International Journal of Pharmaceutics 485 (2015) 261–269
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (A). Mechanisms of interaction Maksim Ionov a, * , Joanna Lazniewska a , Volha Dzmitruk b , Inessa Halets b , Svetlana Loznikova b , Darya Novopashina c, Evgeny Apartsin c , Olga Krasheninina c , Alya Venyaminova c, Katarzyna Milowska a , Olga Nowacka a , Rafael Gomez-Ramirez d,e , Francisco Javier de la Mata d,e , Jean-Pierre Majoral f , Dzmitry Shcharbin b,1, Maria Bryszewska a a
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Poland Institute of Biophysics and Cell Engineering of NASB, Minsk, Belarus Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia d Departamento Química Orgánica y Química Inorgánica, Universidad de Alcalá, Alcalá de Henares, Spain e Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Spain f Laboratorie de Chimie de Coordination, CNRS, Toulouse, France b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 January 2015 Received in revised form 10 March 2015 Accepted 13 March 2015 Available online 16 March 2015
This paper examines a perspective on the use of newly engineered nanomaterials as effective and safe carriers of genes for the therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus and carbosilane) were complexed with anticancer siRNA and their biophysical properties of the dendriplexes analyzed. The potential of the dendrimers as nanocarriers for anticancer siBcl-xl, siBcl2, siMcl-1 siRNAs and a siScrambled sequence was explored. Dendrimer/siRNA complexes were characterized by methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. Some of the experiments were done with heparin to check if siRNA can be easily disassociated from the complexes, and whether released siRNA maintains its structure after interaction with the dendrimer. The results indicate that siRNAs form complexes with all the dendrimers tested. Oligoribonucleotide duplexes can be released from dendriplexes after heparin treatment and the structure of siRNA is maintained in the case of PAMAM or carbosilane dendrimers. The dendrimers were also effective in protecting siRNA from RNase A activity. The selection of the best siRNA carrier will be made based on cell culture studies (Part B). ã 2015 Published by Elsevier B.V.
Keywords: Dendrimer Anticancer siRNA Complex formation Biophysical characteristics Nano-drug-delivery
1. Introduction Gene therapy is one of the most effective ways of treating tumors. One of the new directions of gene therapy is the suppression of malignancy arising in normal cells. During
Abbreviations: CD, circular dichroism; CPD, cationic phosphorus dendrimer; CBD, cationic carbosilane dendrimer; DLS, dynamic light scattering; EB, ethidium bromide; G3, generation 3; G4, generation 4; PALS, phase analysis light scattering; TEM, transmission electron microscopy. * Corresponding author at: Department of General Biophysics, University of Lodz, Pomorska Street 141/143, 90-236 Lodz, Poland. Tel.: +48 426354144; fax: +48 426354474. E-mail addresses:
[email protected] (M. Ionov),
[email protected] (D. Shcharbin). 1 Institute of Biophysics and Cell Engineering of NASB, Akademicheskaja, 27, 220072 Minsk, Belarus. Tel.: +375 172842358; fax: +375 172842359. http://dx.doi.org/10.1016/j.ijpharm.2015.03.024 0378-5173/ ã 2015 Published by Elsevier B.V.
transformation, normal cells start to propagate uncontrollably, loosing the ability to undergo apoptosis. The regulation of apoptosis in cells is due to the family of Bcl-2 proteins (Burlacu, 2003), which is divided into pro-apoptotic and anti-apoptotic proteins. The group of apoptosis inhibitors include Bcl-2, Bcl-xL, Bcl-w, A-1 and Mcl-xL. Synthesis of anti-apoptotic proteins can be suppressed by RNA interference (RNAi) – a process of selective gene silencing (Milhavet et al., 2003; Poeck et al., 2008; Tiemann et al., 2010). A major limiting step for the success of this therapy is the effective delivery and transfection target cells with siRNA (Song et al., 2005). The genetic material can be brought in by some viral and non-viral delivery systems. The former are effective, but their high cytotoxicity, immunogenicity and cancerogenicity in vivo makes it worthwhile finding an alternative delivery mechanism (Córdoba et al., 2013; Creixell and Peppas, 2012; Ionov et al., 2013a, 2015).
