ARTICLE pubs.acs.org/bc
Removable Nanocoatings for siRNA Polyplexes estmír Konak,† Vladimír Subr,† Milena Spírkova,† Yoseph Addadi,‡ Michal Neeman,‡ Libor Kostka,*,† C §,|| Twan Lammers, and Karel Ulbrich† †
)
Institute of Macromolecular Chemistry, v. v. i., Academy of Sciences of the Czech Republic, Heyrovsky sq. 2, 162 06, Prague 6, Czech Republic ‡ Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel § Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands Department of Experimental Molecular Imaging, RWTH - Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany ABSTRACT: To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(L -lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (Mw), the hydrodynamic radius (Rh), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing antiLuciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
’ INTRODUCTION Gene therapy provides a possibility for the treatment of various human diseases in situations where conventional approaches are not feasible or ineffective.1 However, the expansion of gene therapy is limited by the development of safe, sufficiently stable, specific, and efficient gene delivery systems. The delivery systems must be able to protect DNA or RNA from degradation during transport, provide targeted delivery to the pathological site, and promote target cell-specific uptake and intracellular trafficking.2 The gene delivery vectors, such as polyelectrolyte complexes (PEC) of DNA,1 RNA,3,4 or modified viruses, have been developed to overcome these barriers and efficiently mediate in vivo expression of genes. However, major limitations r 2011 American Chemical Society
that still restrict therapeutic use of nonviral vectors relate to their instability in vivo and to nonspecific interactions of PEC with cells of the immune system and blood plasma proteins during their transport to target cells. Both instability and the uptake by macrophages and the resulting rapid elimination of vectors from the bloodstream by cells of the reticuloendothelial system can be successfully suppressed by modifying the surface of polyplexes with covalently linked hydrophilic polymers. Such stealth coatings significantly improve stability, they prolong circulation times Received: April 23, 2010 Revised: December 6, 2010 Published: January 10, 2011 169
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Bioconjugate Chemistry in plasma, and they suppress nonspecific cell entry.5,6 Moreover, the wide tissue distribution of vectors can be restricted by binding of ligands that are able to selectively interact with receptors expressed on the surface of target cells.7,8 However, although such (targeted) polymeric coatings often sufficiently protect the vectors from rapid dissociation and from undesirable interactions with blood components, and although they generally enable efficient and target cell-specific uptake, they often also prevent the escape of DNA (or RNA) from the delivery system to the cytosol, which results in dramatic decreases of transfection efficiency.9 In light of this, novel, highly hydrophilic and multivalent copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) containing reactive carbonylthiazolidine-2-thione (TT) side chain groups bound via reductively cleavable disulfide linkages to the polymer backbone have been developed. These polymers enable surface modification of suitable DNA or RNA nanoparticle carriers with surface nanolayers that are removable by the reductive environment of the cytosol of target cells. The disulfide bridge can be cleaved by an interaction with reductive agents forming two thiol groups, or may interchange their substituents with other thiols or disulfides.10,11 This reductive reaction represents an important function in biological systems, and is especially interesting in the design of drug, protein, RNA, and DNA delivery systems, since the natural reductive agent L-glutathione (L-R-glutamyl-L-cysteinyl glycine, GSH) occurs in concentrations of 0.1-10.0 mmol/L inside living cells, while the extracellular level of GSH is 3 orders of magnitude lower.12 New reactive copolymers containing reductively degradable spacers can be used in the design of new polymer-coated gene delivery vectors that are relatively stable during their transport in the bloodstream, but disintegrate and release siRNA or DNA after cellular internalization. This process increases gene expression and the efficacy of RNA interference. The properties of new, highly hydrophilic polymers bearing reactive TT groups attached via biodegradable spacers and the efficiency of coating of polyplexes with the polymers were compared with those of previously described polymers containing nondegradable spacers, which were successfully used for the coating of both viral and nonviral gene delivery vectors.13 In this paper, as a first step toward the development of polyplexes with improved in vivo stability and prolonged in vivo circulation times, we describe the synthesis and physicochemical characterization of HPMA-based multivalent copolymers containing TT reactive groups bound to the polymer backbone via spacer-containing disulfide bonds. In addition, we also studied their use for the coating of PEC prepared by the self-assembly of antiluciferase siRNA with polyethylene imine (PEI) or poly(HPMA)-grafted poly(L-lysine) (GPL) copolymer, and we extensively analyzed their physicochemical properties. The coating process was monitored by changes in the weight-average molecular weight (Mw), the hydrodynamic radius (Rh), and the zeta potential (ζ) using either static (SLS) or dynamic (DLS) laser light scattering methods, and the stability of the coated (siRNA/ GPL/PHPMA) or uncoated (siRNA/GPL) siRNA complexes was examined by gel electrophoresis and by turbidity measurements in physiological solutions. Finally, to provide some initial experimental evidence for biological activity, we also evaluated the ability of the coated and uncoated complexes to induce target-specific gene silencing; using MDA-MB-231 human breast cancer cells double-transfected with luciferase and red fluorescence protein (RFP).
