An alternative route to dye–polymer complexation study using asymmetrical flow field-flow fractionation

Journal of Chromatography A, 1217 (2010) 4841–4849

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

An alternative route to dye–polymer complexation study using asymmetrical flow field-flow fractionation Susanne Boye, Nikita Polikarpov, Dietmar Appelhans, Albena Lederer ∗ Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Straße 6, 01069 Dresden, Germany

a r t i c l e

i n f o

Article history: Received 12 April 2010 Received in revised form 18 May 2010 Accepted 19 May 2010 Available online 1 June 2010 Keywords: Hyperbranched Encapsulation Asymmetrical flow field-flow fractionation

a b s t r a c t The goal of the present work is to apply the versatile asymmetrical flow field-flow fractionation (AF4) coupled to UV and light scattering detection for the characterization of hyperbranched poly(ethylene imine) decorated with maltose shell (PEI-Mal) and the polar dye Rose Bengal (RB) in respect to their complexation behaviour. The quantitative determination of the non-complexed dye was carried out using the ultra-filtration effect of AF4 during the focussing phase, whereas the non-bound RB is filtrated and transported out of the channel while the complex of RB and PEI-Mal remains inside. A calibration with UV detector (550 nm) was established and different parameters (e.g. membrane material, molecular weight cut-off and stability of both, pure RB and RB@PEI-Mal complexes in solution) were investigated and verified. Successful reproducibility tests were performed. First complexation studies with the developed method were applied successfully with different mixture compositions of RB and PEI-Mal. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Complexation and encapsulation studies are of large importance for (bio-)medical and pharmaceutical development of drug delivery systems. Dendritic macromolecules are promising threedimensional nanostructures, possessing high number of peripheral groups [1], and different cavities within the dendritic scaffold. These polymers were intensively investigated especially in the field of carrier systems for bio-active (macro-)molecules and metal ions [2–4], as templates for metal nanoparticles [5], as artificial enzymes [6] and for the creation of biomimetic structures [7,8]. Even in the early work of Meijer [9] it was shown that high-generation dendritic structures composed of a rigid shell and a flexible core possess internal cavities available for guest molecules (so-called dendritic boxes). At the same time it was shown by Fréchet [10] that dendrimers with a hydrophobic interior and hydrophilic shell are able to solubilize hydrophobic compounds in aqueous solutions. These works underlay the idea to use host-guest dendritic complexes for drug delivery applications, to provide long-term drug release and to preserve drug from aggressive environment or organism from the aggressive drug. Certainly, biocompatibility and non-toxicity of dendritic scaffolds should be also taken into account. One of the ways to decrease toxicity is to modify such macromolecules with sugar, poly(ethylene glycol) or (oligo-) peptide units.

∗ Corresponding author. Tel.: +49 351 4658 491; fax: +49 351 4658 565. E-mail address: [email protected] (A. Lederer). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.05.036

One example is the water-soluble, oligosaccharide modified, hyperbranched poly(ethylene imine), which possesses strongly reduced toxicity and high ability to carry drug molecules, for example, showing enhanced uptake properties for ATP (adenosine triphosphate) molecules in different cell lines [11]. However, those materials can preferably be used in the field of (bio-)medical and pharmaceutical studies [4,12,13]. In the present work such hyperbranched poly(ethylene imine) decorated with maltose moieties (PEI-Mal) was investigated. In order to determine uptake and release properties of highly branched structures, usually their interactions with water- or organic-soluble dyes as model compounds for drugs, partly possessing charge, are tested. One of the most common examples is Rose Bengal (RB), a small, polar dye that was applied in investigations of drug loading and release properties [12,14,15]. It could be easily detected and quantified by the bathochromic shift in the maximum of absorption spectra, when bound to a certain polymer by UV–vis concentration measurements after calibration [16–18]. The qualitative examination of encapsulation or complexation of model compounds can be applied by various techniques such as liquid chromatography [17,19,20], nuclear magnetic resonance (NMR) spectroscopy [21,22], and electrophoresis [23]. The most common methods for the quantification of the amount of bound dye molecules per macromolecule are direct UV–vis measurements of dye and dye–polymer mixture [24,25] and UV–vis measurements with preliminary separation by size-exclusion chromatography (SEC) [17]. However, both methods have limitations and cannot be applied quantitatively for the RB@PEI-Mal complex. The limitations come from the fact that the determination of the

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Fig. 1. Principle of AF4 method for analyte filtration and analyte@polymer complex fractionation.

