pH-Triggered Aggregate Shape of Different Generations Lysine

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pH-Triggered Aggregate Shape of Different Generations LysineDendronized Maleimide Copolymers with Maltose Shell S. Boye,† D. Appelhans,*,† V. Boyko,† S. Zschoche,† H. Komber,† P. Friedel,† P. Formanek,† A. Janke,† B. I. Voit,†,‡ and A. Lederer*,†,‡ †

Leibniz-Institut für Polymerforschung Dresden, Hohe Str. 6, 01109 Dresden, Germany Technische Universität Dresden, D-01062 Dresden, Germany

‡

S Supporting Information *

ABSTRACT: Glycopolymers are promising materials in the field of biomedical applications and in the fabrication of supramolecular structures with specific functions. For tunable design of supramolecular structures, glycopolymer architectures with specific properties (e.g., controlled selfassembly) are needed. Using the concept of dendronized polymers, a series of H-bond active giant glycomacromolecules with maleimide backbone and lysine dendrons of different generations were synthesized. They possess different macromolecular size and functionality along the backbone. Their peripheral maltose units lead to solubility under physiological conditions and controlled aggregation behavior. The aggregation behavior was investigated depending on generation number, pH value, and concentration. A portfolio of complementary analytical tools give an insight into the influence of the different parameters in shaping a rod-, coil-, and wormlike molecular structure and their controlled aggregate formation. MD simulation helped us to understand the complex aggregation behavior of the linear polymer chain without dendritic units.

1. INTRODUCTION Glycopolymer architectures1 are of special interest in the field of polymeric therapeutics,1e,2 diagnostics,3 and glycomimetics,1c,4 but also in the fabrication of supramolecular assemblies with triggered functions.5 In particular, dendritic glycostructures have gained great attention in the molecular recognition for tailoring biological processes, such as hampering the biological activity of viruses and bacteria or increasing the sensitivity against lectins.6,7 Further, dendritic glycostructures have been successfully used as nanosized carrier systems8−10 and as artificial tubulating proteins.9 Besides their successful use in the recognition of individual or assembled sugar units, the sugar decoration of dendritic structures increases their solubility under physiological conditions and enhances their biocompatibility under in vitro and in vivo conditions.10−12 Finally, one can state that glycopolymers are versatile materials with addressable key functions in biology, medicine, and pharmacy. Nevertheless, the design and fabrication of synthetic giant glycopolymers with molecular weight about 1000 kDa is so far a less considered research field and may reveal new possibilities in the formation of supramolecular assemblies with promising functions. Desirable key issues would be the existence of water solubility at room temperature and the presence of H-bonds active shell for undergoing tailored aggregation processes under defined environmental conditions. Motivated by these challenging aspects to realize giant macromolecules, we have successfully applied the concept of dendronized polymers (DenPols) to introduce a new family of sugar-decorated © XXXX American Chemical Society

DenPols (Scheme 1) here in this paper. Along with the perfectly branched dendrimers13 and hyperbranched polymers,14 DenPols15 also belong to the structural family of dendritic macromolecules. They can be described as hybrid macromolecules possessing a linear backbone decorated with dendrons. The dendronization of a linear polymer results in a high concentration of functional groups along the linear backbone. In contrast to dendrimers, where the dendrons are coupled to a core and the size and functionality of the molecules are limited, the fabrication of dendronized linear structures enable us to realize diverse nanoobjects that vary in their length, branch point frequency, and dendron generation. Therefore, the goal of our study was to obtain sugar-decorated DenPols with different molecular shapes such as rod-, coil-, and worm-like by introducing an increasing generation number of the dendrons (from G0 to G3). The tunable variation of the molecular shape of such giant glycopolymers should help us to understand the key functions responsible for the formation of defined supramolecular assemblies. Until now, in most cases, synthetic linear glycopolymers are present in the coil state.1a,d−f In some cases, one can induce their aggregation in spherical shapes with specific biofunctions by external stimuli (e.g., pH).16 Just recently, Schlüter17 reported on the largest synthetic structure based on DenPols. Received: September 21, 2012 Revised: October 29, 2012

A

dx.doi.org/10.1021/bm301489s | Biomacromolecules XXXX, XXX, XXX−XXX

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Scheme 1. Synthetic Route for MI Glycopolymers MI-G0-Mal−MI-G3-Mal