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Fig. 1. Dependence of the fluorescence intensity of the EB–siRNA complex on an increasing concentration of PAMAM – (A), phosphorus – (B) or carbosilane – (C) dendrimers. siRNA concentration 0.35 mM, EB concentration 0.5 mM. Na-phosphate buffer 10 mmol/L, pH 7.4. lex. -480 nm, lem -emission maximum. Results represent mean SD obtained from a minimum of 3 independent experiments.
Although they are less effective than viral systems, non-viral systems are more flexible and safer. Those now being most used are complexes of nucleic acids with liposomes (lipoplexes) and cationic linear polymers (polyplexes). However, the best perspective for cancer gene therapy seems to be nanomaterials (Gardikis et al., 2011; Shcharbin et al., 2014), and foremost are dendrimers. These are synthetic polymers with a diameter of 3–10 nm. Cationic dendrimers can complex with nucleic acids, making complexes called “dendriplexes”. Dendrimers have some advantages over liposomes and linear polymers for gene delivery. They are monodisperse, stable, of low viscosity, a high molecular weight, and the large number of charged end groups for nucleic acid binding (Wang et al., 2010). Polyamidoamine (PAMAM) dendrimers can effectively transfer genes to breast cancer cells (Vincent et al., 2003). Attempts were made to improve PAMAM dendrimers-mediated transfection of green fluorescent protein by conjugation them with carbon nanotubes (Yang et al., 2011). Delivery of anti-cancer drugs by PAMAM dendrimers can come as a modification of dendrimers by folic acid, which significantly increases the selectivity of methotrexate targeting (Mullen et al., 2011; Myc et al., 2010). Carbosilane dendrimers (CBD) have been successfully used as non-viral vectors for transfecting different types of nucleic acids against hepatocarcinoma (de Las Cuevas et al., 2012). Cationic phosphorus dendrimers (CPD) have the ability to complex nucleic acids, and have been widely studied as carriers of DNA and siRNAs for the transfection of cells in vitro (Caminade and Majoral, 2005; Solassol et al., 2004).
We herein report experimental results obtained for 3 different independent groups of dendrimers, complexed with anticancer products, siBcl-xL, siBcl-2, siMcl-1 and siScrambled siRNAs. The complexes were characterized by their fluorescence, zeta potential, dynamic light scattering (DLS), circular dichroism (CD), and transmission electron microscopic (TEM) appearance. We also used a dendriplex disassociation assay involving a polyanionic agent–heparin – to check their ability to release siRNAs and monitor their structure after disassociation. Gel electrophoresis in the presence of RNase-A allowed us to answer a fundamental question as to whether dendrimers protect siRNA from degradation by nucleases. The findings show the potential of dendrimers as carriers of anticancer siRNA into cancer cells, thereby creating an alternative non-viral delivery system for BCL family siRNAs to target cells in gene therapy. 2. Materials and methods 2.1. Dendrimers PAMAM dendrimers (Fig. 1A (SI)); phosphorus dendrimers (Caminade and Majoral, 2005; Solassol et al., 2004) (Fig. 1B (SI)) and carbosilane dendrimers (Bermejo et al., 2007; Ortega et al., 2006; Rasines et al., 2009) (Fig. 1C (SI)) were used in this study. The detailed description of dendrimers is present in the supporting information.
Fig. 2. Zeta average size of the siBcl-xl upon addition of PAMAM – (A), phosphorus – (B) or carbosilane – (C) dendrimers at increasing dendrimer/siRNA molar ratios in 10 mmol/L Na-phosphate buffer, pH 7.4. Results represent mean SD obtained from a minimum of 3 independent experiments.
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Fig. 3. Zeta potential of the siBcl-xl upon addition of PAMAM – (A), phosphorus – (B) or carbosilane – (C) dendrimers at rising dendrimer/siRNA charge ratios in 10 mmol/L Naphosphate buffer, pH 7.4. Results represent mean SD obtained from a minimum of 3 independent experiments.
