Aggregation Behavior of Cationic Nanohydrogel Particles in Human

Apr 3, 2014 - For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. A...
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Aggregation Behavior of Cationic Nanohydrogel Particles in Human Blood Serum Lutz Nuhn,† Sabine Gietzen,‡ Kristin Mohr,‡,§ Karl Fischer,‡ Kazuko Toh,∥ Kanjiro Miyata,∥ Yu Matsumoto,∥ Kazunori Kataoka,∥,⊥ Manfred Schmidt,‡ and Rudolf Zentel*,† †

Institute of Organic Chemistry, Johannes Gutenberg-University Mainz, Duesbergweg 10-14, D-55099 Mainz, Germany Institute of Physical Chemistry, Johannes Gutenberg-University Mainz, Welder Weg 11, D-55021 Mainz, Germany § Max-Planck-Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany ∥ Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan ⊥ Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan ‡

S Supporting Information *

ABSTRACT: For systemic siRNA delivery applications, welldefined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA delivery.

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INTRODUCTION Since its discovery, RNA interference1,2 has become a promising tool to knock-down pathogenic genes, thus, affording novel strategies to a wide field of therapeutic applications.3 However, adequate drug carriers are still required to overcome several barriers after systemic injection of small interfering RNA (siRNA).4 One of the major requirements especially for in vivo delivery is the carrier’s stability under physiological relevant conditions.5 As recently shown, several concepts that have successfully been developed for plasmid DNA (pDNA) delivery in gene therapy could not be adopted to siRNA easily.6 Mostly, lipoplex or polyplex siRNA delivery formulations depend on siRNA as their anionic cargo to stabilize the nanosized object with cationic lipids or (block co)polymers. However, due to its molecular weight of about 14 kDa, siRNA itself is too small to stabilize these nanoparticles especially under biological relevant conditions (e.g., blood serum). Therefore, attempts to introduce covalent cross-linkers after polyplex formation have shown to increase the stability of these carriers.7−10 Alternatively, precise nanoparticular siRNA delivery systems that are predefined in size and shape and independent from their cargo may become useful carriers with improved stability properties for in vivo applications. To that respect, nano© 2014 American Chemical Society

hydrogel particles have recently been attractive to siRNA delivery.11,12 Our group currently developed a new synthetic route to access well-defined cationic nanohydrogel particles via RAFT block-copolymerization of pentafluorophenyl methacrylate (PFPMA) and tri(ethyleneglycol)methylether methacrylate (MEO3MA). In polar-aprotic solvents (e.g., dimethyl sulfoxide) these amphiphilic reactive-ester block-copolymers self-assemble to nanometer-sized aggregates, which can permanently be locked-in by cross-linking with spermine affording cationic nanohydrogel particles of different sizes.13 Although these materials have shown efficient complexation with siRNA as well as proper siRNA uptake into cells, stability assays of these carriers under biologic relevant conditions have not been investigated so far. Generally, a basic understanding of nanomaterial interaction with blood and its components is one of the major challenges for its successful clinical use.14−17 As soon as nanosized particles are applied to in vitro or even in vivo assays, they are exposed to a physiological environment of high protein Received: February 7, 2014 Revised: March 18, 2014 Published: April 3, 2014 1526

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Figure 1. Cationic nanohydrogel particles alone in human blood serum (A) or loaded with siRNA (weight-to-weight ratio NP:siRNA = 25:1) (B). The blue spheres and clouds are representatives of serum protein components occurring in the bloodstream.

performance. Additionally, preliminary in vivo investigations by intravital confocal videography confirmed our results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe complexes of higher stability together proposing systemic siRNA delivery applications.

concentration (e.g., serum proteins) that may be involved in various absorption phenomena, which can change their surface properties and even alter their cellular interaction or biodistribution.18−20 For example, Tenzer et al. looked at the kinetics of protein adsorption onto silica and polystyrene nanoparticles after exposure to human plasma and identified more than 300 different protein components.21 For systemic application, however, the formation of larger aggregates inside the bloodstream or even blood coagulation (blood clot), which can induce thrombosis, is one of the primary investigations one has to consider prior to nanoparticle injection. To that respect, Rausch et al. developed an advanced pre-in vivo screening to sensitively detect the change of size of polymer nanoparticles and formulations in human blood serum.22 Although blood serum is only a part of the whole blood, its analysis can provide basic information about the nanoparticles’ interaction with serum proteins. The size distribution of human blood serum components investigated by Rausch et al. was measured by dynamic light scattering (DLS) and after addition of nanoparticular compounds to the protein solution changes in the light scattering profile could be determined, when aggregates were formed after incubation with the polymeric nanoparticles. To characterize the aggregation behavior of nanoparticular systems under physiological relevant conditions, this method was recently applied to poly(N-(2-hydroxypropyl) methacrylamide) (p(HPMA)) stabilized polystyrene and polylactide colloids to exclude aggregation tendency in serum or cell culture medium.23 Moreover, this method was further adopted to a statistic poly(N-(2-hydroxypropyl) methacrylamide-colauryl methacrylate) system, where the aggregation formation of the polymer with human blood serum was found to be suppressed upon encapsulation of a hydrophobic drug into the polymeric micelles.24 Interestingly, those serum compounds responsible for aggregate formation could be identified by control experiments with single serum components supporting the analytical strength of this novel pre-in vivo screening method. While it cannot completely exclude minor protein adsorption onto the nanoparticles forming monolayers that are beyond the detection limit of dynamic light scattering, it is still a powerful tool as preclinical screening to exclude larger aggregation formation that highly influences the nanoparticles’ stability performance in the bloodstream after systemic application. In the present study, we applied our cationic nanohydrogel particles to DLS measurements in human blood serum to monitor their aggregate formation and found that it could be suppressed at different loading ratios with siRNA (Figure 1). Interestingly, zeta potential measurement as qualitative value for charge could be put into relation with its serum stability