extinction coefficient of complexated dye is dependent on the location of the dye in the core–shell structure. Hence, a calibration curve of dye–polymer complexes with a defined composition cannot be established. Commonly, the separation of the free dye from the complex is performed by means of dialysis, ultra-filtration or ultracentrifugation. However, for our specific system these methods provide non-quantitative information about the composition. Furthermore, the SEC separation does not give the expected performance, since PEI-Mal interacts strongly with the column material and elution retardation takes place. Within the present study a new, alternative strategy for the examination of the complexation between the dye and PEI-Mal is shown, which overcomes the disadvantages of the above mentioned approaches. In asymmetrical flow field-flow fractionation (AF4) technique [26] the separation is achieved along the axis of a thin, long channel in which a laminar flow of mobile phase transports the sample components. The power field is applied perpendicularly to the parabolic flow to force the molecules in different laminar flows. This results in different elution times, due to different velocities depending on size, density and surface properties [27,28]. The combination of AF4 with light scattering (LS) is the most frequently used sub-class of the field-flow fractionation techniques for gentle molecular weight and size determination of synthetic and natural polymers [29], nanoparticles [30], proteins [31] and viruses [32,33]. The advantages using AF4-LS are: - enhanced separation capability in broader molecular weight range (from 103 to above 108 g/mol) in contrast to SEC, - absence of stationary phase ensures the separation almost without interaction, - and the possibility to investigate non-purified solutions and mixtures of different compounds, respectively. The general idea in this work was to apply the AF4 technique for the separation of the free analyte from an analyte@polymer complex. Therefore, the focus flow is used to transport the small free analyte molecules through the ultra-filtration membrane out of the channel (filtration), while the complex remains inside the channel (Fig. 1). This filtration method was described by Giddings et

al. [34,35] for solution purification. The amount of filtered analyte is detected quantitatively by a concentration sensitive detector at the waste line of the cross flow. Within the same measurement, after the filtration, elution within the channel leads to fractionation of the complex according to molecular weight which is detected by molecular weight sensitive detector (light scattering, LS and refractive index, RI detectors). Significant advantage of AF4-LS for complexation studies is that in only one measurement separation and quantification of the non-bound dye together with molecular weight determination of the dye–polymer complex could be performed. Within this work this general method was optimized and tested for the quantitative determination of complexed dye RB with PEIMal polymers. Interaction behaviour of the dye RB and the polymer PEI-Mal with the channel membrane was studied. Reproducibility tests and calibration curve generation for the quantification of RB were carried out. Furthermore, the effect of membrane type (material and molecular weight cut-off) on the separation was examined. Molecular weight characterization of non-complexed PEI-Mal via AF4-LS depending on sample concentration was carried out. Finally, complexation studies with different compositions of RB and PEI-Mal over a defined time period were applied to quantify RB complexed in the environment of PEI-Mal.

2. Materials and methods 2.1. Materials RB was purchased from Sigma-Aldrich® (Germany). Hyperbranched poly(ethylene imine) (PEI) (Mw = 25 000 g/mol) was received under the trade name Lupasol from BASF SE (Ludwigshafen, Germany). The end group functionalization with maltose units was obtained by reductive amination described by Appelhans et al. [11]. Poly(ethylene glycol) as a reference polymer (Mn = 91 300 g/mol; Mw = 95 000 g/mol) was received from Tosoh Corporation (Japan). All samples were dissolved in pure water, which was deionised, UV treated and ultra-filtrated by a Purelab Plus UV/UF equipment (USF Elga, Germany). The water contains 0.02% sodium azide (v/v) to prevent bacteria growth.