2.2. siRNA Three different anticancer siRNAs and one scrambled siRNA were chosen from previously published results (Bellon, 2001; Braicu et al., 2013; Guoan et al., 2010; Jagani et al., 2011, 2013;
Petrova et al., 2012). The sequences of the siRNAs are presented in the supporting information 2.3. Dendrimer-siRNA complex formation Dendrimer/siRNA complexes were formed by mixing equal volumes of siRNA and dendrimer solutions at concentrations giving the desired molar ratio. Ten mmol/L Na-phosphate buffer, pH 7.4, was used for preparation of dendriplexes. The mixture was incubated for 10 min at 22 C. 2.4. Ethidium bromide (EB) intercalation assay EB intercalation assay was used to check dendrimer interaction with siRNAs. EB is a fluorescent dye that intercalates between nucleic acid base pairs (Olmsted and Kearns, 1977). Other molecules interacting with nucleic acids compete with EB. Binding of the nucleic acid by such a molecule leads to displacement of EB and quenching of its fluorescence (Boger et al., 2001; Fischer et al., 2010; Woody, 1985), measured with a PerkinElmer LS-50B fluorescence spectrometer at 25 C. Detailed information describing this technique presented in supplementary data. 2.5. Measurement of particle size The particle size and size distribution of complexes were measured by the dynamic light scattering (DLS) technique using a photon correlation spectrometer Malvern Zeta-Sizer Nano-ZS (UK) in DTS0012 plastic cells (Malvern) (Ayame et al., 2008; Ionov et al., 2012a). The refraction factor was 1.33, at a detection angle of 90 , and a wavelength set at 633 nm. Samples were prepared at 0.3 mmol/l siRNA and molar ratios of dendrimer/siRNA ranging from 0 to 10 in 10 mmol/L Na-phosphate buffer, pH 7.4. Particle size of dendriplexes was measured from the average of 12 cycles at 25 C. To analyze the data Malvern software was used. 2.6. Measurement of zeta potential
Fig. 4. Changes in mean residue ellipticity of the siBcl-xl, at l = 260 nm. (A) – PAMAM, (B) – phosphorus, (C) – carbosilane dendrimers. siRNA concentration 2 mM, wavelength 200–300 nm, bandwidth 1.0 nm, response time 4 s, scan speed 50 nm/min, step resolution 0.5 nm. Na-phosphate buffer 10 mmol/L, pH 7.4. Results represent mean SD obtained from a minimum of 3 independent experiments.
Particle surface charges were measured by phase analysis light scattering (PALS), using a Malvern Instruments Zetasizer Nano-ZS (Malvern, UK). Capillary plastic cells DTS1061 (Malvern) were used to measure the electrophoretic mobility of the samples in an applied electric field by means of the Smoluchowski approximation (Smoluchowski, 1921; Sze et al., 2003). Samples were prepared in 10 mmol/L Na-phosphate buffer, pH 7.4, filtered twice through 0,22 mm filter paper prior to use, and measured at 25 C. From 10 to
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Fig. 5. CD spectra of the siBcl-xl in the presence of PAMAM G3 – (A), PAMAM G4 – (A1); phosphorus CPD G3 – (B), CPD G4 – (B1), carbosilane CBD-CSi – (C), CBD-OSi – (C1) dendrimers and CD spectra of the dendriplexes in the presence of heparin. siRNA concentration 2 mM, wavelength 200–300 nm, bandwidth 1.0 nm, response time 4 s, scan speed 50 nm/min, step resolution 0.5 nm. Na-phosphate buffer 10 mmol/L, pH 7.4. The number of scans varied between 2 and 5 for each sample.
15 measurements of zeta potential were collected and averaged for each sample. The Helmholtz-Smoluchowski equation in Malvern software was used to calculate the zeta potential values.