A remarkable use of sugar-decorated polymers in the field of biomedical applications, varying their backbone structure from linear to perfectly branched, is now available and reported. The knowledge about their pH- and temperature-triggered solution properties will further imply and strengthen their potential use in various application fields. In this work, we describe the solution and aggregation behavior of water-soluble maltosedecorated lysine-dendronized maleimide copolymers (Scheme 1). For future applications in the fabrication of bioactive nanoobjects, we will use the huge reservoir of H-bond active sugar units in DenPols.23,24 Few features are known concerning lysine-based dendrimers,24 highly branched25 or dendrigraft polymers,26 and their application for drug delivery as antimicrobial agents or as vectors in gene therapeutics.27 Recently, detection of bacteria was possible using dendronized

These polymers exist as worm-like structures due to the dense packing of dendrons along the linear chain, which leads to extended chain conformations. Generally, DenPols have essential application potential as nanoscopic building blocks for optoelectronics and biosciences.18 In this context, Fréchet and co-workers19 have tested their potential as drug delivery systems, and Schlüter and co-workers20 applied them as charged polymers for DNA complexation or for the immobilization of peroxidase via avidin−biotin system.21 The increased functionality within DenPol architectures is not only suitable, for example, to control the complexation and solubility in different environments, but it also leads to tunable aggregation phenomena by varying the temperature within the physiologically relevant values.22 B


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polylysine.28 In our work, the control over the organization principles of our DenPols in solution is intensively investigated to clarify to which extent the concentration, the generation number, the temperature, and, especially, the pH value influences their intermolecular interactions. The aggregation of natural polymers is a common effect and was comprehensively investigated. Usually, light scattering techniques lead to quite reliable information on their aggregation.29 Molecular and aggregate shapes could be easily evaluated by molar mass dependencies after sample separation. Size exclusion chromatography (SEC) of such complex structures, which additionally possess extremely dense branching and huge sizes, is not suitable due to different reasons.18 Additionally, SEC does not enable gentle separation of aggregates due to strong, destructive shear forces. A suitable alternative to SEC for multifunctional polymers, especially glycopolymers is the asymmetrical flow field flow fractionation (AF4) in combination with light scattering (LS). This is a powerful tool for size determination in a range from 5 kg/mol up to ultrahigh molecular weights, based on the different diffusion coefficients of the particles. The separation does not proceed in a stationary phase, where adsorption could take place as in the case of liquid chromatography, but in a thin channel through which the sample is transported by a laminar, axial flow.30 The mechanism of AF4 is based on the action of hydrostatic forces using additional vertical cross-flow and gives the possibility for gentle separation of aggregated samples and online detection by static light scattering. Additionally, fast equilibration of the separation system enables easy change of the eluent and the pH value, respectively. The AF4 technique has been shown suitable for the comprehensive analysis of (bio)macromolecules or proteins31 requiring special conditions. The aggregation behavior of natural polymers by extensive AF4 analysis was the subject of investigations by Nilsson et al.32 In our work we use the potential of this technique in combination with complementary microscopy and light scattering techniques for evaluation of the aggregation behavior of maltose-decorated lysine-dendronized maleimide copolymers (Scheme 1).

