Cylindrical Brush Polymers with Polysarcosine Side Chains: A Novel

Article pubs.acs.org/Macromolecules

Cylindrical Brush Polymers with Polysarcosine Side Chains: A Novel Biocompatible Carrier for Biomedical Applications Christian Hörtz,† Alexander Birke,‡ Leonard Kaps,§ Sandra Decker,† Eva Wac̈ htersbach,† Karl Fischer,† Detlef Schuppan,§ Matthias Barz,‡ and Manfred Schmidt*,† †

Institute for Physical Chemistry, Johannes Gutenberg University, Welder Weg 11, D-55099 Mainz, Germany Institute for Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, D-55099 Mainz, Germany § Institute of Translational Immunology and Research Center for Immunotherapy, University Medical Center, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, D-55131 Mainz, Germany ‡

S Supporting Information *

ABSTRACT: Cylindrical brush polymers constitute promising polymeric drug delivery systems (nanoDDS). Because of the densely grafted side chains such structures may intrinsically exhibit little protein adsorption (“stealth” effect) while providing a large number of functional groups accessible for bioconjugation reactions. Polysarcosine (PSar) is a highly water-soluble, nonionic and nonimmunogenic polypeptoid based on the endogenous amino acid sarcosine (N-methyl glycine). Here we report on the synthesis, characterization and biocompatibility of cylindrical brush polymers with either polysarcosine side chains or poly-L-lysine-b-polysarcosine side chains. The latter leads to block copolypept(o)id based core− shell cylindrical brushes with a cationic poly-L-lysine (PLL) core and a neutral polysarcosine corona. The cylindrical brush polymers were prepared by ring-opening polymerization of the respective N-carboxyanhydrides (NCA) from a macroinitiator chain. Preliminary experiments on complex formation with siRNA demonstrate that a core−shell cylindrical brush polymer may complex on average up to 270 RNA molecules amounting to a high loading efficiency of N+/P− = 1.1. No bridging between cylindrical brushes leading to larger aggregates is observed. In vitro studies on the silencing of the expression of ApoB100, which is abundantly expressed in AML-12 hepatocytes, induced by siRNA-cylindrical core−shell brush complexes showed high efficiency, leading to a knock-down efficiency of ApoB100 mRNA of 70%.

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INTRODUCTION

cross-presentation in DEC-positive dendritic cells, only, causing largely enhanced T-cell proliferation. These results motivated us to develop cylindrical brushes that are suitable carriers for siRNA or in general anionically charged immunogens like oligonucleotides, exhibit low cytotoxicity, and eventually would allow for specific cell targeting. Whereas siRNA delivery requires cationic charges in order to complex siRNA, low cytotoxicity and specific cell uptake cannot be easily achieved with cationically charged polymers. In order to resolve this contradiction cylindrical brushes with a cationic core and a biologically inert shell may constitute a promising anisotropic drug delivery system in conceptual analogy to isotropic core−shell micelles.12,13 As compared to spherical micelles the cylindrical brushes were observed to have longer “in vivo” circulation times14 and may exhibit a more pronounced “stealth” effect originating from a stronger stretching of the corona chains at constant grafting

Cylindrical brushes are composed of a normally flexible main chain polymer densely decorated with flexible side chains. Because of the strong steric repulsion of the side chains the main chain is forced into an elongated conformation leading to wormlike structures with a directional persistence similar to DNA.1−6 Biomedical applications of cylindrical brushes are rare despite their anisotropic wormlike shape that could be advantageous for both, cell uptake and circulation time in the blood.7,8 So far, only doxorubicin (DOX) and camptothecin (CT) conjugates with short “bottle-brush” polymers were reported to kill human cancer cells in vitro upon photocleavage of DOX and CT,9,10 but very little is known about cell uptake, circulation time, and biodistribution. Recently, cylindrical brush polymers with poly oxazoline side chains carrying an azide group on each of the many side chains were shown to effectively target dendritic cells when decorated with aDEC205 antibodies.11 Here, additional conjugation of the ovalbumine-derived antigen (SIINFEKL) resulted in antigen © 2015 American Chemical Society

Received: December 11, 2014 Revised: March 4, 2015 Published: March 26, 2015 2074