(JEOL Ltd., Tokio, Japan) microscope. Detailed information describing this technique is present in supplementary data. 2.9. Gel electrophoresis
2.7. Circular dichroism experiments and analysis CD spectra of dendrimer/siRNA complexes were measured with a Jasco, J-815CD spectrometer (Oklahoma, Japan). Concentrations of dendrimers increased over the experiment and concentrationdependent measurements were made in a 10 mmol/L Naphosphate buffer, pH 7.4. Samples at the final dendrimer/siRNA molar ratio were also treated with 0.041 mg/ml heparin. CD spectra were obtained between 300 and 200 nm with a 0.5 cm path-length Helma quartz cell. The recording parameters were as follows: scan speed 50 nm/min, step resolution 0.5 nm, response time 4 s, bandwidth 1.0 nm, and slit -auto. Spectra are given as the average of at least 3 independent scans. CD spectra were corrected against the baseline with the dendrimer dissolved in a buffer without siRNA. The mean residue ellipticity, u , (Böhm et al., 1992; Woody, 1985; Yang et al., 1986) expressed as cm2dmol1, was calculated using software provided by Jasco. Based on maximum ellipticity values for free siRNA (u0), a ratio u/u0 was calculated and plotted on the graph as a function of dendrimer/siRNA molar ratio. 2.8. Transmission electron microscopy – complexes formation The effect of dendrimers on the morphology of siRNA was visualized by TEM, micro-images being obtained with a JEOL-10
Agarose gel electrophoresis was used to study formation of complexes between dendrimers and siRNAs. Gel electrophoresis was used to check whether dendrimers protect siRNA from ribonucleolytic degradation. Detailed information describing this technique is present in supplementary data. 2.10. Statistics Exponential curve fitting and statistical analyses used Origin 8 software (Microcal Software Inc., Northampton, MA). Data were obtained from minimum 3 independent experiments and presented as mean SD (standard deviation). 3. Results 3.1. Ethidium bromide fluorescence Fluorescence intensities of the EB-labeled siRNAs (siBcl-xl, siBcl-2, siMcl-1, siScrambled) in the presence and absence of the PAMAM, phosphorus or carbosilane dendrimers are shown in Fig. 2 SI. Decrease in EB fluorescence indicated binding of dendrimer molecules to siRNA. Dendrimer bounded to siRNA displaced EB from its sites of intercalation. The dendrimers
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was sharp reaching a plateau at a molar ratio of 1–1.5 depending on the siRNA. For CBD dendrimers, this decline in F/F0 was more gentle and the fluorescence curves stabilised at a molar ratio of 2.75–10. Exposure of dendriplexes prepared using PAMAM and carbosilane dendrimers to heparin markedly increased the relative EB fluorescence intensity. For PAMAM, heparin increased the F/F0 ratio from 0.33 0.013–0.46 0.022 to 0.72 0.048–0.83 0.079, while for CBD it was from 0.44 0.015–0.53 0.028 to 0.82 0.024–0.95 0.037, depending on the siRNA, which is comparable to samples at molar ratios of from 0.25 to 3 for PAMAM and 1.5–3.75 for CBD. Heparin addition to the dendriplexes prepared using CPD only slight changed the EB-fluorescence intensity (Fig. 1). 3.2. Zeta potential and size of dendriplexes Fig. 2 gives the zeta sizes (hydrodynamic diameters) of dendriplexes formed after addition of dendrimers to siBcl-xl (results for all siRNAs are seen in Fig. 3 SI). The interaction led to a pronounced increase in the size of particles from 100 nm to 461.2 28.2–734.9 62.9 nm and 830.6 55.1–1190.4 30.2 nm for PAMAM G3 and G4, respectively, 995.6 190.4– 1230 183.6 nm and 814.9 41.2–853 57.7 nm for CPD G3 and G4, respectively and 690.9 59.2–1035.4 157.4 nm and 470.2 21.5–655.9 75.8 nm for CBD-CSi and CBD-OSi, respectively. The hydrodynamic diameters of dendriplexes increased up to a dendrimer/siRNA molar ratio of 2–5 for PAMAM dendrimers, 0.5–1 for CPD, and 5–7 for CBD, and then stabilized. Interactions between dendrimer and siRNAs were also characterized by measurements of zeta potential. Fig. 3 (and Fig. 4 SI for all siRNAs) shows all our tested dendrimers increased the zeta potential from negative to positive values. Addition of dendrimers to siRNAs changed zeta potential from (15)–(20) mV to 10 mV for PAMAM, 20 mV for CPD, and 15–20 for CBD. Zeta potential stabilized at a dendrimer/siRNA charge ratio of 15 for PAMAM and CBD, and 2.75 for CPD. 3.3. Circular Dichroism spectroscopy of siRNA
Fig. 6. Electron micrographs of siRNA [siMcl-1) (top panel) or dendrimer/siRNA mixtures composed using PAMAM G4, CPD G4 or CBD-CSi dendrimers. To obtain the fully saturated complexes, the molar ratios of dendrimers to siRNA were (3–8):1. Dendrimers were dissolved in 10 mmol/L Na-phosphate buffer and immediately mixed with siRNA solution. A magnification of 50,000 was used to examine the siRNAs and their complexes with dendrimers. Bar = 200 nm. To obtain greater contrast, the color of the microphotographs has been inverted.