The NMR experiments were performed on a Bruker DRX 500 NMR spectrometer operating at 500.13 MHz for 1H and at 125.75 MHz for 13C. DMSO-d6 and D2O were used as solvents. For internal calibration the solvent peak of DMSO-d6 was used: δ (13C) = 39.6 ppm; δ (1H) = 2.50 ppm. Sodium 3-(trimethylsilyl)-3,3,2,2tetradeuteropropionate was added for internal calibration (δ (13C) = 0 ppm; δ (1H) = 0 ppm) in D2O. The signal assignment was done by both, 1D and 2D (1H−1H COSY, 1H−13C HMQC, and 1H−13C HMBC) NMR experiments using the standard pulse sequences provided by Bruker. 2.3. Size Exclusion Chromatography (SEC). SEC-MALLS (multi-angle laser light scattering) measurements were performed using PolarGel-M column (Polymer Laboratories, U.K.), Agilent isocratic pump series 1200 (Agilent Tech, U.S.A.), differential refractive index (RI) detector K2301 (Knauer, DE), and a MiniDAWN MALLS detector (Wyatt Technologies, U.S.A.). The eluent was N,Ndimethylacetamide (DMAc) with 3 g/L LiCl and a flow rate of 1.0 mL/min. The dn/dc of 0.09 mL/g was calculated from the measurements for all samples by assuming full mass recovery between injected and detected sample. The molar masses and gyration radii were calculated using the Astra 4.9 software (Wyatt Technologies, U.S.A.). For further interpretations, the z-average of the Rg calculated from the SEC-MALLS or AF4-MALLS measurements was used. 2.4. Asymmetric Flow Field Flow Fractionation (AF4). AF4 measurements were performed on an Eclipse 3 system (Wyatt Technology Europe, DE). The wide channel spacer of poly(tetrafluoroethylene) (PTFE) had a thickness of 350 μm, and the channel dimensions were 26.5 cm in length and from 2.1 to 0.6 cm in width. Membranes with a cut off of 10 kDa consisting of regenerated cellulose were used (Wyatt Technology Europe, DE). The Eclipse 3 system was connected to an Agilent Technologies 1200 series isocratic pump (Agilent, U.S.A.) equipped with vacuum degasser. The detection system consists of RI detector Dn 2010 (WGE Dr. Bures, Germany, λ = 620 nm, 25 °C) and MALLS detector (MiniDAWN, Wyatt Technology, U.S.A.). The channel flow rate, that is, detection flow rate, was maintained at 1.0 mL/min for all AF4 operations. Samples were injected during the focusing/relaxation step with 0.2 mL/min during 2 min. Focusing with 1.5 mL/min over 3 min and elution under linear gradient from 1.5 to 0 in 60 min or exponential cross-flow gradient with factor 1 from 1.5 to 0 mL/min during 15 min were performed. The injection volume was 100 μL. Interpretation of the results was performed using Astra 4.9, Astra 4.7, and Corona software package (Wyatt Technologies, U.S.A.). Deionized, UV treated, and ultrafiltrated by a Purelab Plus UV/UF equipment (USF Elga, DE) water was used as eluent. The water contains 0.02% (w/v) sodium azide to prevent bacteria growth. Adjustment of pH of NaNO3 and acetate buffer was performed with a pH probe using HCl and NaOH. 2.5. Atomic Force Microscopy (AFM). For the AFM measurements of MI-G3-Mal polymer solution with concentration of 0.01 mg/mL was prepared with ultrafiltrated water and was deposited onto a silicon wafer, which was cleaned using liquid CO2 (SnowJet). The deposition was performed by dip coating. The AFM measurements were performed with a Dimension ICON (Bruker-Nano, U.S.A.) in peak force tapping mode with sharp Si-tips (tip radius 15 14400 1980 4810 85900

53 53 87 90

Rg, nm

pH 11

Đ Mw, kg/mol Rh, nm Rg, nm Đ

pH 8.5

Mw, kg/mol Rh, nm Rg, nm pH 7

Đ Mw, kg/mol Rh, nm Rg, nm

pH 5

the polymerization degree of the linear MI-main chain MA with molar mass of 125000 g/mol and the assumption of full modification. The experimental confirmation of these values is hindered by the fact that after deprotection and modification with maltose the polymers get insoluble in organic solvents and, hence, the determination of the molar mass is possible only in water, as desired and predicted. However, due to the extremely high concentration of end groups along the polymer chain, we expected very strong interaction with column material during chromatographic separation. Additionally, the formed aggregates would be destroyed due to shear forces appearing during the liquid chromatographic process. In order to perform reliable separation of these polymers and their aggregates, AF4 as an alternative technique was applied. In the following, the parameters influencing the aggregation behavior of the maltose modified DenPols MI-G1-Mal−MI-G3-Mal are discussed. 3.4. Aggregation Behavior. The molar masses and molar mass distributions of different generations of DenPols determined by AF4-MALLS are listed in Table 3. The strong effect of the pH value on the molar masses and sizes is clearly visible. Furthermore, the pH influences the ratio of the radius of gyration and the hydrodynamic radius determined by AF4MALLS and by dynamic light scattering measurements in batch, respectively, as it will be discussed below. The interactions between the maltose-decorated dendronized macromolecules leading to aggregation could be investigated under different aspects. Here we consider the influence of the following factors: (i) the generation number, which leads not only to larger structures, but particularly to increase of the number of functional end groups; (ii) concentration of the solutions; (iii) pH value of the environment; and (iv) temperature. With respect to the last issue it can be stated, that no effect of temperature on the aggregates in the range of 25−90 °C was observed. The complexity of this investigation is getting straightforward after matrix representation of the first three factors influencing the aggregate size. Figure 5 shows the dependence of the hydrodynamic radius Rh on the concentration at different pH values for MI-G0-Mal to MI-G3-Mal. Generally, a slight increase of the Rh values in the range between 50 and 100 nm with increasing concentration was found. The pH variation leads to a size increase too, as it could be observed for MI-G3-Mal and MIG1-Mal at pH 3.5 even at very low concentrations. The hydrodynamic radii of the residual samples do not show such remarkable effect under the influence of pH. Therefore a pHdependent in situ determination of the Rh for the strongly aggregating MI-G3-Mal and the less aggregating MI-G2-Mal was carried out in comparison. The titration experiment using