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’ MATERIALS AND METHODS Materials. Methacryloyl chloride, 1-aminopropan-2-ol, 3,30 dithio-dipropionic acid (DTDP), 4,5-dihydrothiazole-2-thiol, N,N0 -dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), triethylamine (Et3N), 2,20 -azobis(isobutyronitrile) (AIBN), tetrahydrofurane (THF), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and branched polyethylenimine (PEI, Mw = 25 000, dn/dc = 0.21) were purchased from Sigma-Aldrich. N-(3-Aminopropyl)methacrylamide hydrochloride was from Polysciences, Inc. FlucDsiRNA (siRNA, Mw = 17 930, dn/dc = 0.185, mass/charge = 391 g/mol) was obtained from Integrated DNA Technologies (Leuven, Belgium). Synthesis of cyclic form of the RGD peptide c(RGDfK) and its scrambled analogue was described previously.14 All other chemicals and solvents were of analytical grade. Monomer Synthesis. N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by a modified reaction of methacryloyl chloride with 1-aminopropan-2-ol in dichloromethane in the presence of sodium carbonate.15 Synthesis of 3-(N-methacryloylglycylglycyl)thiazolidine-2-thione monomer (Ma-GlyGly-TT) was performed as described in ref 16. N-3-[3-(Methacryloylamino)propyl]-7-(diethylamino)coumarine-3-carboxamide. This was prepared by reaction of N-(3-aminopropyl)methacrylamide (2.5 mg, 13.9 μmol) and 7-(diethylamino)coumarine-3-carboxylic acid N-succinimidyl ester (5 mg, 13.9 μmol) in 300 μL DMF for one hour at room temperature. Crude product was purified on a Sephadex LH-20 column with methanol as the mobile phase and recrystallized from a mixture of ethyl acetate and diethyl ether. HPLC analysis (Chromolith column, gradient of water in acetonitrile of 5-100%) gave a single peak at 340 nm with a retention time of 13.6 min. Synthesis of N-1-3-[(3-[3-Oxo-3-(2-thioxo-1,3-thiazolan3-yl)propyl]disulfanylpropanoyl)amino]propyl-2-methylacrylamide (Ma-SS-TT). 2-Methyl-N-[3-[3-[3-oxo-3-(2-thioxothiazolidin-3-yl)propyl]disulfanylpropanoylamino]propyl] prop-2-enamide (DTDP-(TT)2, 1.77 g, 4.3 mmol; synthesis described previously17) was dissolved in 20 mL of THF. To this solution, 0.36 g (2 mmol) of N-(3-aminopropyl)methacrylamide hydrochloride dissolved in a mixture of 5 mL THF and 5 mL methanol containing 0.33 mL (2.4 mmol) Et3N was added dropwise, and the reaction mixture was stirred for 30 min at room temperature. Product Ma-SS-TT was purified on a silica gel column in a mobile phase of dichloromethane and acetone (1:1), and then recrystallized from a mixture of dichloromethane and diethyl ether. Yield: 0.34 g (47%). HPLC analysis (Chromolith column, gradient of water in acetonitrile of 5-100%) gave a single peak at 305 nm with a retention time of 8.6 min. Elemental analysis: calcd/found C = 44.11/44.35%; H = 5.78/5.71%; N=9.65/9.45%; S=29.44/28.92%. 1H NMR 300 MHz (CDCl3, 296 K) ppm: 1.66-1.69 (m, 2H), 1.97 (s, 3H), 2.61 (t, 2H), 2.92-2.99 (m, 4H), 3.27-3.35 (m, 6H), 3.67 (t, 2H), 4.58 (t, 2H), 5.34 (s, 1H), 5.76 (s, 1H), 6.37 (s, 1H), 6.67 (s, 1H). Synthesis of Copolymers. All copolymers were prepared by radical solution copolymerization of HPMA and the respective comonomers in DMSO using AIBN as the initiator. Polymerization was carried out under a nitrogen atmosphere in a sealed ampule at 60 °C for 6 h.15 Structures of all copolymers are shown in Scheme 1. Polycationic graft copolymer PLL-g-poly(HPMA) (GPL, Mw = 2 105, dn/dc = 0.18, mass/charge = 344.5 g/mol) was 170
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Scheme 1. Structure of HPMA Copolymers Containing TT Reactive Groups in Side Chains
Table 1. Characteristics of Reactive HPMA Copolymers cp
Mw (g/mol)
Mw/Mn
TT (mol %)
POL 1
85 300
1.7
9.9
POL 2
56 000
1.9
8.2
POL 3
52 800
1.4
9.0
NaCl) to the filtered solution of GPL (8 mL, 0.584 mg/mL, 0.01 M NaCl). Surface Modification of PEC. A freshly prepared aqueous solution of HPMA copolymer containing TT groups was added to a stirred solution of PEC prepared with an excess of amino groups (j = 1.5). Optimal conditions for aminolysis were achieved by addition of 1 M HEPES buffer (pH 8.7, 40 μL per mL) to reach the final pH of 8.2. The reaction was carried out at room temperature, typically for 0.5 to 1.0 h. An excess of polymer TT groups in comparison to free amino groups on the PEC surface was used and the concentrations of the coating polymers ranged from 0.5 to 2.0 mg/mL. After the coating reaction, the excess of the unreacted polymer was removed by centrifugal filtration, repeated 5 times (Millipore Amicon Ultra-4 with Ultracel-100 membrane). PEC Characterization Using Light Scattering Methods. Weight-average molecular weight was determined by static light scattering (SLS) carried out with an ALV goniometer. The instrument was equipped with an intensity-stabilized 22 mW He-Ne laser as the light source and a PC for data recording. The accuracy of the measurements was found to be approximately 1%. The scattering curves were measured 2-3 times after preparation of PEC and 2-3 times after their surface modification. The refractive index increments, dn/dc, of the individual components of the complexes were taken from the literature. The complex concentrations (cpec) and refractive index increments (dn/dc)pec of the complexes were calculated as a function of the molar mixing ratio, j (positive to negative charge ratio), on the basis of the model of complex formation given in our previous paper.4 The static light scattering data were analyzed by a Zimm plot