without siRNA had no effect on the intensity of EB fluorescence, due to lack of any EB/dendrimer interaction. After adding dendrimers to EB-siRNA, the relative fluorescence intensity (F/F0) decreased along with the increasing dendrimer/siRNA molar ratio (Fig. 1 shows the interaction of dendrimers with siBcl-xl). With PAMAM and CPD, decrease in the fluorescence intensity F/F0
To investigate interactions between siRNAs and dendrimers, CD spectroscopy was used. The CD spectra for siRNAs were typical for A-form the secondary structure of RNA, with characteristic peaks at 210 nm and 260 nm (Kypr et al., 2009). The elipticity of complexes decreased in all cases, along with the increasing siRNA/ dendrimer molar ratio (Fig. 5 SI and 6 SI), reaching values close to 0 at molar ratios of 2–4 for PAMAM dendrimers, 2.5–5 for CPD, and 6–10 for CBD. The change in u/u0 after addition of dendrimers to siBcl-xl siRNA is shown in Fig. 4. After addition of dendrimers to siRNAs, the complexes had a red-shift in their CD spectra maximum from 260 to 280 nm for the positive peak, and from 210 nm to 220 nm for the negative peak (data shown only for PAMAM dendrimers, Fig. 4 SI). The general shape of the siRNA spectra was unaltered by dendrimers. Heparin treatment of saturated dendriplexes formed with PAMAM and CBD gave CD spectra similar to those of free siRNA, an effect that was considerably weaker for phosphorus dendrimers (Fig. 5). 3.4. Dendriplexes formation – TEM observations The effect of dendrimers on the morphology of siRNA was analyzed by TEM. For the detailed study, only one dendrimer from each group was chosen as an example. Fig. 6 shows the structure of non-complexed (top panel) and complexed siRNA, indicating the ability of dendrimers to form dendriplexes. Characteristic branched structures were seen for all siRNAs/ dendrimer mixtures (Fig. 7 SI). The shape and size of these
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Fig. 7. Analysis of the formation of dendrimer/siRNA complexes between siBcl-xl and PAMAM, CPD and CBD dendrimers. Dendrimers were complexed with fluoresceinlabeled siRNA in 10 mmol/L Na phosphate buffer, pH 7.4. Complexes were analyzed by electrophoresis on 3% agarose gels with siRNA migration identified by labeled siRNA staining. The complexes were formed at different dendrimer/siRNA molar ratios. The first lane shows the migration of non-complexed siRNA.
complexes depended on the kind of siRNA and the kind of dendrimer. The biggest structures were found when siMcl-1 was complexed with CBD-CSi. The most homogenous complexes of the same size were seen for siBcl-2. In all cases, the size of dendriplexes that formed varied from 200 to 800 nm. Dendrimer/siRNA complexes were formed at molar ratio (3–8):1. In the presence of excess of dendrimers, some single (uncomplexed) nanomolecules of 5–10 nm were seen in the images, confirming the correctness of dendrimer/siRNA molar ratios calculated using previously described methods.
at (1.5–2):1, CPD dendriplexes at (2.5–3.5):1 and CBD at (5.5–8):1 molar ratios. To test a possible protective effect of dendrimers on siRNA against degradation by RNase, agarose gel electrophoresis was used. Naked siRNA was completely digested in the presence of RNase A (Fig. 8, second line of each panel). When siRNA was incubated with dendrimers, treatment with RNase did not lead to degradation, since addition of heparin led to its release from the complex. This effect is shown for PAMAM dendrimers and CBD (Fig. 8. A, A1 and Fig. 8C, C1, respectively). Dendriplexes formed with CPD showed no migration of free (undegraded) siRNA (Fig. 8 B, B1).