DOI: 10.1021/ma502497x Macromolecules 2015, 48, 2074−2086

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Macromolecules

polypeptide chains onto a polymer chain.38,39 On the basis of our own experience, grafting from a macroinitiator appeared to be most promising in order to achieve both, long main and long side chains. Poly[N-(6-aminohexyl)methacrylamide] was successfully applied as the macroinitiator for the preparation of cylindrical brushes with PLL side chains resulting in structures with a nondegradable main chain.35 In the present work, also a PLL macroinitiator chain is going to be utilized as a proof of concept for the preparation of an all-polypept(o)id cylindrical brush structure. Upon successful synthesis, the following questions are going to be addressed: (1) Does siRNA complexation lead to single brush polymer complexes or do interbrush aggregates form? (2) How many siRNA molecules can be loaded into the cylindrical brush core? (3) Is siRNA released from the cylindrical brush core when taken up by cells?

density in a 2-D particle geometry. For good biocompatibility such cylindrical brushes should consist of polypept(o)id side and main chains,15 since polypept(o)ids may be enzymatically degradable. First data also indicates the degradation of polypept(o)ids in the presence of reactive oxidant species (ROS) to occur already after a few hours.16 In the present work poly-L-lysine (PLL) side chains were chosen for the cationically charged core, shielded by a highly water-soluble nonionic polysarcosine (PSar) corona (see Chart 1). Chart 1. Sketch of a Cylindrical Core−Shell Brush: Main Chain (Black), Poly-L-lysine Core (Yellow), and Polysarcosine Corona Chains (Blue)

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EXPERIMENTAL PART

Synthesis. Poly[N-(6-aminohexyl)methacrylamide] (PAHMA). Poly[N-(6-aminohexyl)methacrylamide] (PAHMA) was synthesized according to the literature with minor modifications.35 For RAFTpolymerization benzyl-2-hydroxyethyl-trithiocarbonate40 was used as chain transfer agent and recrystallized azo-bis(isobutyronitrile) (AIBN) as initiator. In a typical polymerization 2.5 g (8.8 mmol) of N-boc-N′-methacryloyl-1,6-diaminohexane, 3.1 mg (1.25 × 10−2 mmol) of benzyl-2-hydroxyethyl trithiocarbonate and 0.3 mg (1.85 × 10−3 mmol) of AIBN were dissolved in 7.5 mL anisole within a Schlenk flask. Oxygen was removed by four freeze−pump−thaw cycles, and the polymerization was performed at 90 °C for 3 days. Subsequently, the product was precipitated in diethyl ether and redissolved in THF for a second precipitation. After drying in high vacuum for 24 h, 1.1 g (44% yield) protected macroinitiator was obtained. For BOC-deprotection 410 mg (1.5 mmol repeat units) of PAHMA was dissolved in 2 mL of methylene chloride and 2 mL trifluoroacetic acid and stirred overnight. The resulting gel was centrifuged and washed three times with benzene and methylene chloride. After lyophilization in water, 410 mg (1.4 mmol repeat units; 93% yield) of deprotected PAHMA·TFA macroinitiator were retrieved. 1 H NMR (300 MHz, D2O): δ [ppm] = 2.0−0.6 (13H; aliphatic main and side chain overlaid); 3.2−2.85 (4H; 3.06 −CH2−NH3+ and 2.96 CO−NH−CH2−), (see Supporting Information, Figure S1 for the spectrum). Sarcosine N-Carboxyanhydride. The synthesis of sarcosine-NCA was adapted from the literature24 and modified as follows: A total of 9.0 g (101 mmol) sarcosine, dried in vacuo for 1 h, was weighed into a predried, three-neck, round-bottom flask and suspended with 100 mL of abs. THF under dry argon. After heating to 60 °C, 10.5 g (35.4 mmol) of freshly recrystallized triphosgene was added and the suspension was mildly refluxed for 2 h, resulting in an almost clear solution. During the reaction a steady flow of dry argon was passed through the reaction flask and subsequently led into aqueous NaOH solution in order to remove gaseous HCl and phosgene. After evaporation of the solvent under reduced pressure, yellowish oil was obtained as crude reaction product. The oil was heated to 70 °C and dried under high vacuum for 15 min yielding an amorphous solid, which was dissolved in THF and precipitated in abs. hexane. After filtration the precipitate was dried in high vacuum for 2 h and sublimated at 90 °C under reduced pressure (90%. A minor amount of linear polysarcosine was formed as a side product due to traces of impurities in the macroinitiator, which in case of PAHMA is easily removed by ultrafiltration (see Figure 1). Assuming that every PAHMA repeat unit has started a side chain the theoretical degree of polymerization of the polysarcosine side chain is Pns = 50. Although it has been demonstrated that a close to 100% initiating efficiency may be obtained by grafting NCA from a solid support45,46 grafting from a linear chain macroinitiator most probably leads to a lower initiating efficiency of approximately 50% resulting in a true side chain Pns = 100. Since there is no easy way to cleave off the polysarcosine side chains without side chain degradation, the initiator efficiency remains unknown. Therefore, in the following only the theoretical side chain degree of polymerization is discussed. Static light scattering (Figure 2a) yields Mw = 5.8 × 106 g/mol, Rg = 64 nm, and A2 = 2.1 × 10−5 mol cm3/g2, and angular and concentration dependent DLS (Figure 2b) leads to Rh = 40.4 nm. The measured molar mass is