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MATERIALS AND METHODS

Materials. All chemicals and solvents were purchased from Acros Organics or Sigma-Aldrich and used as received unless otherwise indicated. Oregon Green 488 cadaverine was obtained from Invitrogen. Pentafluorophenol was obtained from Fluorochem (Great Britain, U.K.). Phosphate buffered saline (PBS) was obtained from Fisher BioReagents containing 137 mM NaCl, 11.9 mM phosphates and 2.7 mM KCl. Dialysis was performed using Spectra/ Por 3 membranes obtained from Carl Roth GmbH+Co.KG (Germany) molecular weight cut off 8000−10 000 g/mol. Nonlabeled siRNA (sequence is directed against enhanced green fluorescent protein EGFP) was purchased form IBA GmbH (Göttingen, Germany). Sense strand: 5′-GCAAGCUGACCCUGAAGUUCAU-3′; Antisense strand: 3′-GCCGUUCGACUGGGACUUCAAG-5′. Alexa 647 labeled siRNA (sequence is directed against Firefly GL3 luciferase) was purchased from Hokkaido System Science Co., Ltd. (Hokkaido, Japan) with dye label attached to the 5′-end of the sense strands. Sense strand: 5′-CUUACGCUGAGUACUUCGAdTdT-3′; Antisense strand: 3′-dTdTGAAUGCGACUCAUGAAGCU-5′ siRNA duplexes were formed by mixing labeled sense and unlabeled antisense strands in PBS and incubating for 2 min at 95 °C followed by 1 h hybridization period at 37 °C. Instrumentation. All 1H-, 13C- and 19F-NMR spectra were recorded on a Bruker 400 MHz FT NMR spectrometer. Chemical shifts (δ) are given in ppm relative to TMS. Samples were prepared in deuterated solvents and their signals referenced to residual nondeuterated solvent signals. The polymers’ molecular weight was determined by gel permeation chromatography (GPC) in tetrahydrofuran (THF) as solvent and with the following parts: pump PU 1580, auto sampler AS1555, UV-detector UV 1575 (detection at 254 nm), and RI-detector RI 1530 from JASCO. Columns were used from MZ-Analysentechnik: MZ-Gel SDplus 102 Å and MZ-Gel SDplus 106 Å. Calibration was done using polystyrene standards purchased from Polymer Standard Services. UV−vis spectra were recorded using a Jasco V-630 Spectrophotometer (1 cm × 1 cm quartz cell). Syntheses. The syntheses of poly(tri(ethylene glycol) methyl ether methacrylate)-block-poly(pentafluorophenyl methacrylate) P(MEO3MA)-b-P(PFPMA) block copolymers P1 (Mn = 9600 g/mol, PDI = 1.23) and P2 (Mn = 14 200 g/mol, PDI = 1.24) were performed as recently described.13 Analytical data can be found in the Supporting Information (Scheme S1 and Figures S1−S5). Synthesis of Cationic Nanohydrogel Particle13 NP. P(MEO3MA)12-b-P(PFPMA)25 block copolymer P1 (49.6 mg; 5.17 μmol polymer or 129.2 μmol reactive ester) was transferred into a round-bottom flask equipped with a stir bar and anhydrous DMSO was added (5.0 mL). Supported by sonication for 1 h the polymer 1527