prepared by reaction of poly(HPMA) grafts (M n = 4 300) terminated with succinimidyloxycarbonyl end groups with highmolecular-weight PLL (Mw = 130 000).3 Characterization of Reactive Polymers. The content of TT groups in copolymers was determined spectrophotometrically using UV absorption with a Specord 205 (Analytik Jena, Germany) spectrophotometer (ε305 = 9 500 L/mol.cm, methanol). Weight (Mw) and number (Mn) average molecular weights were determined by size exclusion chromatography on a HPLC system LC-10 (Shimadzu, Japan) equipped with a DAWN 8 multiangle light scattering detector, an Optilab rEX (Wyatt Technology Corp., USA), a UV detector, and a Superose 6 column (300 10 mm, GE Healthcare, USA) using 0.3 M sodium acetate buffer, pH 6.5, as a mobile phase at a flow rate of 0.5 mL/min.16 Characteristics of reactive polymers are summarized in Table 1. Preparation of siRNA/PEI Complexes. Formation of siRNA/ PEI complexes (w/w = 1) was achieved by flash addition of filtered PEI solution (40 μg/mL) to siRNA solution (40 μg/mL) with vigorous stirring. Formed complexes were filtered over 0.22 μm PVDF filters (saturated with PEI). Preparation of siRNA/GPL Complexes. The siRNA/GPL PECs were prepared with various charge ratios (j) from 20 to 1. We found that the complexes prepared with j = 1.5 are the most suitable for subsequent coating reactions and their stability was reasonable. Since low concentrations of salt may decrease the level of aggregation by 2 orders of magnitude,18 all these complexes were prepared in the presence of 0.01 M NaCl. These PECs were prepared by slow addition from a linear dispenser (rate of 15 mL/h) of siRNA solution (4 mL, 0.69 mg/mL, 0.01 M
Rg 2 q2 Kcpec 1 ¼ þ pec Mw RðqÞ 3Mw pec
ð1Þ
where R(q) is the Rayleigh ratio of the scattering intensity, q = (4π/λ)sin θ/2, λ is wavelength in the medium, θ is the 171
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scattering angle between the incident and the scattered beam, K is a contrast factor containing the optical parameters, cpec is the complex concentration, Mw is the weight-average of the molar mass of the complex particles, and Rg is their radius of gyration (calculated from the z-average of the square). The concentration dependence was neglected, which seemed to be justified because of the low concentrations of the PEC solutions (∼10-5 g/mL). Extrapolation to the zero scattering angle was carried out by linear or quadratic fits of the scattering curves. The experimental error of Mw determination for the complexes was typically about 2%. The hydrodynamic radius of particles (Rh) was determined by dynamic light scattering (DLS) carried out on the same ALV instrument in the angular range 30-140°. An ALV 6000 multibit, multitau autocorrelator covering approximately 12 decades in delay time (τ) was used for measurements of time autocorrelation functions. The autocorrelation functions were analyzed using the method of cumulants (a quadratic fit). From the first cumulant, Γ, the diffusion coefficient, D, was calculated from the equation D = Γ(q)/q2, where q is the scattering vector q = 4πn sin(θ/2)/λ, n is the refractive index of a solvent, θ is the scattering angle, and λ is the wavelength of the incident light. The polydispersity index (PDI), which reflects the size polydispersity of the complexes, was obtained from the second moment (cumulant), μ2, where PDI = μ2 /Γh 2 . The inverse Laplace transform using the REPES19 method of constrained regularization, which is similar in many respects to the inversion routine CONTIN,20 was used for an analysis of the time autocorrelation functions obtained from multicomponent solutions. REPES directly minimizes the sum of the squared differences between the experimental and calculated intensity time correlation functions using nonlinear programming. This method uses an equidistant logarithmic grid with fixed components (here, a grid of 20 components per decade) and determines their amplitudes. As a result, a scattered light intensity distribution function, A(τ), of delay times was obtained (τ = 1/Γ) that could be easily transformed in a distribution function of hydrodynamic sizes. The hydrodynamic radius (Rh) was calculated from the diffusion coefficient (D) using the Stokes-Einstein equation Rh ¼ kT=6πηD
scattering amplitude from DLS. This procedure could be successfully used when the scattered intensities of different particle types were comparable and their contributions to the distribution of particle sizes (e.g., Rh) were sufficiently distinct. Since both of these requirements were fulfilled in the systems under investigation, the weight-average molecular weight of coated PEC (Mwcc) could be estimated even in the presence of coating polymers and small amount of high-molecular-weight PEC aggregates (nanogels formed by the reaction of excessive PEI and multivalent polymer) in the solution. If the light scattering intensity is comparable to that of PEC, the weight fraction of the aggregates must be on the order of 0.01 (1 wt %) and Mwpec can be calculated using the scattering intensity generated from coated PEC alone at their original concentration, cpec. The weight-average molecular weight of PEC is then calculated using the regular Zimm plot procedure. ζ-Potential. The ζ-potentials of uncoated and coated PEC were measured using a Nano-ZS, model ZEN3600 (Malvern Instruments, UK). At least 10 measurements of each sample were carried out to check reproducibility. The measurements of the electrophoretic mobility were converted to ζ-potentials (mV) using the Smoluchowski approximation. A reference measurement using the Malvern ζ-potential standard was run prior to each sample analysis to check for correct instrument operation. Atomic Force Microscopy (AFM). The nanoparticles (concentration 1 mg/mL) were deposited on fresh mica substrate, and the surface morphology (height image) and the sum of tip-sample interactions (phase image) were characterized by AFM. All measurements were performed under ambient conditions using a commercial atomic force microscope (NanoScope Dimension IIIa, MultiMode Digital Instruments, Santa Barbara, CA, USA) equipped with a SSS-NCL probe, Super Sharp Silicon SPM-Sensor (NanoSensors Switzerland; spring constant 35 Nm-1, resonant frequency 173 kHz). The tapping mode AFM technique was used for collecting the images. This technique allowed two- and/or three-dimensional information to be obtained of both height and material heterogeneity contrast with high resolution when recording height and phase shifts simultaneously. Kinetics of Complex Coagulation. The kinetics of coagulation of the siRNA/polycation complexes was measured by turbidity in 0.15 M NaCl solutions. The complexes were prepared and coated with polymer, as described above, at standard conditions in 0.01 M NaCl solutions. The turbidity of