Figure 3. AFM height micrographs of the (a) PSarPAHMA brush spincast from methanolic solution and (b) PSarPLL brush spin-cast from aqueous solution. 2079


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nm, qualitatively agree with those previously determined on other cylindrical brush polymers with similar side chain length1−6 and confirm the large directional persistence of the structures. The somewhat larger chain stiffness obtained for the PLL brush assuming a helical main chain conformation could well result from the smaller distance between neighboring side chains. The smaller value, lk = 40 nm, calculated for the PLL brush assuming a coiled main chain conformation, appears to be unrealistically small in comparison to published lk values of cylindrical brush polymers with similar side chain length,1−6 thus corroborating the CD-data that the PLL main chain may adopt a helical conformation. It should be mentioned that in the above analysis excluded volume effects are ignored which, however, are known to be negligible as long as Lw/lk < 10.49 This condition is fulfilled except for the coiled PLL main chain calculation where Lw/lk = 25. Considering excluded volume would even further reduce the Kuhn length rendering a coiled conformation of the PLL main chain even less likely. Since each side chain end carries a secondary amine (Nterminus) the cylindrical brushes are cationically charged. However, as shown below the cationic charge of the cylindrical brush polymers is not high enough in order to form complexes with DNA at physiological conditions. Therefore, the next goal was to prepare core−shell cylindrical brush polymers50,51 with a cationic core comprising lysine repeat units and a neutral corona of polysarcosine which may be utilized to complex, shield and finally deliver small interfering ribonucleic acid (siRNA) into the cytosol of cells. The block copolypept(o)id brushes were synthesized in a “one pot” reaction, starting with the TFA protected polylysine block and sequential addition of sarcosine-NCA according to the 2 procedures shown in Scheme 2. The protonated amine reaction proceeds slower than the deprotonated amine polymerization and produces more linear impurities. Isolation of the PLL brushes was avoided because often, but not always Z-protected as well as TFA-protected PLL brushes were difficult to dissolve after precipitation and drying/ lyophilization. Some of the samples dissolved molecularly in DMF and HFIP as reported before,35 some formed aggregates and some did not dissolve at all. The origin of this problem could not be clarified. Even worse, no hypothesis can be offered to explain this observation, since partial deprotection of the lysine would lead to cylindrical brushes with hyperbranched side chains, which should be soluble as well. Chemical crosslinking reactions are unlikely to occur. Possibly, some β-sheet formation may be responsible for aggregation/insolubility, but could not be detected by CD measurements.