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mixture of serum of seven healthy donors was used for all measurements. Sample Preparation for Light Scattering Measurements. All solutions for light scattering experiments were prepared in a dust free flow box. Cylindrical quartz cuvettes (20 mm diameter, Hellma, Mühlheim) were cleaned by dust-free distilled acetone. Human blood serum solutions were filtered through Milex GS filters, 220 nm pore size (Millipore) with negligible losses of serum proteins. All PBS solutions (nanohydrogel particles alone or loaded with siRNA, siRNA only, or buffer only) were filtered through Milex LCR filters, 450 nm pore size (Millipore) with negligible sample losses. Single protein solutions of bovine serum albumin (BSA) (80 g/L in PBS) and immunoglobuline G (IgG) (20 g/L in PBS) were filtered through Milex AA anotop filters, 20 nm pore size, prior to use. For light scattering of nanohydrogel particles (alone or loaded with siRNA at given weight-to-weight ratios) in PBS or protein solution, 0.5 mL of each sample (0.15 g/L nanohydrogel particle in PBS) was filtered into the light scattering cuvette containing 1.0 mL of filtered PBS buffer or protein solution. For the measurements of nanohydrogel particles (alone or loaded with siRNA at given weight-to-weight ratios) or siRNA alone in serum, 0.5 mL of each sample (0.15 g/L nanohydrogel particle in PBS or 0.01 g/L siRNA in PBS) was filtered into the light scattering cuvette containing 1.0 mL of nondiluted filtered human blood serum, thus resulting in a dilution factor of 2/3 for serum components only. All cuvettes were incubated for 30 min on a shaker at room temperature before measurement. Light Scattering Setup. All light-scattering experiments were performed with an instrument consisting of a HeNe laser (632.8 nm, 25 mW output power), an ALV-CGS 8F SLS/DLS 5022F goniometer equipped with eight simultaneously working ALV 7004 correlators, and eight QEAPD Avalanche photodiode detectors. All correlation functions were measured at T = 293 K in the cross-correlation mode at scattering angles between 30 and 150° in steps of 30°. Intravital Confocal Videography. This experiment was performed in analogy to an early reported procedure.25 All steps were done in accordance with the Guidelines for the Care and Use of Laboratory Animals as stated by The University of Tokyo. All picture/ movie acquisitions were taken by a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1 (Nikon Corp., Tokyo, Japan) equipped with a 20× objective, 640.1 nm diode laser with a band-pass emission filter of 700/75 nm and a 487.3 nm diode laser with a band-pass emission filter of 525/50 nm. The pinhole diameter was set to result in a 10 μm optical slice. Eight-week-old female BALB/c nude mice (Oriental Yeast Co., Ltd., Tokyo, Japan) were anesthetized with 2.0−3.0% isoflurane (Abbott Japan Co., Ltd., Tokyo, Japan) using a Univenter 400 Anaesthesia Unit (Univentor Ltd., Zejtun, Malta). Mice were then subjected to lateral tail vein catheterization with a 30-gauge needle (Becton, Dickinson and Co, Franklin Lakes, NJ, U.S.A.) connected to a nontoxic medical grade polyethylene tube (Natsume Seisakusho Co., Ltd., Tokyo, Japan). Anesthetized mice were placed onto a temperature-controlled pad (Thermoplate; Tokai Hit Co., Ltd., Shizuoka, Japan) integrated into the microscope stage and maintained in a sedated state throughout the measurement. Ear lobe dermis was observed without surgery and was easily fixed beneath a coverslip with a single drop of immersion oil. Data was acquired in video mode for 3 min (30 frames/sec), followed by snapshots every 1 min thereafter. Oregon Green labeled nanohydrogel particles NP* only (105 μL of a 10 mg/mL NP* solution in PBS + 95 μL PBS) as well as Alexa647-labeled siRNA only (50 μL of 60 μM or 0.84 mg/mL Alexa647-siRNA + 150 μL PBS) and siRNA loaded nanohydrogel particles (50 μL of 60 μM or 0.84 mg/ mL Alexa647-siRNA + 105 μL of a 10 mg/mL NP* solution in PBS + 45 μL PBS, complexed 1h prior to experiment) were injected as 200 μL total volume via tail vein 10 s after the start of video capture.

could be dispersed in DMSO under nitrogen atmosphere forming selfassembled micellar aggregates. They were cross-linked by addition of spermine (135.5 μL of a 0.1 g/mL solution in DMSO; 67.0 μmol) and triethylamine (111.3 μL; 802.9 μmol). The flask containing the reaction mixture was then immersed in an oil bath at 50 °C under vigorously stirring. After 19 h, a 19F-NMR sample (0.2 mL dissolved in 0.4 mL DMSO-d6) was taken showing complete reactive ester conversion (compare Supporting Information Figure S6). Yet, to remove further traces of polymer bound pentafluorophenol below the NMR detection limit, excess of noncross-linking methoxy triethylene glycol amine (218.5 μL of a 0.1 g/mL solution in DMSO; 133.6 μmol) was added and the reaction mixture was stirred for additional 20 h at 50 °C. To remove small molecular byproducts, the reaction mixture was afterward purified by dialysis against Millipore water for several days (including water exchange twice every day) and subsequent lyophilization affording NP (31.8 mg, 85%) as a voluminous colorless powder. Synthesis of Fluorescently Labeled Cationic Nanohydrogel Particle13 NP*. P(MEO3MA)25-b-P(PFPMA)38 block copolymer P2 (30.0 mg; 2.11 μmol polymer or 80.3 μmol reactive ester) was transferred into a round-bottom flask equipped with a stir bar and anhydrous DMSO was added (3.0 mL). Supported by sonication for 1 h the polymer could be dispersed in DMSO under nitrogen atmosphere forming self-assembled micellar aggregates. For fluorescent labeling, Oregon Green Cadaverine (80.0 μL of a 2.5 mg/mL solution in DMSO; 0.4 μmol) was added first, followed by triethylamine (56.0 μL; 404.0 μmol), and then spermine (8.1 mg; 40.1 μmol) for cross-linking. The flask containing the reaction mixture was then immersed in an oil bath at 50 °C under vigorously stirring. After 64 h, a 19F-NMR sample (0.2 mL dissolved in 0.4 mL DMSOd6) was taken showing complete reactive ester conversion (compare Supporting Information Figure S6). Yet, to remove further traces of polymer bound pentafluorophenol below the NMR detection limit, excess of noncross-linking methoxy triethylene glycol amine (13.1 mg; 80.3 μmol) was added and the reaction mixture was stirred for additional 25 h at 50 °C. To remove small molecular byproducts the reaction mixture was afterward purified by dialysis against Millipore water for several days (including water exchange twice every day) and subsequent lyophilization affording NP* (19.7 mg, 83%) as a voluminous orange powder. Cationic Nanohydrogel Particle Loading with siRNA. The lyophilized cationic nanohydrogel could easily be redispersed in PBS buffer at given concentrations supported by sonication. For loading with siRNA the samples were mixed at the given concentrations with the corresponding weight-to-weight ratios of siRNA (15:1, 25:1, or 50:1 nanohydrogel particle to siRNA) and incubated for 30 min at room temperature. To visualize siRNA loading agarose gel electrophoresis was performed after complexation. Agarose Gel Electrophoresis. For agarose gel electrophoresis experiments, a 0.5% agarose gel containing GelRed (Biotium) was used. Samples were prepared of 70 ng siRNA with nanohydrogel particles at given weight-to-weight ratios in PBS and incubated for 30 min before mixing with 6× loading puffer (30% glycerol, 0.25% bromophenol blue, and 0.25% xylene cyanol). Electrophoresis was performed in TBE buffer (89 mM tris-(hydroxymethyl)-aminomethane, 89 mM boric acid, and 2 mM Na2EDTA, pH 8) at 120 V for 30 min, and upon excitation at 365 nm, fluorescence was imaged by a conventional digital camera. Zeta Potential Measurement. For zeta potential analysis, a Malvern Zetasizer Nano ZS was used. Samples of nanohydrogel particles (alone or loaded with siRNA at given weight-to-weight ratios) were prepared at 0.05 g/L in Millipore water and applied to zeta potential measurement after incubation for 30 min. For each sample, 15−25 independent measurements were performed and analyzed by their mean average and standard deviation as mean error. Preparation of Human Blood Serum. Human blood serum used for this study was obtained from the University Medical Center Mainz (Germany) and was prepared according to the standard guidelines of the University Clinic Mainz. Because of the high variation in protein composition by different patients,22 a pool of serum obtained by the