the complexes was measured spectrophotometrically at λ = 500 nm and started immediately after increasing the ionic strength of the complex solutions up to 0.15 M NaCl by the fast addition of 4 M NaCl stock solution. Electrophoresis. The ability of coating copolymers to covalently bind on the PEC surface to retard the release of siRNA was determined by horizontal agarose gel electrophoresis. The coated complexes of siRNA/GPL with POL1 or POL3 (siRNA/ GPL/PHPMA) were formed as described above at j = 1.5. The samples were loaded on E-Gel agarose gel (2%) with SYBR Safe and run for 15 min in an E-Gel iBase from Invitrogen. The gel images were made by a Canon EOS 400D digital camera at 280 nm. Luciferase Assay. MDA-MB-231 human epithelial breast adenocarcinoma cells expressing both luciferase and red fluorescent protein (RFP) were kindly provided by Prof. Yoram Salomon and were grown in RPMI media supplemented with 10% FCS L-glutamin and antibiotics. Cells were plated in 96
ð2Þ
where k is the Boltzmann constant, T is the absolute temperature, and η (0.894 cP) is the viscosity of water at 25 °C. The experimental error of Rh determination for the complexes was typically about 3%. Evaluation of the Weight-Average Molecular Weight of PEC in the Presence of Coating Polymer and Particle Aggregates. In the presence of aggregates and coating polymer
molecules in solution, the weight-average molecular weight of polyplexes (Mwpec) was estimated using a combination of dynamic and static light scattering experiments.21 If several kinds of particles were present in the solution under investigation, the total scattered intensity (Ist) could be expressed as the sum of their individual contributions. The analysis of dynamic light scattering data provided, in addition to hydrodynamic sizes of particles, corresponding relative scattering amplitudes of the distinguished particle types present in the tested solution. Thus, the scattering intensity generated by particles of special interest (e.g., PEC) could be extracted from the total scattering intensity of the mixed solution by multiplication of Ist by their relative 172
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described in the literature.22 We then compared the stability of the PEI-based vectors with the stability of poly(HPMA)-grafted poly(L-lysine) (GPL) vectors, and we evaluated their suitability for further improvement of vector properties by coating their surfaces with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. It is known that PEI-siRNA complexes are not suitable for in vivo application,23 especially because of their improper stability,24 and coating of this complex does not result in improvement of its stability (see Figure 1). This result reinforced our focus on further development of siRNA complexes based on GPL polycations. It is well-known, however, that PLL-a polycation routinely used for DNA delivery-does not form compact complexes with short oligonucleotides, such as siRNA.24 With the aim of improving the ability of PLL to form stable complexes with siRNA, we decided to use a new type of PLL-based polycation, grafted with short chains of hydrophilic poly(HPMA) developed and studied in our laboratory.3 In this paper, a comparison of the properties of uncoated siRNA/PEI and HPMA copolymer-coated complexes with the properties of siRNA/GPL complexes and their coated analogues is presented and discussed. Properties of siRNA/PEI Polyplexes. Basic physical characteristics of siRNA/PEI complexes prepared as reference PECs and models for the study of the coating reaction of PECs with multivalent reactive copolymers bearing carbonylthiazolidine-2thione (TT) reactive groups attached to the polymer backbone via degradable and nondegradable spacers were determined by light scattering methods (Table 2). The Rhpec and Mwpec of complexes coated with various copolymers differed only slightly, which demonstrated a good reproducibility in PEC preparation. The size of the PEI-based PECs was found to be close to that published in the literature.22 The ratio Rh/Rg = 0.8 found for the PECs corresponded with that predicted for solid spheres (0.78).26 The polydispersity of PEI PEC (PDI=0.14) was slightly higher than that of DNA polyplexes.13 As the PECs were prepared with an excess of polycation, their zeta potentials were positive (Table 2). These PEI-based complexes were then coated with HPMA copolymers containing nondegradable (POL 1) and degradable (POL 2) spacers terminating with TT reactive groups in buffer at pH 8.2. We incorporated the fluorescent probe coumarin into the copolymer structure of POL 2, which enabled better monitoring of the coating polymer during the purification step. Particle Rh distributions found in solutions of uncoated PECs and PECs coated with POL 2 were measured at θ = 150°, and data are shown in Figure 1. While Rh distribution of PECs dominates before coating, as shown in Figure 1, the distribution of particle size in solution after the coating procedure was more complex. The size distribution showed, besides the coated particles (35 nm), new
well plates (at a density of 55 000 cells per well), and 24 h after plating, various amounts of siRNA-containing particles equivalent to 1, 10, and 20 μg of siRNA were added to a final volume of 125 μL per well. In some experiments, siRNA/Lipofectamine complex was included as a control, and it was used according to the instructions of the manufacturer (i.e., Invitrogen). Plates were analyzed after 48, 72, and 96 h of incubation, using a Victor plate reader (PerkinElmer, USA), and both luminescence and fluorescence were quantified. The luminescent signal was produced by means of the Bright-Glo Luciferase Assay System (Promega, USA), and its reduction (as compared to untreated controls) upon incubation with the complexes was used as a measure for overall gene silencing. The reduction of RFPmediated fluorescence (as compared to untreated controls) was used as a measure for nonspecific gene silencing/toxicity. To determine target-specific gene silencing, the percentage of reduction in fluorescence was subtracted from the percentage of reduction in luminescence.