by light scattering (see Supporting Information, Figure S11, and Table 1). For this sample a small shoulder toward smaller molar masses was observed by GPC (see Supporting Information, Figure S12) the origin of which is unclear. AFM shows polydisperse cylindrical brush structures as expected from GPC analysis but also reveals smaller “nonbrush” structures that may originate from structures seen in the GPC-trace as the smaller molar mass shoulder. The light scattering and GPC results are summarized in Table 1. Again, the experimentally determined molar mass Mw = 12.2 × 106 g/ mol is somewhat larger than the calculated molar mass Mw = 11 × 106 g/mol assuming 92% sarcosine conversion (according to GPC), which results into 52 sarcosine units per main chain initiator, i.e., Pns = 52, and Pw = 2812 of the PLL macroinitiator. CD measurements in water indicate the PLL main chains of the cylindrical brushes to adopt a helical conformation although the CD signal is noisy due to the low mass fraction of the main chain (see Supporting Information, Figure S13). A quantitative comparison of the dimensions of the two cylindrical brushes is difficult, because the contour length needs to be known. For vinylic main chains like PAHMA the length of one repeat unit, l, is calculated to l = 0.25 nm, whereas for a peptide repeat unit l = 0.363 nm results, if the polypeptide adopts a coiled conformation. For an α-helix the pitch per repeat unit (relevant for the contour length of the helix) has been determined to h = 0.15 nm. Thus, the contour length of the PLL main chain strongly depends on the main chain conformation. Application of the wormlike chain model47 to the light scattering data and accounting for polydispersity in terms of a Schulz−Zimm chain length distribution48 yields the Kuhn statistical segment lengths as given in Table 2. The larger values, lk = 80 nm and lk = 120 Table 2. Kuhn Statistical Segment Length, lk, and Hydrodynamically Effective Diameter, d, Derived by Application of the Wormlike Chain Model to the Measured Rg and Rh Values of the Cylindrical Brush Polymers with Polysarcosine Side Chains brush main chain

Lwa/nm

lk/nm

d/nm

PAHMA·TFA PLL·HBr (α-helix) PLL·HBr (coiled)

310 420 1012

80 120 40

20 35 25

a

Calculated from the respective degrees of polymerization utilizing the length per repeat unit, l = 0.25 nm (PAHMA), l = 0.15 nm (PLLhelix) and l = 0.363 nm (PLL-coil).

Scheme 2. Different Pathways To Obtain Core−Shell Cylindrical Brushes Consisting of Polysarcosine and Polylysine

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a

GPC

GPC+AAA

CD

brush

WSar /%

XSar /%

PLys

PSar

ΓSar,Lys

PLL-b-PSar copolymer 1 PLL-b-PSar copolymer 2

76 58

85 71

17 ± 2 31 ± 3

96 ± 10 77 ± 8

5.6 2.5

a

b

c

WSara/% 78 52

Sarcosine weight fraction. bMolar sarcosine fraction. cMolar ratio sarcosine/lysine.

Also, Z-protected lysine could not be utilized because deprotection of the Z-group caused severe degradation of the PSar block as demonstrated by the following experiment: To a soluble precursor brush prepared by copolymerization of Zlysine−NCA and sarcosine−NCA (molar ratio 1:1) was added sarcosine−NCA as the second block in order to yield soluble (PLL(Z)-stat-PSar)-b-PSar brushes (see Supporting Information, Figure S14 for an AFM image). However, deprotection of the PLL(Z) with HBr caused severe degradation of the PSar block (see Supporting Information, Figure S5) in agreement with recent literature.28 In order to avoid the problems above, TFA protected lysine− NCA was polymerized from the protonated macroinitiator PAHMA·TFA as the first block and after full conversion a 6fold excess of sarcosine−NCA was directly added to the PLL(TFA) brush solution, i.e. the small amount of unavoidable linear polylysine homopolymers was not removed. Accordingly, the resulting core−shell cylindrical brush 1 was contaminated with linear PLL(TFA)−PSar block copolymers (see Supporting Information for GPC-traces, Figure S15) that could be largely removed by Amicon 50K centrifugation filters after deprotection (see Supporting Information, Figure S15). Linear impurities could be reduced by grafting from a deprotonated PAHMA macroinitiator that resulted in the PLLb-PSar copolymer brush 2 (see Supporting Information for GPC-traces, Figure S16). Both routes led to soluble core−shell cylindrical brushes with polylysine-b-polysarcosine side chains that according to DLS might be slightly aggregated after deprotection with hydrazine depending on subtle drying effects and dissolution time. For the deprotected and lyophilized PLL-b-PSar copolymer brush 1 Rh = 85 nm was observed in PBS after a dissolution time of 2 days which decreased to Rh = 70 nm after standing for 2 weeks. A more detailed DLS investigation was performed on the amine-initiated PLL−PSar brush 2. Measurements on the reaction solution determined the size of the protected PLL(TFA) precursor brush to Rh = 55 nm which increased to Rh = 59 nm after addition of the PSar block, i.e. for PLL(TFA)−PSar brush 2. After lyophilization the protected PLL(TFA)−PSar brush 2 dissolved well in DMF without aggregate formation (Rh = 62 nm) which remained constant after deprotection of the Lys(TFA) (Rh = 60.5 nm, measured on the dialyzed reaction solution against water and PBS added subsequently). However, after lyophilization the size of the deprotected PLL-b-PSar copolymer brush 2 increased to Rh = 75 nm in PBS even after 2 weeks dissolution time. Static light scattering was measured on the nondried PLL-bPSar copolymer brush 2 yielding Mw = 12.5 × 106 g/mol. Rg = 81 nm and A2 = −1.5 × 10−4 mol cm3/g2 (see Supporting Information, Figure S17). The slightly negative virial coefficient confirms the poor solvent quality of PBS buffer. PLL-b-PSar