Đ

Mw of starting polymer MA ∼125000 g/mol (poly(ethene-alt-maleic anhydride). a

Mw, kg/mol

∼830000 ∼1700000 ∼3250000 ∼6600000

Rh,nm

total Mw g/mol

832.85 1714.73 3276.28 6656.17

Rg, nm

Mw of repeating units g/mol

992 992 992 992

Đ

MI-G0-Mal MI-G1-Mal MI-G2-Mal MI-G3-Mal

DPa

pH 3.5

Table 2. Theoretical Molar Masses of the Different Generations

Mw, kg/mol

Table 3. Molar Masses, Molar Mass Distributions (Đ = Mw/Mn), Radius of Gyration, and Hydrodynamic Radius of the Different MI Copolymers MI-G0-Mal−MI-G3-Mal, Depending on pH

The theoretically expected molar masses of the maltose modified copolymers are listed in Table 2 as calculated from

Rh, nm

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Figure 5. Dependence of the Rh on the concentration measured at different pH for MI-G0-Mal (filled circle), MI-G1-Mal (open triangle), MI-G2Mal (open square), and MI-G3-Mal (filled square).

tion of the functionalities in all generations of DenPols. Hence, the aggregation number Mw/Mw,0 can be plotted against the generation number as shown in Figure 7. Whereas MI-G1-Mal

pH range between 2 and 9, is shown in Figure 6, confirming the strong dependence of the size of MI-G3-Mal on the pH value.

Figure 6. Dependence of the hydrodynamic radius from pH using titration at 1 mg/mL.

Figure 7. Aggregation number Mw/Mw,0 of the different generations DenPols MI-G0-Mal−MI-G3-Mal, depending on the pH at 1 mg/mL.

Whereas MI-G2-Mal remains nearly constant with hydrodynamic radius of approximately 70 nm, the size of MI-G3-Mal increases at acidic conditions passing through a maximum between pH 4 and 5 and strongly decreasing at alkaline pH. Quantification of the aggregation degree should be possible knowing the molar mass of the samples and normalizing it with the molar mass of the single macromolecule. The weight average molar masses were determined by AF4 coupled to MALLS. They are listed in Table 3. Information on single macromolecules can be achieved from the theoretically calculated molar masses given in Table 2. Furthermore, the structural characterization confirms nearly complete modifica-

and MI-G2-Mal show marginal aggregation, MI-G3-Mal is not available as a single macromolecule at any pH and reaches highest aggregation degree at pH 3.5. The fact that the molar mass of MI-G1-Mal does not increase, indicates the appearance of suppressed aggregation which contradicts the result from Figure 5. This behavior could be explained with nonstable aggregates build by the second generation DenPol, which are detected by static DLS measurements but are destroyed under slight shear during the AF4 measurements. In contrast, MI-G3Mal forms stable associates, which should be ascribed to the strongly increasing number of maltose end-groups with the H


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density can be expressed using the molar mass and the radius of the species. Here, the volume was determined by the hydrodynamic radius (eq 1).

generation number. Thus, H-bonds interactions are mainly responsible for the aggregate formation in MI-G3-Mal. This assumption is undoubtedly supported by the similar charge of the DenPols, as found by zeta-potential measurements (Figure SI2). Furthermore, this means that the driving parameters for intermolecular interaction are not Coloumb forces but Hbonds. The strength of the H-bonds is depending on the number of end-groups and is correspondingly stronger at higher generations. This type of interaction was found to be valid also between these polymers and cellulose nanocrystals.23 Truly surprising was the aggregation behavior of MI-G0-Mal, the generation with lowest number of end functionalities. Unexpectedly, MI-G0-Mal possesses stronger intermolecular interactions than MI-G1-Mal and MI-G2-Mal, with aggregation number dependence on pH similar to that of MI-G3-Mal. This fact should be ascribed to the helical conformation of this polymer as shown and discussed in a previous work.35 To confirm this, MD simulation of this structure with a polymerization degree up to 100 alternating monomer units was performed showing helical structure with densely packed maltose units at the surface (Figure 8).