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RESULTS AND DISCUSSION 1. Synthesis and Characterization of siRNA-Loaded Nanohydrogel Particles. Predefined nanoparticular carriers like nanogels are generally considered to have improved 1528

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Figure 2. Synthesis of nanohydrogel particle NP and loading with siRNA.

Figure 3. SiRNA loaded nanohydrogel particle NP characterized by agarose gel electrophoresis (A) and zeta potential measurement (B).

stability for siRNA delivery.11,12 We have recently established a new method to synthesize well-defined cationic nanohydrogel particles via RAFT block-copolymerization of pentafluorophenyl methacrylate (PFPMA) and tri(ethyleneglycol)methylether methacrylate (MEO3MA). In polar-aprotic solvents (e.g., dimethyl sulfoxide), these amphiphilic reactive-ester block copolymers self-assemble to nanometer-sized aggregates with the fluorinated reactive ester as inner core and the MEO3MA block stabilizing corona. These structures can permanently be locked-in by cross-linking with spermine affording cationic nanohydrogel particles with a cationic core.13 For DLS measurements in human blood serum, an amphiphilic P(MEO3MA)-b-P(PFPMA) copolymer P1 was synthesized (compare Supporting Information Scheme S1 and Figures S1− S5) and utilized for nanohydrogel particle synthesis (Figure 2). After micellar self-assembly in DMSO it could successfully be cross-linked by spermine (as monitored by 19F NMR; compare Supporting Information Figure S6) affording nanohydrogel particle NP. Purified nanohydrogel particle NP could nicely be redispersed in Millipore water or PBS buffer supported by sonication. First, loading capacity with siRNA was tested in PBS buffer in analogy to earlier reports.13 To verify siRNA loading for NP agarose gel experiments were performed using a 0.5% agarose gel (containing GelRed as nucleic acid stain) in TBE buffer and applying 120 V for 30 min for nucleic acid separation. For each sample, 70 ng of siRNA were incubated with increasing amount of NP (1.05, 1.75, and 3.5 μg, weightto-weight ratio NP/siRNA 15:1, 25:1 and 50:1) for 30 min prior to loading onto the gel (Figure 3A). At the 15:1 weight-

to-weight ratio NP/siRNA, we observed that still a lot of siRNA was not complexed by the nanogel and thus penetrated into the gel-like free siRNA. At the 25:1 ratio, however, hardly any free siRNA was visualized anymore. Instead, a diffuse smearing suggested compensated loading of siRNA onto the nanogel. At 50:1 ratio, all siRNA was complexed by NP remaining in the sample well. Additionally to the agarose experiments, zeta potential analyses of the nanohydrogel particles alone and loaded at the three different ratios with siRNA were performed (Figure 3B). To that respect, samples were prepared at 0.05 g/L for NP in Millipore water and incubated with siRNA (1.00 mg/L, 2.00 mg/L and 3.33 mg/L − weight-to-weight ratio NP:siRNA 50:1, 25:1 and 51:1) for 30 min before each measurement. For each sample, 15−25 independent measurements were performed and analyzed by their mean average and standard deviation as mean error for representative interpretation. Interestingly, the nanohydrogel particles themselves alone in water showed a strong positive zeta potential of about +36 mV due to the highly cationic spermine units incorporated into the nanogel system. This value could be reduced by increasing amount of siRNA complexed by the gel in analogy to the results of the agarose gel electrophoresis. While at the 50:1 ratio the zeta potential was still positive (+20 mV) with no free siRNA, at the 25:1 ratio it reached a slightly negative value that could hardly be increased at the 15:1 ratio, where free siRNA was still present. Summing up, the 25:1 ratio seemed to be the ideal state of almost complete charge neutralization of the nanohydrogel with hardly any free siRNA. 1529

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Figure 4. ACF of nanohydrogel particle NP in human blood serum (A), IgG solution (B), and BSA solution (C), scattering angle 30°. In serum (A) or BSA (C) aggregates were found with Rh = 300 and 190 nm, respectively.