copolymer brush 1 could not be measured by SLS due to the lack of sufficient sample. The exact composition, i.e., the ratio of lysine/sarcosine, of both dialyzed PLL-b-PSar copolymer brushes was determined by amino acid analysis (AAA) performed by Genaxxon Bioscience, Ulm, Germany, by conducting a hydrolytic degradation of the polypeptide side chains and subsequent quantification by ion chromatography (see Supporting Information, Figure S18, for details). Next, the theoretical PLL block length of the precursor brush was determined by the ratio the GPC elution peak areas of the precursor brush with PLL side chains and the linear PLL chain fraction, i.e. the fraction of the added lysine which was incorporated into the cylindrical brush was determined. This procedure yields reliable results with an experimental uncertainty of ±10% as long as brush, linear PLL and solvent peaks are well separated. (See Figures S15a, S16a.) The results are summarized in Table 3. The AAA results were confirmed within experimental error by CD measurements of the copolymer brushes in water utilizing a calibration curve obtained from linear PLL solutions of known concentrations (see Supporting Information, Figure S19). As was shown earlier the CD spectra of linear PLL and cylindrical brushes in water, i.e., when PLL is coiled, are similar.35 The compositions of the two block copolymer brushes differ somewhat, i.e., for PLL-b-PSar copolymer brush 1 17 lysine and 96 sarcosine per main chain initiator group was obtained and PLL-b-PSar copolymer brush 2 exhibits 31 lysine and 77 sarcosine units per main chain initiator. The AFM pictures shown in Figure 4 confirm the wormlike nature of both core− shell brushes. Complex Formation with DNA and siRNA. To a filtered solution of cylindrical brushes with polysarcosine side chains in 5 mmol phosphate buffer a filtered solution of pUC DNA was added and the size of the resulting complexes was monitored by DLS. As evident from the data shown in Figure 5 large complexes well above the size of either brush (Rh = 45 nm) or

Figure 4. AFM micrographs (height mode) taken from spin-cast aqueous solution onto Mica: (a) PLL-b-PSar copolymer brush 1 and (b) PLL-b-PSar copolymer brush 2. 2081


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Figure 7. (a) Relative increase in molar mass, Mc/Mb (Mc the complex molar mass, Mb the brush molar mass; left scale, black filled circles), and hydrodynamic radius, Rh (right scale, blue full sqares), as a function of charge stoichiometry P−/N+ for complexes formed by PLL-b-PSar copolymer brush 2 and siRNA. The open circles represent the expected relative molar mass for single cylindrical brush complexes, i.e., no bridging. (b) PAGE of the corresponding solutions showing the appearance of free siRNA: P−/N+ = 0.05; lane 2, P−/N+ = 0.21; lane 3, P−/N+ = 0.38; lane 4, P−/N+ = 0.58.

