer Society, and the gene therapy program at the Norwegian Radium Hospital.

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REFERENCES

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