3.5. Gel electrophoresis 4. Discussion To evaluate complexes formed between siRNA and dendrimers, we also ran gel electrophoresis of samples. Saturation of complexes was measured by the charge neutralization of the dendriplexes as the migration of siRNA became retarded on 3% agarose gels. Fig. 7 shows the electrophoregrams with different patterns of migration of siBcl-xl (labeled with fluorescein) depending on the kind of dendrimer. Complexes were formed at different dendrimer/siRNA molar ratios with siRNA being mixed with increasing concentrations of PAMAM (Fig. 7 top panels), CPD (Fig. 7 middle panels) or CBD (Fig. 7 bottom panels). Under the same conditions, dendrimers could fully retard siRNA mobility in gels, being complete when in excess. PAMAM dendriplexes were fully saturated by dendrimers
There are many reports on the application of dendrimers as vectors for nucleic acids, and RNAi-based anticancer therapy is also gaining much attention (Dudek et al., 2014; Li et al., 2013). Using dendrimers PAMAM, phosphorus and carbosilane as potential carriers of anticancer siRNAs (siBcl-2, siMcl-1, and siBcl-xl), which silence anti-apoptotic genes, and siScrambled siRNA sequence, we have monitored the formation of dendrimer/siRNA complexes, characterized them, and assessed their ability to protect oligonucleotide duplexes against nucleases. EB intercalation assay shows that addition of dendrimers to siRNA strongly decreased EB fluorescence intensity ratio F/F0
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Fig. 8. Dendriplex stability. Dendrimers have a protective effect on siRNA in the presence of RNase. PAMAM G3 – (A), PAMAM G4 – (A1); phosphorus CPD G3 – (B), CPD G4 – (B1), carbosilane CBD-CSi – (C), CBD-OSi – (C1) dendrimers. The first lane shows migration of non-complexed siRNA. The second lane shows migration of siRNA previously incubated with RNase A. The third line is the migration of siRNA due to its release from dendrimers in the presence of heparin 0.041 mg/ml, representing the protective effect of dendrimers to siRNA against RNase.
Fig. 1), which indicates that dendrimers competed with EB for binding to siRNAs, and therefore the formation of dendrimer/ siRNA complexes took place. The interaction between dendrimers and siRNA molecules resulted in a reduced number of EB binding sites, manifested by a decrease in EB fluorescence intensity, data which accords with previous findings demonstrating the ability of these dendrimers to interact with DNA or siRNA (Ionov et al., 2012b; Shakhbazau et al., 2010; Suzuki et al., 1997). While fluorescence reached a plateau at a molar ratio of 1.0–1.5 for PAMAM dendrimers and CPD, it was 2.75–10 for CBD, which might be due to CBD being of lower generation (G2) than PAMAM dendrimers and CPD (G3 and G4). It therefore has considerably fewer positive charges at the surface (16) as compared to PAMAM dendrimers (32 and 64 for G3 and G4, respectively) and CPD (48 and 96 for G3 and G4, respectively). Therefore, it is possible that siRNA have to bind more molecules of CBD to form a saturated complex. Since an effective siRNA carrier cannot alter the structure of the transported nucleic acid and should easily release its cargo after internalization into the cell (Schaffer et al., 2000), we assayed dendriplex disassociation in the presence of heparin. This partly
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restored the initial fluorescence of EB-siRNA in the case of PAMAM and CBD, indicating that siRNA was disassociated from dendrimers. This effect was much weaker for CPD, which might be explained by the strength of electrostatic interactions between dendrimers and siRNAs resulting from its smaller number of positive charges. Since CPD has more surface positive charges than PAMAM (of the same generation) and CBD. Hence, it is likely that the same concentration of heparin is ineffective in displacing siRNA from the complex. For further characterization of dendriplexes, zeta size and potential were measured. The former showed that, upon binding of dendrimers to siRNAs, large dendriplexes are formed (Fig. 2, Fig. 3 SI), possessing hydrodynamic diameters of 461.2 28.2– 1230 183.6 nm, depending on the dendrimer and siRNA. These complexes are big and their endocytosis may be limited. Based on the use of fluorescent microspheres, particles up to 200 nm are preferentially taken up by cells by clathrin-mediated endocytosis. For larger particles, caveolae-mediated internalization occurs and this is a predominant pathway for 500 nm particles; 1000 nm size particles do not enter cells (Rejman et al., 2004). Nevertheless, dendriplexes >500 nm can transfect cells efficiently, with low toxic effects (Morales-Sanfrutos et al., 2011; Zinselmeyer et al., 2002). Fig. 3 (and Fig. 4 SI) shows that along with the increasing dendrimer/siRNA charge ratio, zeta potential changed significantly from negative to positive values, confirming formation of complexes between dendrimers