Figure 5. Correlation functions recorded at a scattering angle 64° of PSarPAHMA cylindrical brushes with pUC19 DNA. Key: red, PSarPAHMA in 150 mM PBS, Rh = 45.9 nm; dark blue, PSarPAHMA/DNA complexes (P−/N+ = 0.3) in 150 mM PBS, Rh = 45.2 nm; light blue, PSarPAHMA in 5 mM PBS, Rh = 44.5 nm; black, PSarPAHMA/DNA complexes (P−/N+ = 0.15) in 5 mM PBS, Rh = 160 nm.

pUC19 DNA (Rh = 43 nm) are being formed as soon as DNA is added. However, in physiological PBS buffer no complexes could be detected. Obviously, the cationic charge density originating from the secondary amines at the each of polysarcosine side chain ends in the cylindrical brushes is not high enough to form stable complexes at high salt. This result is advantageous for the goal of the present work aiming to achieve single brush complexes with small siRNA and core−shell cylindrical brushes with a cationic core as shown in detail below. The corresponding experiment with the PLL-b-PSar core− shell brushes was not repeated with high molar mass DNA but rather with small double-stranded siRNA with a total of 46 bases (23 on each strand). Again, the RNA solution was titrated into the light scattering cuvette containing the filtered core− shell brush solution. The results for both core−shell brushes are shown in Figures 6 and 7 where the relative masses (left scale) as well as the hydrodynamic radii (right scale) of the complex

solutions are plotted against the charge ratio P−/N+52 of the mixture. For PLL-b-PSar copolymer brush 1, the relative molar mass, i.e., the complex molar mass Mc divided by the brush molar mass Mb, increases linearly with P−/N+ until it remains constant at P−/N+ > 0.9. The concomitantly measured hydrodynamic radii do not change, thus indicating the formation of single brush complexes. Obviously, the siRNA molecules are complexed by the cationic core of the brush and are perfectly shielded by the sarcosine corona, thus prohibiting interbrush bridging. At P−/N+ > 0.9, the relative molar mass remains constant; i.e., the added RNA is not complexed anymore but merely dilutes the cylindrical brush solution. At this point gel electrophoresis detects free RNA in solution (see Figure 6b). Note that the contribution of free siRNA to the total scattering intensity is negligible. The maximum siRNA loading of the brush reached at P−/N+ ≈ 0.9 corresponds on average to 270 double-stranded siRNA molecules complexed by one cylindrical brush polymer estimated from the theoretical molar mass Mn of the PLL-b-PSar copolymer brush 1 utilizing the side chain composition determined by AAA/GPC and Mw/ Mn of the PAHMA macroinitiator. In contrast the PLL-b-PSar coplymer brush 2 with 31 lysine and 77 sarcosine units per main chain repeat unit does not yield single brush complexes as evidenced by the increase of the hydrodynamic radii for P−/N+ > 0.4 and the increasing deviation of the experimentally determined molar masses to the theoretically expected ones. Obviously the polysarcosine corona is not long enough to effectively prohibit “inter cylindrical brush bridging” by siRNA, although the estimated (unperturbed) end-to-end distance, ⟨R2⟩1/2, of the copolymer brush 1 (⟨R2⟩1/2 ≈ 9.1 nm) is only 10% larger than for copolymer brush 2 (⟨R2⟩1/2 ≈ 8.2 nm). These values ought to be compared to the length of the siRNA double strand, LRNA = 7.8 nm. Possibly, two copolymer brushes can penetrate each other to some extend indicating a soft repulsion potential of the outer polysarcosine corona. Whether or not the interbrush bridging observed for the copolymer brush 2 is indeed caused by the small difference in corona thickness remains to be investigated.

Figure 6. (a) Relative increase in molar mass of the complexes, Mc/Mb (Mc the complex molar mass, Mb the brush molar mass; left scale, black filled circles), and hydrodynamic radius, Rh (right scale, blue filled squares), as a function of charge stoichiometry P−/N+ for complexes formed by PLL-b-PSar copolymer brush 1 and siRNA. (b) PAGE of the corresponding solutions showing the appearance of free siRNA: lane 1, P−/N+ = 0.31; lane 2, P−/N+ = 0.62; lane 3, P−/N+ = 0.93; lane 4, P−/N+ = 1.24. 2082


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Figure 8. Autocorrelation function g1(t) recorded from PSarPAHMA (a) and PLL-b-PSar brush 1 (b) in human serum (○ circles). Blue line: Force fit with eq 1. Red line: Fit with eq 2. Scattering angle 30°, brush concentration cb = 0.05 g/L, serum concentration cs = 30 g/L, and T = 30 °C. Note the systematic deviation of the residues around 1 ms in part b.