’ RESULTS AND DISCUSSION An important limitation of RNAi-based therapy is the difficulty in achieving effective delivery of RNAi-bearing vectors to target cells and tissues in vivo. Many laboratories developing PEC-based delivery vectors for small oligonucleotides, like siRNA, are designing oligonucleotide delivery vectors using commercially available branched polyethylene imine (PEI). With the aim of using these complexes as controls in our studies, we prepared these PEI based PECs using standard methodologies
Figure 1. Rh distributions for uncoated (---) and coated (___) siRNA/ PEI complexes with POL 2 copolymer (ccp = 1 10-3 g/mL) after incubation for 220 min; cpec = 2 10-5 g/mL, θ = 150°.
Table 2. Effect of Coating on the Characteristics of siRNA/PEI Polyelectrolyte Complexes Coated with the Reactive Copolymers POL 1 and POL 2 reactive copolymer cpec 105 (g/mL) ccp 10-3 (g/mL) Mwa 10-6 (g/mol) ΔMct 10-6 (g/mol) Fh g/mL nct Rh (150°) (nm) ζ potential (mV)
a
-
2
-
4.4
POL 1 POL 1
1.85 1.85
2 0.5
10.7a 10.0a
3.1 2.9
0.065
POL 2
1.85
1
17.2a
5.1a
0.14
30 36 34
>29 >29
91
35
þ12
-5
Contribution of the coating polymer and “new particles” to the SLS was subtracted. 173
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Figure 3. Dependence of molecular weight Mwpec (a) and Rh (b) of siRNA/GPL complexes on the mixing ratio j. The concentration of starting polycation solution was 3.91 10-4 g/mL.
Figure 2. Zimm plot of scattering curves of the siRNA/GPL complexes in relation to the mixing ratio, j (top, j = µ; bottom, j = 1).
particles with Rh of about 120 nm. These particles (nanogel) were probably formed by chemical reaction between amine groups of free PEI polymers (used in excess) and TT reactive groups of the coating copolymers. The formation of similar nanogels was also observed in PEC solutions after coating with reactive copolymer POL 1 containing a nondegradable spacer. The small peak at Rh ≈ 8 nm corresponds with the peak of free coating copolymers, which were used in excess. Unfortunately, purification of complexes from excessive polycations turned out to be impossible, because of low stability of the generated complexes, and disintegration of the PEI-based polyplexes was observed after dialysis, column filtration, and centrifugation (results not shown). In the case of such complicated particle mixtures as those obtained after the coating procedure, the weight-average molecular weights of coated polyplexes (Mwcc) could be estimated using a combination of dynamic and static light scattering experiments.21 The increase in the molecular weight of PEC due to coating with reactive copolymer POL 1 and POL 2, ΔMct, is shown in Table 2. In order to characterize the compactness of complexes, the structural density (Fh) of PEC was calculated from the hydrodynamic volume of particles (VH, in cm3) and from their corresponding molecular weight, Mwcc (Mwcp þ ΔMct). These results are shown in Table 2. The value of Fhcc = 0.14 obtained for coated siRNA/PEI complexes (Table 2) was comparable with those found for micelles in organic solvents,27 for DNA/PLL complexes coated with HPMA copolymer13 and oligonucleotide/ (PLL-g-PHPMA) complexes. 3,4 Values for Fh pec of uncoated siRNA/PEI complexes were approximately 2-fold smaller (Table 2). This result meant that the coating procedure increased the observed density of PEC. The coating efficiency was characterized by the number of molecules attached to the PEC surface, nct, as calculated from ΔMct, using the formula nct = ΔM/Mwcp, where Mwcp is the weight-average molecular weight of used coating polymer. The results obtained for this parameter for POL 1 and POL 2 are shown in Table 2. The nct was found to be higher for the coating copolymer POL 2 due to its lower molecular weight.
Figure 4. Zimm plot for uncoated (9) and coated siRNA/GPL complexes with copolymers POL 1, POL 3, POL 3 þ c(RGDfK), and POL 3 þ c(RDGfK) (j = 1.5, ccp = 1 10-3 g/mL) after an incubation time of 24 h; cpec = 5.5 10-4 g/mL.
On the basis of these findings, it can be concluded that the purification of polymer-coated siRNA/PEI complexes is impossible due to their poor stability and the formation of large particles by chemical reaction of an excess of PEI with reactive coating polymers. This represents a serious problem for the proper characterization of coated siRNA/PEI complexes for possible future medical applications. Therefore, we have designed and tested another polyelectrolyte system (i.e., siRNA/GPL) more suitable for medicinal in vivo applications. Properties of siRNA/GPL Polyplexes. First, we studied the formation of the complexes as a function of the molar mixing ratio (j). Typical results are shown in Figure 2, where the Zimm plot of the siRNA/GPL complexes is shown for a variety of mixing ratios. 174
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The behavior of Mwpec and Rhpec as a function of j is shown in Figure 3a,b. The Mwpec was found to increase with increasing j, with the increase accelerated in the vicinity of the charge compensation point, j ≈ 1. Since the size of PEC was too small for reliable determination of the radius of gyration by static light scattering, the hydrodynamic radius (Rh) of the complexes was estimated by DLS. The j-dependence of Rhpec was more complex than that of Mwpec, while Rhpec of complexes was practically independent of j at j > 2.5. The size of the complexes was strongly increased by enhanced hydrophobic aggregation at j ≈ 1. No flocculation was observed in the investigated region of j = 10-1.05. The obtained Rhpec and Mwpec of siRNA/GPL complexes corresponded well with results obtained earlier for other oligonucleotides. 3,4 For all the following experiments, we decided to use complexes prepared at j = 1.5, which was in the region of suitable Rh and Mw values of the complexes. The presence of a free polycation in the PEC solution was also not observed at this j; in contrast to PEI, the chemical coating reaction was not contaminated by the reaction of excessive coating copolymers with free polycations resulting in formation of nanogel. From the Mwpec values of the complexes, their aggregation number (nagg = Mwpec/Mws) could be estimated, where Mws was the molecular weight of the unimolecular siRNA/GPL complex. Assuming full charge compensation of GPL macromolecules by siRNA at j = 1, Mws was calculated using mass/charge data and