Knowing the ACF of the human blood serum (eq 1) and the respective nanohydrogel particle ACF (alone or loaded with siRNA) (eq 2), the correlation function of the serum nanohydrogel mixtures could be analyzed. If no or negligible aggregation occurs, the resulting ACF of the serum nanohydrogel mixture should perfectly be fitted by the sum of the individual correlation functions keeping all parameters of the two compounds (serum/nanohydrogel) in eq 1 and eq 2 fixed and leaving the intensity contributions for serum fs and nanohydrogel particle f np as only fit parameters affording

To test the stability of these polyelectrolyte complexes in protein solution and to exclude that high concentration of negatively charged proteins like albumin26 would already release siRNA out of the nanogel competitively, additional gel electrophoresis experiments of siRNA loaded nanohydrogel particles in BSA solution were performed (compare Supporting Information Figure S8): For both BSA concentrations at 50 and 150 g/L no release was detectable, instead all siRNA was still complexed by the nanoparticle at both 25:1 and 50:1 weight-toweight ratios NP/siRNA. Similar experiments were even performed in human blood serum at the 25:1 ratio (Supporting Information Figure S9), where still no free siRNA was detectable. In this context, it is important to note that free siRNA in serum as control sample was not completetly digested by serum RNases in the time frame of the experiment (30 min serum incubation) (Supporting Information Figure S9, lane 4) and thus still detectable by agarose gel electrophoresis. 2. Dynamic Light Scattering Studies in Human Blood Serum. The behavior of the well-characterized nanohydrogel particle NP alone and loaded with siRNA in human blood serum was studied by dynamic light scattering (DLS) by applying an established method of Rausch et al.22 The electric field autocorrelation function (ACF) of human serum can nicely be described by the sum of three exponentials

g1,m(t ) = fs g1,s(t ) + fnp g1,np(t )

This eq 3 is called forced fit function. However, in case of aggregation formation by interaction of the nanohydrogel particles with serum components resulting in larger sizes than the serum components themselves, the forced fit function of eq 3 does not overlap with the obtained data points and thus needs to be extended by an additional longer ACF relaxation time to get statistically distributed residuals by the fitting function g1,m(t ) = fs g1,s(t ) + fnp g1,np(t ) + fagg g1,agg (t )

(4)

In this so-called aggregate fit function fagg represents the intensity contribution of the formed aggregates and g1,agg(t) the unknown correlation function of the aggregates with

⎛ t ⎞ ⎛ t ⎞ ⎛ t ⎞ g1,s(t ) = a1,s exp⎜⎜ − ⎟⎟ + a 2,s exp⎜⎜ − ⎟⎟ + a3,s exp⎜⎜ − ⎟⎟ ⎝ τ1,s ⎠ ⎝ τ2,s ⎠ ⎝ τ3,s ⎠ (1)

⎛ t ⎞⎟ g1,agg (t ) = a1,agg exp⎜⎜ − ⎟ ⎝ τ1,agg ⎠

with the amplitudes ai and the decay times τi = 1/(q2Di) including scattering vector q = (4πn/λ0)sin(θ/2) and Brownian diffusion coefficient Di for each component i. The ACFs for the slightly disperse nanohydrogel particles alone or loaded at different ratios of siRNA in PBS can successfully be fitted by a sum of two exponentials ⎛ ⎞ ⎛ t ⎞ ⎟⎟ + a 2,np exp⎜⎜ − t ⎟⎟ g1,np(t ) = a1,np exp⎜⎜ − ⎝ τ1,np ⎠ ⎝ τ2, np ⎠

(3)

(5)

It should be noted that the applied DLS method developed by Rausch et al. is extremely sensitive for detection of high molar mass or larger-sized aggregates being formed in complex multicomponent serum mixtures.22 The term “larger sized” in this case refers to larger than the largest component present in either pure serum or nanohydrogel particle solution. Changes within the size distribution of serum and polymers are only detectable if the amplitudes (i.e., the intensity fractions) of the newly formed sizes are sufficiently large and thus detectable by DLS. Preliminary tests (data not shown) typically revealed intensity fractions between 3 and 20% of newly formed particles to be necessary in order to become detectable by the described fitting procedure.24 In a first study, a sample of nanohydrogel particle NP alone in human blood serum was prepared and analyzed by the force fit method. Figure 4A clearly shows that it failed and only the additional aggregation function in eq 4 was able to describe the obtained data points successfully. To further investigate this aggregation behavior, samples of NP in albumin (BSA) and IgG

(2)

As reported earlier,27 eq 2 can be used to calculate the first cumulant resulting in an angle dependent diffusion coefficient D or reciprocal hydrodynamic radius Rh, respectively, according to formal application of Stokes−Einstein law. By extrapolation of Rh/q2 to q2 = 0, z-average hydrodynamic radius Rh = ⟨1/ Rh⟩−1z can be obtained. Interestingly, for the nanohydrogel particle NP alone or loaded with siRNA their hydrodynamic radii were conserved at each state (⟨1/Rh⟩−1z = 26 nm) and thus, independent from the cargo (compare Supporting Information Figure S10). 1530