Biocompatibility. Polymeric nanocarriers for biomedical applications ideally should not interact with serum proteins. Such an interaction could interfere with cell targeting because some serum proteins such as complement factors may redirect the polymer to be taken up by certain cell types. Even worse, bridging of polymers into much larger structures (i.e., >500 nm) could result, which might affect not only in vivo distributions of polymers but also in vitro cell uptake results. The identification of “hard” and “soft” protein coronae around nanoparticles is a tedious and complex procedure and beyond the scope of the present work.53,54 Rather, the formation of larger aggregates in human serum is investigated for cylindrical brushes with polysarcosine side chains. As described recently in detail elsewhere,44 DLS represents a powerful method to sensitively monitor the formation of larger aggregates in highly concentrated human blood serum. In short, the intensity-time correlation function of polymer, g1(t)polymer, and serum, g1(t)serum, are measured separately. The correlation function of the serum-polymer mixture, g1(t)mix is then subjected to a force fit by the sum of the correlation functions of each of the components g1(t )mix = ag1(t )polymer + bg1(t )serum

shown below, serum proteins do adsorb onto cylindrical brushes, thus prohibiting unspecific cell uptake. Since PLL-bPSar copolymer brush 2 has an even shorter polysarcosine block and tends to form intercylindrical brush bridging in the presence of siRNA, the polymer was not investigated in serum. Cytotoxicity. Besides forming no larger structures in serum a polymeric nanoDDS should also not be toxic for cells up to concentrations well above those that are reached in vitro as well as in vivo experiments. Toxicity of the PLL-b-PSar brush 1 was tested with AML-12 hepatocytes that were utilized for siRNA induced knock-down experiments described below. The results of Figure 9 reveal an IC50 value for cell viability of 300 mg/L.

(1)

If the fit with a and b being the only fit parameters does not show systematic residues, the size distribution of each of the components did not change in size upon mixing. The formation of larger aggregates would require an additional exponential term, g1(t)agg, to the fit function (eq 2), the decay rate of which would yield the size of the newly formed aggregates g1(t )mix = ag1(t )polymer + bg1(t )serum + cg1(t )agg

Figure 9. AML-12 cell viability as a function of PLL-b-PSar copolymer brush 1 concentration normalized to PBS (n = 3). Incubation time: 48 h.

This value is lower than that obtained for cylindrical brushes with nonionic polyoxazoline side chains11 but is much higher than the concentrations applied in the present work. It is probable that the cationic charges of the polysarcosine side chain ends are responsible for the slightly increased cytotoxicity. Cell Uptake and In Vitro Knockdown Experiments. To demonstrate the potential application of the core−shell cylindrical brush as siRNA carrier for biomedical applications,55,56 preliminary in vitro knockdown experiments were conducted on AML-12 hepatocytes targeting the siRNA that encodes the highly expressed hepatocyte protein ApoB100. The complex composition was fixed to P−/N+ = 0.55. As expected from the data shown in Figure 6, no free RNA could be detected by PAGE. The results obtained with full and reduced serum concentration are shown in Figure 10.

(2)

As shown in Figure 8a, no formation of larger aggregates is observed upon mixing the cylindrical brush polymers with polysarcosine side chains and human serum whereas the PLL-bPSar copolymer brush 1 does induce aggregates with Rh = 230 nm (Figure 8b). Given the small intensity amplitude of 15%, only, the weight fraction of brushes involved in aggregate formation is estimated to amount to less than 5%. Ignoring this small fraction of protein induced aggregates (originating from interbrush bridging) the results do not suggest that the “stealth” effect of the major fraction of nonaggregated cylindrical brush polymers is sufficiently strong to effectively repel proteins, since a monolayer of proteins adsorbed onto cylindrical brushes is not detectable by DLS in concentrated serum solution. As 2083


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qualitatively correlates with the observed mRNA knock-down efficiency. The cationic charge of the cylindrical brushes originating from the N-termini of the polypept(o)id side chains seems to be sufficient to induce unspecific cell uptake, coupled with a significant in vitro release of siRNA out of the complexes and an efficient mRNA knock-down. In high protein media the cationic charges are most probably reduced by protein adsorption, thus reducing cell uptake and mRNA knockdown, accordingly. Although not favorable for the goal of the present work the lack of cell uptake caused by protein adsorption is mandatory for future cell targeting where unspecific cell uptake must be minimized in order to optimize cell selectivity. Chemical conversion of the cationic N-termini into uncharged functional groups like azides would not only further reduce unspecific cell uptake, but additionally allow for subsequent conjugation reactions with substrates that selectively bind to certain cell receptors.