data used for Figure 2, with nagg calculated to be 6.7 and the number of siRNA molecules per PEC was found to be nrn ≈ 10. Static and dynamic light scattering methods were also used to monitor the efficiency of the coating reaction of siRNA/GPL complexes. Changes in the weight-average molecular weight (Mw) and hydrodynamic radius (Rh) of the PEC were examined. The sensitivity of the methods is demonstrated in Figures 4 and 5, where Zimm plots and the Rh-distribution functions for uncoated and coated siRNA/GPL (siRNA/GPL/PHPMA) complexes are shown. The complexes were prepared at j = 1.5 and were coated with copolymer POL 1, which was attached to the complex surface via nondegradable spacers, and polymer POL 3, which was connected with degradable linkages (siRNA/GPL/PHPMA) (Scheme 1). To provide some initial proof-of-principle for the feasibility of developing polymer-coated PECs for targeted delivery to certain receptor-overexpressing cells, we also incorporated the standard targeting moiety c(RGDfK) and a control oligopeptide (i.e., c(RDGfK)) into the copolymer structure, by means of a polymer analogous reaction (i.e., POL3þc(RGDFK) and POL3þc(RDGfK)), and these copolymers were used for coating of complexes. Zimm plots of uncoated and coated siRNA/ GPL complexes measured after 24 h are shown in Figure 4. The concentration of coating copolymer was again 1 mg/mL in these experiments. Results of light scattering measurements obtained for all copolymer-coated complexes are shown in Table 3. The efficiency of coating with POL 1 was slightly higher than that obtained with POL 3 due to its higher molecular weight (Table 1). The number of siRNA molecules incorporated in a complex was between 3 and 3.5. It was calculated using data on molecular weight of the complexes, their density, known molecular weight of used polycation, and the N/P (j) ratio. In principle, the coating reaction with a multivalent polymer can result in aggregation of complexes. Inadequate increases in Rh of complexes after coating can be used as evidence of such aggregation. Additional evidence of particle aggregation can be represented by increased polydispersity (broadening of Rh distribution) of the coated particles, which should be close to that of uncoated particles. It can be seen in Figure 5 that the width of the Rh distribution of the coated complexes was slightly smaller than that of the original complexes. Therefore, particle aggregation was practically negligible. The maximum of the distribution curve of coated PECs was found to be shifted by 5 nm to higher Rh values. Thus, the mean thickness of the coating layer (ΔRhct) was found to be 5 nm. The number of molecules attached to the PEC surface (nct) was 28 after 24 h of coating. The results above also show that attachment of targeting oligopeptide does not significantly influence the coating reaction and such copolymers are suitable for preparation of coated and oligopeptide-targeted delivery vectors.
Figure 5. Rh distributions for siRNA/GPL complexes (j = 1.5) and coated PEC after 24 h; coating copolymer POL 1, ccp = 1 10-3 g/mL and cpec = 4.2 10-4 g/mL.
Table 3. Characteristics of siRNA/GPL PECs Modified by Reactive Copolymers at j = 1.5, Obtained after 24 h Mwa 10-6 coating polymer
-1
(g mol )
ΔMwct 10-6 -1
(g mol )
nct
28 27
Fpec,cc
Rh (θ = 0°)
ζ potential
Nrn
(g mL-1)
nm
(mV)
3
0.007
37
þ7
0.042 0.032
42 40
-3
-
0.80
POL 1 POL 3
7.92 5.32
2.04 1.45
POL 3 þ c(RGDfK)
6.69
1.66
0.031
44
POL 3 þ c(RDGfK)
6.65
1.65
0.029
45
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Figure 6. AFM amplitude images of siRNA/GPL complexes (a,b) (j = 1.5) and their POL 3-coated analogues (c,d) (ccp = 1 10-3 g/mL): (a,c) field of view 2 μm; (b,d) field of view 500 nm.
Figure 7. (a) Time dependence of Mwpec and Rhpec (θ = 0) of PEC prepared at j = 1.5. (b) Time dependence of Mwcc and Rhcc (θ = 0) of PEC prepared at j = 1.5 and coated with POL 3 (ccp = 1 10-3 g/mL).
Figure 8. Time dissociation of siRNA/GPL complexes from horizontal electrophoresis.
Atomic Force Microscopy (AFM). AFM was used as a direct method for characterization of PEC particle morphology, which consisted of size, shape, and size polydispersity. The AFM amplitude images of siRNA/GPL complexes (j = 1.5) obtained in the tapping mode are shown in Figure 6a,b. The sizes of dried polyplexes obtained by the analysis of AFM images were only slightly higher (the radius was in the range 60-70 nm) than the Rh values presented in Table 3. Surprisingly, the uncoated polyplexes exhibited a regular angular structure (Figure 6b). Time Stability of PEC. Despite the slow speed of preparation of siRNA/GPL complexes (15 min), the final complexes were still in a nonequilibrium state. Therefore, we tested the
Figure 9. Time dependence of the turbidity of uncoated and coated PEC as investigated in 0.15 M NaCl solvent.
medium-term stability of their parameters (Rh, Mw). The results of stability for uncoated PECs (siRNA/GPL) and PECs coated with copolymer POL 3 (containing a degradable spacer) obtained within three weeks are shown in Figure 7a,b. 176
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Figure 10. Concentration (A) and time (B) dependence of target-specific gene silencing in vitro in MDA-MB-231 human epithelial breast adenocarcinoma cells expressing both luciferase (to determine siRNA-specific gene silencing) and RFP (to determine unspecific gene silencing/toxicity) induced by uncoated and POL3-coated antiluciferase siRNA/GPL complexes. Values were compared to those of untreated controls and to those induced by Lipofectamine/siRNA complexes. In B, lipofectamine was used at an optimal dose of 0.5 μg of siRNA (according the manufacturer’s instructions), while the coated and uncoated polyplexes were used at doses corresponding to 10 μg of siRNA.