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2.2. Methods

2.3. Data treatment

The UV–vis measurements were carried out on a Cary 100 spectrophotometer (Varian, USA) at ambient conditions using a precision glass cuvette with a thickness of 10 mm and a volume of 3.5 mL. The molecular weight determination of the pure PEI was carried out on a modular build SEC-system coupled to a light scattering ( = 632 nm) detector (Dawn Tristar from Wyatt Technologies, USA) and a RI detector (Knauer, Germany) in combination with a PolarGel C column (PL, UK) using a flow rate of 1 mL/min. N,NDimethylacetamide (DMA) with lithium chloride (3 g/L) was used as eluent. All AF4 measurements were performed on an Eclipse 3 system (Wyatt Technology Europe, Germany). The wide channel spacer made of poly(tetrafluoroethylene) (PTFE) had a thickness of 350 ␮m and the channel dimensions were 26.5 cm in length and from 2.1 to 0.6 cm in width. The membranes used as accumulation wall and filtration device had different molecular weight cut-offs (MWCO, 5 and 10 kDa) and consist of regenerated cellulose or poly(ether sulfone) (PES, Wyatt Technology Europe, Germany). Flows were controlled with an Agilent Technologies 1200 series isocratic pump equipped with vacuum degasser. The detection system consists of a variable wavelength UV–vis detector (UV) (Knauer, Germany) tuned at 550 nm, a RI detector Dn 2010 (WGE Dr. Bures, Germany,  = 620 nm, 25 ◦ C) and a three-angle laser light scattering detector (MiniDAWN from Wyatt Technology, USA). The UV detector was used at the channel outlet or the cross flow outlet for the determination of free dye amount. All injections were performed with an autosampler (1200 series, Agilent Technologies). The channel 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. Method A: For RB filtration the focus flow was set to 3 mL/min for 20 min. The fractionation of the complex within this measurement followed in elution mode with linear cross flow gradient from 2 to 0 mL/min during 20 min. Method B: Determination of the molecular weight of the complex was performed after focussing at 1.5 mL/min focus flow over a time period of 2 min and elution under a linear cross flow gradient from 2 to 0 mL/min during 20 min. At the end of the elution step the cross flow rate was maintained at zero for 5 min in order to ensure that the entire sample has been eluted. Two measurements of each sample were carried out for molecular weight determination and afterwards, a blank run was performed, necessary for the baseline subtraction of the pressure sensitive RI signals.

After collection of the LS, RI and UV profiles using the ASTRA 4.9.1 and 4.7 software (Wyatt Technologies, USA), subsequent baseline subtraction of RI signals of samples and pure eluent was applied with CORONA 1.4 software (Wyatt Technologies, USA). For Mw determination the dn/dc values were externally determined at 25 ◦ C using RI detector Dn 2010 (WGE Dr. Bures, Germany,  = 620 nm). Different sample concentrations in the range of 0.4, 0.8, 1.6, 2.0, 2.4 and 3.2 mg/mL were prepared for this purpose. 400 ␮L were injected at a flow rate of 0.15 mL/min. Refractive index increments, dn/dc of 0.167 mL/g for pure RB, 0.14 mL/g for all PEIMal samples, 0.100 mL/g for RB–PEI-Mal B complex (at different molar ratios), and for pure PEI −0.125 mL/g in DMA + LiCl were determined for the light scattering calculations. The calculation of the molecular weight distributions and the determination of Mn , respectively, were carried out by linear fit of the molecular weight/elution volume dependence. 3. Results and discussion 3.1. UV measurements Accurate determination of the absorption maximum of RB is essential for subsequent quantitative investigations. As it was mentioned above RB is commonly used for the investigation of encapsulation properties because the location of the RB spectrum maximum is influenced by the dye’s environment. Additionally to that, RB exists in two forms depending on pH value: at pH below 3 in neutral lactonic form, which is characterized by broad spectrum of very low intensity, while at pH above 4 it exists in negatively charged quinoid form with well pronounced maximum in the spectrum at 550 nm. Static UV–vis measurements of our samples showed highest absorption at a wavelength of 550 nm for the pure RB at pH 6.7 (Fig. 2a). Similarly, the influence of hyperbranched environment on the properties of complexed molecules was proved by changes in their UV–vis spectra. The absorption spectra of RB in the presence of PEIMal (1:1 mixture) are also shown in Fig. 2a. At pH 6.7 the appearance of a bathochromic shift to longer wavelength in the spectrum is an evidence of the RB@PEI-Mal complex formation. The absorption maximum of this complex is located at 558 nm. Under strong basic conditions (pH > 11, Fig. 2b) no shift was observed due to negative charge of both, RB and PEI-Mal B molecules and their electrostatic repulsions, respectively. Otherwise, in acidic solutions (pH < 3, Fig. 2c) the spectrum of bound

Fig. 2. UV–vis spectrum of pure RB and mixture of RB and PEI-Mal B at pHs of (a) 6.7, (b) 11.5 and (c) 2.

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RB strongly differs from the spectrum of the non-bound one. This finding proves the presence of strong interactions between RB and PEI-Mal B. The results of the UV measurements give clear qualitative information about the affinity of RB to the PEI-Mal polymer. However, quantitative determination of the amount of RB cannot be performed accurately by this technique, due to the presence of two different chemical structures in the polymer. In the present work the investigations with AF4 are focussed on solutions with pure water, due to their simplicity for the method development.