and siRNAs. The dendriplexes were saturated at a charge ratio of 3 for CPD and 6–10 for PAMAM and CBD. Positive zeta potentials were also obtained for CBD interactions with anti-HIV oligonucleotides (Ionov et al., 2013b), as well as for PAMAM and CPD complexation with siRNA (Suzuki et al., 1997 Ziraksaz et al., 2013). The positive charges on the surface of dendriplexes favor interactions with cell membranes and the internalization process. Indeed, cationic dendrimers enter cells more effectively than anionic or neutral ones (Albertazzi et al., 2012). However, positive surface charges are also responsible for cytotoxic effects of dendrimers (Jevprasesphant et al., 2003). The CD spectra for siRNAs was characteristic for A-type RNA duplexes, with peaks at 210 and 260 nm (Kypr et al., 2009). Increasing dendrimer/siRNA molar ratios led to the decrease in ellipticity to values around 0 (Fig. 5 SI and Fig. 6 SI), indicating formation of complexes. This reduction can be explained by the decrease in the absorbance of nucleosides in siRNA-dendrimer complexes (Law et al., 2008). Moreover, a red shift of CD spectra occurs upon addition of all 3 types of dendrimers (data shown only for PAMAM dendrimers, Fig. 4 SI). The changes in CD spectra can, to some degree, result from stacking interactions of base pairs (Breunig et al., 2008). Nevertheless, the general A-form pattern of CD spectra was maintained, suggesting that dendrimers do not seriously alter the structure of siRNAs. These results accord with previous findings showing interactions of dendrimers with RNA (Reyes-Reveles et al., 2013; Suzuki et al., 1997). Similarly, as in the
Fig. 9. Schematic representation of interactions between dendrimers and siRNA. Protective properties of dendrimers against RNase degradation.
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EB assay, heparin disassociated siRNA from complexes formed with PAMAM and CBD, but did not cause so pronounced siRNA release form CPD/siRNA dendriplexes (Fig. 5). Analysis of electron microphotographs indicates that all kinds of cationic dendrimers can form complexes with siBcl-xl, siBcl-2, siMcl-1 siRNAs, which correlates with the results from circular dichroism, fluorescence and zeta techniques (Fig. 6, Fig. 7 SI). The microscopic results slightly differ from those using the zeta average size technique. The smaller sizes of complexes in microphotographs are likely to result from a different way of sample preparation, i.e. the size distribution measurements by DLS were made in a solution, whereas the samples for TEM were dried. Similar discrepancies in sizes of dendriplexes measured by DLS and atomic force microscopy were previously obtained (Ionov et al., 2013b). Interactions between dendrimers and siBcl-xl were also analyzed on agarose gel electrophoresis (Fig. 7), showing complex formation between siRNA and dendrimers. Fluorescinated siRNA attached to the dendrimer cannot migrate in the gel. This confirms that the dendriplexes are positively charged, and the data is in accord with the results from the zeta potential technique. Gel electrophoresis in the presence of RNase A and heparin used to check if dendrimers can protect siRNA from nucleolytic showed that it can effectively prevent digestion of Bxl-xl (Fig. 8). In the case of CPD, this result is less clear due to strong association of siRNA with the dendrimers and its poor release from complexes in the presence of heparin, as described above. The protective properties of dendrimers against nucleases were also shown in other studies for CBD and PAMAM dendrimers (Weber et al., 2008; Abdelhady et al., 2013). In summary, formation of complexes occurs between anticancer siRNAs, siBcl-2, siBcl-xl and siMcl-1 and 3 groups of dendrimers, PAMAM, CPD and CBD. Besides the ability to form stable dendriplexes, these dendrimers protect siRNAs against nucleolytic degradation (Fig. 9), which is a property of utmost importance for effective gene transfection, making the dendrimers suitable candidates for gene delivery vectors. The selection of the best siRNA carrier will be made based on cell culture studies (Part B). Conflict of interest The authors confirm that the content of the paper entails no conflict of interest. Acknowledgements The authors thank Ms. S. Michlewska, Ms. L. Balcerzak and Dr. S. ska for their assistance during microscopic measurements. Glin This work was supported by a Marie Curie International Research Staff Exchange Scheme Fellowship within the 7th European Community Framework Programme, project No. PIRSES-GA-2012-316730 NANOGENE, co-financed by the Polish Ministry of Science and Higher Education (grant No. W21/7PR/ 2013), by the Belarusian Republican Foundation for Fundamental Research, Project No. B13MS-004, by the CTQ2011-23245 (MINECO), NANODENDMED ref S2011/BMD-2351 (CAM) and CIBER-BBN awarded to R. G. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.03.024
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