As can be seen, the Mw of both types of complexes decreased in the first four days of observation. After that, coated complexes approached equilibrium, but the Mw of uncoated PEC continued to decrease slightly until the experiment ended. The Rhcc of coated PEC decreased more rapidly than their Mwcc, probably due to their internal restructuring. While the ζ-potential of PEC was positive after the coating reaction, similar to uncoated complexes, the polarity of the ζ-potential of coated complexes switched overnight to become negative (see Table 3). The drop in ζ-potential was evidently a result of hydrolysis of the remaining reactive TT groups not participating in the coating reaction (formation of -COOH groups). If the excessive unreacted coating polymer was not removed from the PEC solution, the drop in ζ-potential continued to a value of -22 mV after 22 days. SiRNA molecules were fixed inside the complexes by electrostatic interactions, therefore, they could be undesirably displaced from complexes by an exchange reaction with polyanions.3 The remaining coating copolymers were hydrolyzed, and their reactive TT groups, which had not been aminolyzed in the coating reaction, were changed into carboxylic acid groups. SiRNA could then be released from the complexes during longer storage times in their solutions by interaction with hydrolyzed coating copolymers. Indeed, the release of siRNA from the unpurified complexes was proven by agarose gel electrophoresis, and the results of the measurements of 22-day-old samples are shown in Figure 8. In order to avoid this problem, we have developed a cleaning procedure for freshly prepared coated PECs. To purify the PECs from excessive coating polymer, the PEC solution was filtered through the Millipore Amicon Ultra-4 centrifugation device 5 times with an Ultracel-100 membrane from regenerated cellulose (cutoff 100 kDa) and diluted with 0.01 M NaCl buffer to the original concentration. The concentration and size of the complexes was verified by light scattering. The weight losses of the PEC were less than 10% after this cleaning procedure. The Rh of complexes was conserved during the cleaning procedure, and results obtained after 22 days of storage of the purified complexes are shown in Figure 8. Only the purified complexes, therefore, were used for all subsequent experiments and for biological testing. Stability of Complexes in Saline Solution. To prove the stability of the studied PECs under conditions mimicking
physiological environments, we measured the time dependence of turbidity in saline solution (0.15 M NaCl). These measurements are related to the kinetics of PEC aggregation followed by phase separation. Results are shown in Figure 9 for uncoated and polymer-coated complexes. Complexes prepared from PEI polycations showed the fastest coagulation rate, while the rate of coagulation was lower for PECs prepared by self-assembly of siRNA with GPL. Furthermore, both types of polymer-coated PEC were stable in saline solution; the turbidity did not change with time. Thus, it can be concluded that coating the PEC with HPMA copolymers substantially increased the colloidal stability of the polyplexes, with stability of GPL complexes being superior to those of PEI, and it seems reasonable to assume that also, in vivo, the stability of the coated PEC might be higher than that of uncoated PEC. Initial in Vitro Evaluation of Gene Silencing Activity. To provide some initial experimental evidence for the retention of biological activity upon coating the siRNA-containing complexes, their silencing efficacy was analyzed using MDA-MB-231 human epithelial breast adenocarcinoma cells stably expressing luciferase and red fluorescent protein (RFP). To this end, both coated and uncoated GPL-based antiluciferase PECs were prepared, and their ability to specifically reduce the expression of luciferase was compared to that of the standard transfection agent Lipofectamine. Here, luciferase knockdown upon exposure to different concentrations (i.e., 1, 10, and 20 μg siRNA) and different incubations times (i.e., 48, 72, and 96 h) was correlated with loss of RFP signal (i.e., nonspecific silencing/toxicity), findings were normalized to those of untreated controls, and they were compared to those obtained for Lipofectamine (which according the instructions of the manufacturer was always applied at an optimal siRNA concentration of 0.5 μg). As shown in Figure 10, it was found in these initial proof-of-principle analyses that, in the majority of cases, luciferase-specific gene silencing induced by the polymer-coated PEC was more or less comparable to that induced by Lipofectamine, and that it in some cases even turned out to be significantly better. Also, as compared to uncoated PEC, the silencing efficacy of the polymer-coated PEC turned out to be superior in the majority of cases (likely mostly because of a reduction in nonspecific gene silencing/toxicity, rather than because of an increase in specific gene silencing). These findings are by no means conclusive, and additional in vitro efficacy, in vitro uptake, and in vitro toxicity analyses are necessary before 177
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Bioconjugate Chemistry transferring these systems to the in vivo situation. Nonetheless, these initial in vitro insights already demonstrate quite clearly that polymer-coated siRNA-containing PEC are able to specifically induce target-specific gene silencing in vitro, and they thereby exemplify that even systems (physico-) chemically optimized to eventually perform well in vivo compare well to unstable and nonstealth systems primarily designed for gene silencing in vitro.