Figure 10. Expression of normalized ApoB100 mRNA in AML-12 hepatocytes in low and high protein media. CB1 ≙ PLL-b-PSar copolymer brush 1; + control: maximum knock down using siRNA to ApoB100 (100 nM) with INTERFERin transfection agent, ***≙ p < 0.0001 versus PBS-control and negative (−) control siRNA/CB1 100 nM (n = 3).

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CONCLUSION Polypept(o)id based core−shell cylindrical brush polymers with a cationic poly-L-lysine core and a polysarcosine corona have been successfully prepared and single brush complexes comprising on average up to 270 double-stranded siRNA molecules were obtained. The cylindrical brush complexes were observed to knock-down ApoB100 mRNA in AML-12 hepatocytes when the transfection was conducted in low protein medium whereas the complexes were hardly taken up by AML-12 cells in high protein medium, thus causing no mRNA knock-down. The present results do not only highlight the importance of serum protein adsorption onto polymeric and nanoscopic carriers for cell uptake but in addition draw attention to the role of protein adsorption for cell targeting experiments. For future selective cell targeting, unspecific cell uptake needs to be minimized while specific uptake should be achieved via antibodies or antibody fragments conjugated to the nanoDDS. In this respect, protein adsorption may be considered to have a positive effect unless the adsorbed proteins do not readdress the nanocarriers to other (unwanted) cell populations. Since the latter risk may quite high a better strategy might be to chemically convert the cationic N-termini into uncharged functional groups like azides that are well suitable for subsequent conjugation reactions with substrates specific to certain cell receptors.

The highest knock-down efficiency was observed for the highest siRNA−cylindrical brush complex concentration in low protein medium. This is in contrast to the results obtained in high protein media where no knock-down of ApoB100 was detectable. Since it is well-known that siRNA and DNA may be released from polyelectrolyte complexes by a large excess of albumin,57,58 PAGE was conducted on siRNA−brush complexes incubated with increasing concentrations of albumin. As shown in the Supporting Information, Figure S20, albumin concentrations as high as 40% (w/w) could not release siRNA out of the complex. In order to further trace the origin of this discrepancy, cell uptake experiments were conducted with AlexaFluor 488 2,3,5,6-tetrafluorophenyl ester labeled PLL-bPSar copolymer brushes 1 into AML-12 cells in low and high protein media, respectively. The results are shown in Figure 11 and FACS data are shown in the Supporting Information (Figure S21). As shown in Figure 11b in low protein media cell uptake increased from 50% for the lowest polymer concentration (corresponding to 25 nM siRNA) to 70% for the highest cylindrical copolymer brush concentration (corresponding to 75 nM siRNA). In contrast, in high protein media cell uptake was reduced to less than 10% (Figure 11a). Thus, cell uptake

Figure 11. In vitro cellular uptake of bare polymer brushes in AML-12 liver cells (n = 3): Double positive cells (live+, particle+) incubated with 1, 2, and 3 mg/L AlexaFluor 488 labeled PLL-b-PSar brush 1 for (a) 0.5, 1, 6, and 12 h in high protein media and (b) for 24 h in low protein media. * ≙ p < 0.05. The concentrations 1, 2, and 3 mg/L correspond to 25, 50 and 75 nM siRNA. 2084


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ASSOCIATED CONTENT

S Supporting Information *

NMR and CD spectra, Zimm Plots, GPC elugrams, FACS-data, Gelelectrophoresis data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the Deutsche Forschungsgemeinschaft (SFB1066 Nanodimensional Polymer Therapeutics for Tumor Immune Therapy, Projects A6, B3, and Q1) is gratefully acknowledged by M.B., D.S., and M.S. We are indebted to Alexander Brose for preparing cell cultures and for conducting FACS experiments.

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