ρapp =

Mw V (R )

(1)

In this work the z-average Rh was determined by DLS in batch. One should keep in mind, that for this calculation molar masses, which were determined by online measurements in combination with radii from batch DLS are used. In this way, only tendencies in the apparent density could be established as shown in Figure 9A. Slight density increase could be observed with increasing generation number from G1 to G3. MI-G0-Mal shows generally higher densities with maximum value at pH 3.5. In contrast, MI-G3-Mal shows highest density at pH 11. This fact is a first indication that the aggregation mechanism of MI-G0-Mal and MI-G3-Mal deviate from each other. Usually the shape of the aggregates is proven by the techniques used for determination of spatial parameters as the ratio Rg/Rh. As mentioned above, calculations based on online separation and LS detection and batch DLS detection are not fully correct, since average values of aggregates of MI-G0-Mal are compared with single macromolecules of MI-G1-Mal and MI-G2-Mal. Additionally, the LS detector used for the online Rg determination approached its limits, due to limited detection range of scattering angles, though certain trends of this ratio could be observed, as shown in Figure 9B. According to Burchard,41 Rg/Rh is in the range from approximately unity to 2 for linear or branched polymers in a good or θ solvent. Lower values correspond to dense globular particles as microgels. Higher values were found for rod-like natural macromolecules.41,42 The values of this ratio for our DenPols are in the expected range of linear coils or branched structures. Taking into account the very broad dispersity and complex architecture, it is not surprising, that this ratio approximates 2, which could be related to broadly distributed linear coil or to randomly branched macromolecule. However higher values were obtained for MI-G0-Mal corresponding to an anisotropic shape of the aggregates. The existence of rigid structures should be ascribed to the fact that the contour length of the chain is short in relation to the Kuhn segment length. Quantitative determination of the chain stiffness could be performed by conversion of Kratky in Cassasa-Holtzer Plot.43 However, here

Figure 8. MD simulation of (A) longitudinal view along the polymeric backbone and (B) view on the end of rod-like structure of MI-G0-Mal with 100 alternating monomeric units of maltosylated N-4-aminobutylsuccinimide and ethane.

One possibility to understand the type of aggregates is the determination of their apparent density, whereas usually the density decreases with increasing aggregation.40 The apparent

Figure 9. Apparent density (eq 1; A) and the ratio Rg/Rh (B) of the different generations DenPols MI-G0-Mal−MI-G3-Mal, depending on the pH at 1 mg/mL. I


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MI-G0-Mal corresponding to the helical structures and extended, thinner chains with length of more than 500 nm are visible in the image of MI-G1-Mal (Figure 10B). The third generation is significantly thicker (up to 12 nm) at a similar chain length. The sizes of the DenPols in solution and in solid state differ from each other, as shown already in previous studies.44 Correlation to the proportions in solution could be achieved by cryo-TEM measurements. Cryo-TEM investigations of the three different generations (Figure 11) show that G0 is significantly shorter (approximately 200 nm) than G3 (approximately 500 nm) which corresponds to disordering and extension of the macromolecules with dendronization as shown in Scheme 2. This effect was observed on a similar manner for dendronized poly(L-lysine).45 However, before reaching worm-like character, the polymer chain exists as an unordered coil in the first and second generation, which turns into a cylindrical, worm-like chain stabilized by the third-generation dendrons.15 One powerful feature of AF4-MALLS is the separation according to molar mass and simultaneous detection by static light scattering. This technique has two advantages: first, molar mass dependent determination of the radius of gyration can be performed in one measurement, because narrowly distributed fractions are detected. Secondly, the scaling law could be calculated as far as the separation leads to enough fractions of different size. This data can give another indication for the shape of the aggregates. Figure 12 shows Rg and molar mass values from fractions selected from the AF4 separation of MIG3-Mal. The separation of MI-G3-Mal led at high pH to broad range of fractions (Figure 12A), while at pH 3.5, the radii of gyration are strongly shifted to very high values. Hence, different separation methods were applied to cover different molar mass ranges (Figure 12B). Generally, applying exponential cross-flow gradient of broadly distributed samples, the lower molar mass species are well detected. Higher molar masses are separated during linear cross-flow gradient showing significantly denser species. At pH 7 and 11, nonlinear dependence of Rg can be observed with particular character of the aggregation mechanism. The scaling curve at pH 7 can be divided into two parts; at lower molar masses, up to approximately 6000 kg/mol, linear behavior with a slope of 0.6 can be calculated. Taking into account the scaling law