’ CONCLUSIONS The synthesis and physicochemical properties of reactive copolymers suitable for the coating of PEC and the physical properties of coated and uncoated siRNA-containing PECs are described. Results of stability measurements showed that oftenstudied PEI-based PECs are not suitable for coating reactions and consequently are likely suboptimal vectors for in vivo siRNA delivery. We therefore set out to develop siRNA-containing PECs based on GPL, as well as GPL PECs coated with HPMAbased reactive hydrophilic copolymers. The synthesis and physicochemical characterization of HPMA-based copolymers containing TT reactive groups bound to the polymer chain via spacers containing biodegradable disulfide bonds was described. The use of the HPMA copolymers for siRNA/GPL complex coating reactions resulted in attachment of (on average) 28 polymer molecules to the nanoparticle surface, forming a 5-nm-thick hydrophilic polymer protective layer that was selectively removable in reducing environments. Coating the siRNA/GPL vectors with the stealthy hydrophilic polymer substantially enhanced the stability of the vectors in aqueous media, and it might thereby improve their potential for passive targeting to solid tumors by means of the EPR effect. Preliminary results on the in vitro gene silencing efficacy of the coated complexes furthermore showed that they were able to specifically reduce target gene expression in human epithelial breast adenocarcinoma cells, thereby suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
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dct - surface density of coating polymer (g/nm2 mol) nct - number of coating molecules fixed on PEC surface tct - characteristic reaction time of the slow coating process (min)
’ MEANING OF SUBSCRIPTS AND SUPERSCRIPTS cp - coating polymer pec - uncoated polyelectrolyte complexes cc - coated PEC ct - coating layer of PEC ’ DEFINITIONS ΔMwct = Mwcc - Mwpec ΔRhct = Rhcc - Rhpec ’ REFERENCES (1) Seymour, L. W., Kataoka, K., and Kabanov, A. V. (1998) (Kabanov, A. V., Felgner, P. L., and Seymour, L. W., Eds.) pp 219, Wiley, New York. (2) Lechardeur, D., and Lukacs, G. L. (2002) Intracellular barriers to non-viral gene transfer. Curr. Gene Ther. 2, 183. ., Reschel, T., Zintchenko, A., and (3) Dautzenberg, H., Ko nak, C Ulbrich, K. (2003) Cationic graft copolymers as a carriers for delivery of antisense-oligonucleotides. Macromol. Biosci. 3, 425–435. ., Reschel, T., Subr, (4) Dautzenberg, H., Zintchenko, A., Ko nak, C V., and Ulbrich, K. (2001) Polycationic graft copolymers as carriers for oligonucleotide delivery. complexes of oligonucleotides with polycationic graft copolymers. Langmuir 17, 3096–3102. (5) Green, N. K., Herbert, C. W., Hale, S. J., Hale, A. B., Mautner, V., Harkins, R., Hermiston, T., Ulbrich, K., Fisher, K. D., and Seymour, L. W. (2004) Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus. Gene Ther. 11, 1256–1263. (6) Oupicky, D., Ogris, M., Howard, K. A., Dash, P. R., Ulbrich, K., and Seymour, L. W. (2002) Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation. Mol. Ther. 5, 463–472. (7) Fisher, K. D., Stallwood, Y., Green, N. K., Ulbrich, K., Mautner, V., and Seymour, L. W. (2001) Polymer-coated adenovirus permits efficient retargeting and evades neutralising antibodies. Gene Ther. 8, 341–348. (8) Dash, P. R., Read, M. L., Barrett, L. B., Wolfert, M. A., and Seymour, L. W. (1999) Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Ther. 6, 643. (9) Walker, G. F., Fella, C., Pelísek, J., Fahrmeir, J., Boeckle, S., Ogris, M., and Wagner, E. (2005) Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo. Mol. Ther. 11, 418–425. ., Subr, V., and Ulbrich, K. (2007) Coating of (10) Laga, R., Ko nak, C nanoparticles bearing amino groups on the surface with hydrophilic HPMA-based polymers. Colloid Polym. Sci. 285, 1509–1514. (11) Bernkop-Schn€urch, A. (2005) Thiomers: A new generation of mucoadhesive polymers. Adv. Drug Delivery Rev. 57, 1569–1582. (12) Chawla, R. K., Lewis, F. W., and Kutner, M. H. (1984) Plasma cysteine, cystine, and glutathione in cirrhosis. Gastroenterology 87, 770–776. ., Laga, R., and Ulbrich, K. (2006) Coating (13) Subr, V., Ko nak, C of DNA/Poly(l-lysine) complexes by covalent attachment of poly[N-(2hydroxypropyl)methacrylamide]. Biomacromolecules 7, 122–130. (14) Pola, R., Studenovsky, M., Pechar, M., Ulbrich, K., Hovorka, O., íhova, B. (2009) HPMA-copolymer conjugates Vetvicka, D., and R targeted to tumor endothelium using synthetic oligopeptides HPMA copolymer-based conjugates targeted with specific oligopeptides. J. Drug Targeting 17, 773–786. (15) Ulbrich, K., Subr, V., Strohalm, J., Plocova, D., Jelínkova, M., íhova, B. (2000) Polymeric drugs based on conjugates of synthetic and R and natural macromolecules I. Synthesis and physico-chemical characterisation. J. Controlled Release 64, 63–79.
’ AUTHOR INFORMATION Corresponding Author
*To whom correspondence should be addressed. E-mail: kostka@ imc.cas.cz. Fax: þ420 296809410. Tel.: þ420 296809209.
’ ACKNOWLEDGMENT The authors acknowledge financial support by the Grant Agency of Academy of Sciences of the Czech Republic (IAAX00500803), Academy of Sciences of the Czech Republic (KAN 200200651) and by the European Union (grant LSHB-CT-2004-512087 and MediTrans - Targeted delivery of Nanomedicines). ’ LIST OF SYMBOLS Mw - weight-average molecular weight (g/mol) Mwa - apparent weight-average molecular weight (g/mol) Rh - hydrodynamic radius (nm) Rg - radius of gyration (nm) ζ - zeta potential of PEC (mV) F - particle density of PEC (g/mL) c - concentration (g/mL) dn/dc - refractive index increment q - scattering vector (m-1) j - ratio of positive to negative charges 178
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