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Figure 5. ACF of siRNA-loaded nanohydrogel particle NP in human blood serum at 50:1 (A), 25:1 (B), and 15:1 (C) weight-to-weight ratio NP/ siRNA, scattering angle 30°. At the 50:1 ratio (A), aggregates were found with Rh = 220 nm. At the 15:1 ratio (C), aggregates were found with Rh = 190 nm.

not overlap with the obtained data points. The siRNA loaded but still highly cationic nanohydrogels caused, in the same way like the nanohydrogel particles alone in serum (Figure 4A), larger aggregates that require the additional aggregate fit function from eq 4. On the other hand, when looking at the behavior of siRNA loaded nanohydrogel particles at the 25:1 ratio in serum in Figure 5B, the forced fit method successfully described the obtained data points, thus, no aggregation occurs in human blood serum at this state. Taking into account the nanohydrogel’s zeta potential at this loading ratio being rather neutral to slightly negative (compare Figure 3B), electrostatic interaction between negatively charged proteins like albumin and the nanohydrogel particles are at this state not strong enough to from larger aggregates detectable by the DLS set up. Similar results were obtained for siRNA loaded nanohydrogel particles in BSA solution, which mimic this charge based aggregation behavior (compare Supporting Information Figure S11). While at 50:1 weight-to-weight ratio of NP/siRNA aggregation with albumin was still slightly detectable, the forced fit functions overlap with the obtained data points of the ACFs at the 25:1 and the 15:1 ratio excluding albumin derived aggregate formation. To that respect, nanogels loaded at the 25:1 and the 15:1 ratio should display a similar behavior in serum as a consequence of their almost neutral zeta potential. However, experiments on serum demonstrate that nanohydrogels loaded with siRNA at the 15:1 ratio lead to aggregation, as an additional aggregation fit function was required for data fitting (Figure 5C). The samples differ from the 25:1 ratio only by the presence of free siRNA detectable by agarose gel electrophoreses (compare Figure 3A). To that respect, the behavior of free siRNA in human blood serum was finally studied by preparing a sample of siRNA in serum at the same concentration like the 15:1 ratio (3.33 mg/ L) (compare Supporting Information Figure S12). Similar to the results in Figure 5C, it was only possible to describe the obtained data points by applying an additional aggregate fit function. On the basis of these results, free siRNA itself was able to induce aggregates in human blood serum in the time frame of the experiments we performed (30 min serum incubation), which may also explain aggregate formation at the 15:1 ratio shown in Figure 5C. We hypothesize that as long as siRNA is not yet completely degraded by serum RNases (as shown in Supporting Information Figure S9, lane 4) and thus able to play out its immunostimulatory effect, it may probably be recognized more specifically by, for example, immunoprotein fractions in the serum affording larger immunocomplexes or aggregates detectable by dynamic light scattering. Taking these results into account, both cationic carrier (nanohydrogel

protein solution as two of the most abundant protein components in serum28 were prepared. In analogy to human blood serum, single protein solutions alone can again be described by a single exponential decay function ⎛ t ⎞⎟ g1,prot (t ) = a1,prot exp⎜⎜ − ⎟ ⎝ τ1,prot ⎠

(6)

(Thereof, the obtained extrapolated hydrodynamic radii Rh = ⟨1/Rh⟩−1z for BSA and IgG were 3.0 and 6.0 nm, respectively). Mixing protein solution with nanohydrogel solution allows applying the forced fit function or the aggregation functions of eqs 4 and 5 replacing fsg1,s(t) by f protg1,prot(t) with f prot as the intensity contribution of the protein solution. Using these analytical tools, nanohydrogel particle NP in IgG solution showed no aggregation (Figure 4B) as the forced fit function described all data points perfectly. However, for NP in BSA solution it was necessary to assume additional aggregation for optimal data fitting. The ACF curve in Figure 4C clearly visualizes a second decay for the data points at higher correlation times representing an additional aggregation component of larger size occurring. Taking into account that the isoelectric point of albumin is in the acid pH regime,26 these proteins are partially negatively charged in PBS, which may promote charge-based attraction with cationic nanohydrogel particles affording larger aggregates. However, for IgG with an isoelectric point rather averaged in the neutral pH regime,26 less interaction between proteins and nanohydrogel particle occurs, which prevents detectable aggregation. This may also explain the behavior of NP in human blood serum. Mostly albumin and other negatively charged protein fractions may cause aggregates based on the cationic charge of the nanohydrogel. Although the newly appearing aggregation fraction cannot be visualized by eye in the data points of ACF as easy as for the BSA experiment (compare Figure 4A with Figure 4C), the forced fit method clearly fails and only the presence of additional aggregate function allows perfect data fitting. In a second study, this aggregation phenomenon was further characterized after nanohydrogel loading with siRNA. Therefore, mixtures of NP with siRNA at the three different weightto-weight ratios NP/siRNA (15:1, 25:1, and 50:1) were prepared similarly to the agarose electrophoresis and zeta potential experiments and then incubated with human blood serum prior to DLS analysis. At the 50:1 ratio, where the nanohydrogel particle still had a highly cationic zeta potential (compare Figure 3B), the forced fit function in Figure 5A did 1531

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Figure 6. Intravital confocal videography images of nanohydrogel particle NP* only (A) and siRNA loaded nanohydrogel particle NP* (weight-toweight ratio NP*/siRNA = 25:1) (B) at given time points after sample injection. Oregon Green labeled NP*: green. Alexa647 labeled siRNA: red. While nanohydrogel particles alone (A) cause larger aggregates and even minor blood clotting (indicated by white arrows), siRNA loaded nanohydrogel particles (B) enable blood circulation without large aggregates detectable by intravital confocal videography.