the limited light scattering data cannot allow for this determination. Nevertheless, we could estimate that in the case of MI-G0-Mal the regular and unperturbed main chain seems to possess long persistence length compared to its dendronized relatives. The reason for this surprising effect can be found in the tendency for G0 to form helical structures.35 Molecular dynamic simulations of MI-G0-Mal confirm this effect as shown in Figure 8. Percec and Schlüter showed that helical backbone can be induced and even stabilized by dendronization.45,46 However, in our case the dendronization is disturbing the helical order in the polymeric main chain leading first to coil-like structures until in the third generation sufficient density of dendrons is reached for worm-like behavior as shown in Scheme 2. Visualization of the single macromolecules was Scheme 2. Molecular Shape Transformation with Increasing Generation Number of DenPols

possible by AFM of MI-G0-Mal, MI-G1-Mal, and MI-G3-Mal performed after dip coating onto a substrate (Figure 10). The preparation of the solutions for AFM investigation is accompanied by different processes including drying of buffer salts, which led to the spherical islands. Spin coating led to preparation of single chains clear of salt; however, the complete chain length was not detectable due to strong shear forces leading to chain break. The averaged aspect ratio gained by cross-section of the AFM images of the different macromolecules in Figure 10D can be easily compared. Shorter macromolecules with high/ thickness ratio of approximately 1/8 nm were observed at

Figure 10. AFM images of dendronized MI-G0-Mal (A), MI-G1-Mal (B), and MI-G3-Mal (C) prepared by dip coating. Peak Force Error image, size 400 × 1000 nm and (D) averaged AFM cross-section profiles corresponding to the indicated, white lines in (A), (B), and (C). J


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Figure 11. Cryo-TEM images of single macromolecules of MI-G0-Mal (A), MI-G1-Mal (B), and MI-G3-Mal (C) at pH 3.5 and 0.1 mg/mL. Scale bar corresponds to 100 nm.

Figure 12. Dependence of the Rg on the molar mass extracted from AF4-MALLS measurements of MI-G3-Mal at pH 7 and 11 (A) and pH 3.5 (B) using exponential cross-flow gradient for the low molar masses and linear gradient for the high molar masses. The fits are performed with a correlation factor R2 > 0.95. The scattering data are fitted with a linear fit.

R g = K ·M ν

Nonlinear scaling behavior was observed for MI-G0-Mal at pH 3.5, as plotted in Figure 14. Already the single macromolecules represented by the low molar mass region possess rod-like shape with ν = 0.98. Fitting the curve to higher molar masses, the anisotropic shape remains, but again dense structures are indicated. Obviously at acidic conditions, MI-G0Mal exists as rigid rod with fractal dimension of 1 and forms aggregates with very high density. Cryo-TEM visualization of these aggregates show that the rigid rods are assembling radial to each other forming dense core with a star-like halo (Figure 13A). In contrast to the behavior at pH 3.5, at higher pH values, the aggregates do not possess pronounced rigidity. The dependence of Rg/molar mass is linear up to high molar mass values. At pH 7 it corresponds to a fractal dimension of 1.5, which is closer to coil-like structures in a good solvent. Strong contraction of the aggregates of MI-G0-Mal can be observed at pH 11, contrary to MI-G3-Mal. At these conditions, the scaling exponent corresponds to a fractal dimension of 4. This value of df is rather unusual for polymers, and it was already predicted for highly uniform shape of particles with a dense surface.47 The aggregates which were found by cryo-TEM do not show such uniform shape but a very small and dense core with coil-like arms. Summarizing, MI-G0-Mal forms dense star-like aggregates from helically structured rigid rods at pH 3.5, which are getting