(compare Supporting Information Movie S1). This process is obviously faster than probable aggregation tendencies with murine serum protein compartments as observed by the DLS studies in human serum after incubation for 30 min (compare Supporting Information Figure S12). Moreover, immunodeficient BALB/c nude mice were used that may not be able to provide RNA sensitive immuno-components in their blood serum. However, the siRNA loaded nanohydrogel particles NP* continue circulating longer (Supporting Information Movie S3). Similar results were obtained for nanohydrogel particle NP* only, yet over time larger aggregates could be found in the bloodstream that even formed minor blood clottings (Supporting Information Movie S2). Figure 6 clearly summarizes these circulation properties after tail vein injection. While light green and red fluorescence representing siRNA loaded nanohydrogel particles NP* can still be detected circulating in the blood vessels after 60 min in Figure 6B, bright intense green spots of NP* aggregates are formed already within 15 min and can still be detected after 60 min (Figure 6A) proving the results found by dynamic light scattering in serum.

particle only) and anionic cargo (siRNA) themselves have high potential to induce large aggregates in human blood serum detectable by dynamic light scattering. Only a reasonable loading ratio, which guarantees nanoparticular formulation of almost neutral zeta potential as well as hardly any free siRNA cargo, can exclude large aggregate formation with human blood serum compartments and thus enables potential for systemic siRNA delivery applications. 3. Comparison with Preliminary in vivo Data. Although the light scattering experiments in human blood serum provide only primary information about the nanohydrogel particles with the serum protein components and further interaction of the nanohydrogel particles with blood compartments like cells or coagulation factors still have to be studied carefully, we were interested in the reliability of the obtained pre-in vivo results. To that respect preliminary in vivo experiments by intravital confocal videography of siRNA loaded nanohydrogel particles were performed in mice. This method has been shown as a powerful tool to determine aggregation behavior of polyplexes in the bloodstream as well.29 For successful in vivo monitoring, fluorescently labeled nanohydrogel particles were synthesized as earlier reported.13 Self-assembled amphiphilic P(MEO3MA)b-P(PFPMA) copolymer P2 micelles in DMSO were sequentially aminolyzed first by Oregon Green cadaverine and then cross-linked by spermine affording nanohydrogel particle NP* (compare Supporting Information Figure S7) with similar properties like nonlabeled NP (e.g., ⟨1/Rh⟩−1z = 21 nm). They were successfully loaded with Alexa647 labeled siRNA again at the 25:1 weight-to-weight ratio NP*/siRNA in PBS and then injected into the tail vein of a BALB/c nude mouse. As control experiments, NP* alone as well as pure Alexa647 labeled siRNA only were injected into mice and then studied by intravital confocal videography. Blood vessels of the mice’s ear lobe could easily be imaged during the experiment showing fluorescently labeled samples circulating through the bloodstream. The injected siRNA alone quickly disappeared out of the bloodstream because of its low molecular weight affording vast body distribution and rapid kidney clearance

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CONCLUSION Small interfering RNA (siRNA) requires safe and stable carriers for in vivo application, for example, nanogel particles. In former studies, we introduced a novel synthetic approach for cationic nanohydrogel particles and demonstrated that they were able to transport siRNA into cells successfully.13 In this work, we investigated their behavior under biologically relevant conditions via dynamic light scattering in human blood serum as novel pre-in vivo characterization method.22 It provides a sensitive and efficient screening for monitoring larger aggregate formation of nanoparticles in contact with complex body fluids like serum or single serum components. To that respect, we observed aggregation formation occurring for the nanohydrogel particles alone in serum or in albumin solution, one of the most abundant serum proteins with an anionic charge. Because of the nanogel’s high cationic zeta 1532