(2)

the slope ν = 0.6 corresponds to coil-like structure in good solvent conditions.41 This could be the case for the single macromolecules in the lower molar mass range. With increasing molar mass, the logarithmic function of Rg/molar mass dependence starts to deviate from the linearity. The decreasing slope (ν = 0.24) at high molar masses corresponds to dense sphere shape. Similar behavior can be observed for pH 3.5 in Figure 12B. In contrast to lower pH values, at pH 11, significantly higher slope can be observed corresponding to rigid rod macromolecules with fractal dimensions approximating unity (df = 1/ν) in the low molar mass range. Again, deviation from the linearity occurs at higher molar masses. In this case, formation of sphere like objects out of rigid rods is rather unlike. Such scaling behavior has been already observed in the process of fibrin clot formation.46 Using light scattering data in combination with results of molecular dynamic modeling, the authors calculated that such kind of aggregation corresponds to lateral arrangement of the macromolecules in aggregates. Comparing these calculations to cryo-TEM visualization of the aggregates (Figure 13), full agreement of the results was achieved. The images C and D in Figure 13 correspond to the aggregates at pH 3.5 and 11. The aggregates at pH 7 showed globular shape as those at pH 3.5 and in accordance to the scaling investigations. K


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Figure 13. Cryo TEM images of MI-G0-Mal aggregates at pH 3.5 (A) and 11 (B) and of MI-G3-Mal at pH 3.5 (C) and 11 (D). Large structures in images belong to TEM grids.

Mal, at pH 3.5. Furthermore, MI-G3-Mal is less rigid at pH 7 and possesses a coil-like structure that aggregates to dense spheres. In contrast, MI-G0-Mal remains coil-like at the same conditions.

4. CONCLUSIONS In summary, we demonstrated a synthesis of maltose-modified maleimide copolymers with lysine dendrons in three different generations with a high degree of substitution. Starting from side-chain functionalized alternating maleimide copolymers, only a polymer-analogous reaction is needed to establish dendronized glycopolymers. These glycopolymers can be considered as H-bond active giant macromolecules that are water-soluble at room temperature. The functionality of the polymers increases dramatically with the generation number, which leads to pronounced aggregation as a result of Hbonding interactions. This aggregation is strongly dependent on generation number, concentration, and pH value, as shown by DLS, zeta-potential, and molar mass measurements by AF4. The mechanism of aggregation depends strongly on the shape of the macromolecules. Thus, linear lysine-copolymers possess helical, rigid shape at low pH, which changes at high pH values. Low-generation dendrons disturb the helical structure without aggregate formation due to lower concentration of maltose endgroups at the surface of the macromolecules. In the third generation, the high density of functional groups leads to extended, cylindrical type of macromolecules. The lateral aggregates of these big macromolecules can be transformed

Figure 14. Dependence of the Rg on the molar mass extracted from AF4-MALLS measurements of MI-G0-Mal at pH 3.5, 7, and 11. The fits are performed with a correlation factor R2 > 0.95. The scattering data are fitted with a linear fit.

globular at higher pH values (Table 4). Dendronization of this structure with first- and second-generation dendrons leads to disordering of the helix, resulting in a coil-like shape, which does not lead to significant aggregation. But modification with third-generation dendrons (MI-G3-Mal) leads to dense, wormlike macromolecules that have pronounced association to lateral aggregates at pH 11. This fact could explain the very high apparent density of MI-G3-Mal at pH 11, as well as of MI-G0L


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Table 4. Generalized Structure of Single Macromolecules and Aggregates of DenPols, Depending on pH

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to globular changing the pH value from 11 to 3.5. The control over aggregate formation in the different generations was proven by the aggregate density, scaling behavior, and microscopy visualization. In a future study we will use the addressable key functions of the giant glycomacromolecules in the design and fabrication of biohybrid structures with specific functions. The first promising study23 showed us that especially the worm-like, giant glycomacromolecule MI-G3-Mal forms uniquely defined biohybrid architectures with cellulose nanocrystals (CNC). The formation of single supramolecular entities23 between CNC and MI-G3-Mal is to be ascribed only to (i) the controlled H-bond driven interaction features of the macromolecule MI-G3-Mal against the surface of CNC and (II) the worm-like shape of MI-G3-Mal.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details. 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]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Prof. Walther Burchard for the fruitful discussions on aggregate shape interpretation. Anja Caspari is greatly acknowledged for performing the DLS and zeta potential measurements and Josef Brandt for drawing Scheme 2, Table 4, and TOC for better clarity in these quite complex DenPols.

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REFERENCES

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pH-Triggered Aggregate Shape of Different Generations Lysine

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