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(7) Christie, R. J.; Miyata, K.; Matsumoto, Y.; Nomoto, T.; Menasco, D.; Lai, T. C.; Pennisi, M.; Osada, K.; Fukushima, S.; Nishiyama, N.; Yamasaki, Y.; Kataoka, K. Biomacromolecules 2011, 12, 3174−3185. (8) Schaffert, D.; Troiber, C.; Salcher, E. E.; Fröhlich, T.; Martin, I.; Badgujar, N.; Dohmen, C.; Edinger, D.; Kläger, R.; Maiwald, G.; Farkasova, K.; Seeber, S.; Jahn-Hofmann, K.; Hadwiger, P.; Wagner, E. Angew. Chem., Int. Ed. 2011, 50, 8986−8989. (9) Christie, R. J.; Matsumoto, Y.; Miyata, K.; Nomoto, T.; Fukushima, S.; Osada, K.; Halnaut, J.; Pittella, F.; Kim, H. J.; Nishiyama, N.; Kataoka, K. ACS Nano 2012, 6, 5174−5189. (10) Fröhlich, T.; Edinger, D.; Kläger, R.; Troiber, C.; Salcher, E.; Badgujar, N.; Martin, I.; Schaffert, D.; Cengizeroglu, A.; Hadwiger, P.; Vornlocher, H.-P.; Wagner, E. J. Controlled Release 2012, 160, 532− 541. (11) Ramos, J.; Forcada, J.; Hidalgo-Alvarez, R. Chem. Rev. (Washington, DC) 2013, 114, 367−428. (12) Smith, M. H.; Lyon, L. A. Acc. Chem. Res. 2012, 45, 985−993. (13) Nuhn, L.; Hirsch, M.; Krieg, B.; Koynov, K.; Fischer, K.; Schmidt, M.; Helm, M.; Zentel, R. ACS Nano 2012, 6, 2198−2214. (14) Dobrovolskaia, M. A.; McNeil, S. E. Nat. Nanotechnol. 2007, 2, 469−478. (15) Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nat. Mater. 2009, 8, 543−557. (16) Liu, Z.; Jiao, Y.; Wang, T.; Zhang, Y.; Xue, W. J. Controlled Release 2012, 160, 14−24. (17) Walkey, C. D.; Chan, W. C. W.; Liu, Z.; Jiao, Y.; Wang, T.; Zhang, Y.; Xue, W.; Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M.; Dobrovolskaia, M. A.; McNeil, S. E. Chem. Soc. Rev. 2012, 41, 2780−2799. (18) Aggarwal, P.; Hall, J. B.; McLeland, C. B.; Dobrovolskaia, M. a; McNeil, S. E. Adv. Drug Delivery Rev. 2009, 61, 428−437. (19) Walczyk, D.; Bombelli, F. B.; Monopoli, M. P.; Lynch, I.; Dawson, K. A. J. Am. Chem. Soc. 2010, 132, 5761−5768. (20) Miller, T.; Hill, A.; Uezguen, S.; Weigandt, M.; Goepferich, A. Biomacromolecules 2012, 13, 1707−1718. (21) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Nat. Nanotechnol. 2013, 8, 772−781. (22) Rausch, K.; Reuter, A.; Fischer, K.; Schmidt, M. Biomacromolecules 2010, 11, 2836−2839. (23) Kelsch, A.; Tomcin, S.; Rausch, K.; Barz, M.; Mailänder, V.; Schmidt, M.; Landfester, K.; Zentel, R. Biomacromolecules 2012, 13, 4179−4187. (24) Hemmelmann, M.; Mohr, K.; Fischer, K.; Zentel, R.; Schmidt, M. Mol. Pharmaceutics 2013, 10, 3769−3775. (25) Matsumoto, Y.; Nomoto, T.; Cabral, H.; Matsumoto, Y.; Watanabe, S.; Christie, R. J.; Miyata, K.; Oba, M.; Ogura, T.; Yamasaki, Y.; Nishiyama, N.; Yamasoba, T.; Kataoka, K. Biomed. Opt. Express 2010, 1, 1209−1216. (26) Jin, Y.; Luo, G.; Oka, T.; Manabe, T. Electrophoresis 2002, 23, 3385−3391. (27) Schmidt, M. In Dynamic Light Scattering, The Method and Some Applications; Brown, W., Ed.; Clarendon Press: Oxford, 1993. (28) Krebs, H. A. Annu. Rev. Biochem. 1950, 19, 409−430. (29) Nomoto, T.; Matsumoto, Y.; Miyata, K.; Oba, M.; Fukushima, S.; Nishiyama, N.; Yamasoba, T.; Kataoka, K. J. Controlled Release 2011, 151, 104−109.

potential aggregate formation was induced by those anionic serum components via charge interaction, yet, could be suppressed after loading the nanohydrogel particles with siRNA. Even at high albumin concentration siRNA was not released from the gel competitively. Only the nanoparticle’s zeta potential was thereby lowered to almost neutral affording no aggregate detectable by the DLS. Therefore, cationic zeta potential values may be helpful as primitive parameter to predict aggregation tendency in blood serum. Interestingly, excess of free siRNA afforded additional aggregate formation probably caused by the RNA oligonucleotides. The nanohydrogels’ stability performance could further be reproduced by preliminary in vivo experiments using intravital confocal videography: While the nanohydrogel particles alone caused larger aggregates in the bloodstream, siRNA loaded particles continued circulating without any larger aggregate visible. To that respect, both nanohydrogel carrier and siRNA cargo may have only limited stability on their own but their complexes show high serum stability at an ideal weigth-toweight ratio with no free siRNA and an almost neutral zeta potential. At this state siRNA loaded nanohydrogel particles may provide perfect properties for various siRNA delivery applications in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

Syntheses and analytical data of P(MEO3MA)-b-P(PFPMA) block copolymers and cationic nanohydrogel particles, agarose gel electrophresis experiments in BSA and serum solution, dynamic light scattering data of siRNA loaded cationic nanohydrogel particles in PBS and BSA solution, dynamic light scattering data of siRNA alone in human blood serum, and intravital confocal videography movies. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +49-6131-3924778. Tel: +49-6131-3920361. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Fonds der Chemischen Industrie (FCI), the Max Planck Graduate Center with the Johannes GutenbergUniversität Mainz (MPGC) and the DFG Sonderforschungsbereich SFB 1066 for financial support. Moreover, Mark Helm and his group (Institute of Pharmacy and Biology, Johannes Gutenberg-University Mainz) are gratefully acknowledged for providing siRNA.

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REFERENCES

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