3.2. Rose Bengal calibration of AF4 3.2.1. Optimization of quantitative determination Calibration curve was established for quantification of free, noncomplexed RB at a fixed wavelength of 550 nm, and for further calculations of the complexed RB amount. Therefore an UV detector (fixed wavelength 550 nm) was connected to the cross flow waste line of the AF4 channel. The filtration of the small molecules of free RB from the AF4 channel takes place during the focussing step. Commonly, the focus step of AF4 technique is applied to concentrate the sample after injection and is performed prior to separation. During this focusing process (Fig. 1), the sample material (mixture of RB and RB@PEI-Mal complex) is forced into a narrow “band” from which the separation is started. Therefore, an opposite stream of the carrier liquid is pumped into the channel from the channel outlet additionally to the flow through the inlet port. As a consequence, the sample molecules are focussed at a certain point close to the sample injection port and the carrier liquid leaves the channel passing through the membrane. During this phase the small RB molecules, with a molecular weight of 1017.6 g/mol, are flushed out by the focus flow and permeate through the membrane. The RB@PEI-Mal complex retains inside of the channel, because of its larger dimensions and the limited pore size of the membrane (MWCO with 5 and 10 kDa). Using this method, we are aware of the unusually strong forces applied to remove the free dye from the polymer mixture. In fact, these filtration conditions are comparable to the ultra-filtration technique, commonly used for this purpose, as described above. Additional advantage of the focus flow is that elimination of weak interactions between dye and polymer takes place, which leads to separation only of the stable RB@PEI-Mal complex from the residual dye molecules. In order to develop a suitable separation method, different influencing parameters such as focussing times and flows were taken into account. The best combination of these parameters was found to be focus flow with 3mL/min over a time period of 20 min (Method A, see Section 2.2). During this time the whole amount of injected RB, which is non-bound, can be filtered through the channel and detected. The tests were carried out with pure RB solutions of different concentrations including variation of the injected volume (described in Section 3.2.2). The limiting factor was that the entire amount of RB has to be removed from the channel. Thus, while switching from focus to elution mode for the determination of molecular weights, the free dye is completely removed. This was proved by UV detection at the channel outlet resulting in the absence of any response. Further efforts to isolate the RB during the elution step showed that during this step a separation of free RB and the complex is not as efficient as during the focussing. In the following method development combination of filtration of non-complexed RB and additionally the fractionation of the complex for molecular weight determination in a single measurement was aimed (Method A, see Section 2.2). In order to test the potential of this method for simultaneous dye quantification and molecular weight determination of the complex, after the focussing of 20 min at 3 mL/min, cross flow of 20 min at a linear decrease from 2 to 0 mL/min was applied.

3.2.2. Influence of the ultra-filtration membrane and establishment of calibration curve The subsequent investigations were applied in order to check the influence of the ultra-filtration membrane on the concentration detection of RB during the focussing step. This concerns additional parameters such as material, molecular weight cut-off and measurement conditions. Two different membrane materials were investigated: regenerated cellulose and poly(ether sulfone) (PES). The tests with the developed method (see Section 3.2.1) demonstrated low application ability of PES for the quantitative determination of RB, since no detectable amount of dye was filtered through the membrane. The entire injected sample retained during the focussing step inside the channel and exited the channel through the longitudinal outlet. Apparently, repulsion effects between the RB and the surface of the PES membrane reduce the permeation of RB through the membrane. Undersized pores in the membrane can be excluded, since the MWCO of 10 kDa is almost the decuple of the molecular weight of RB. Regenerated cellulose membranes are well suited for the RB determination because of their anionic properties. However, the stronger affinity of RB to the cellulose membrane leads to modification of the membrane as typical for regenerated cellulose [36]. Therefore, saturation of the membrane with RB before starting quantitative measurements and separation of free RB from RB@PEIMal complex is essential for reproducible and accurate results. The saturation point of the membrane was determined by 15 successive injections with the same sample load of 50 ␮g of pure RB solutions. Fig. 3 shows that after the 4th injection, the detected peak area remains constant confirming complete modification of the fresh membrane. The deviation calculated between this and the following 10 runs is below 2%. The experiment was repeated with a new membrane by injection of higher sample mass (100 ␮g) leading to standard deviation between the first two runs of 50%, decreasing to 0.7% after the 4th injection and leading to reproducible peak areas in the next injections (Fig. 3). This leads to the conclusion, that RB amount of at least 200 ␮g is necessary to saturate the surface of a fresh membrane of regenerated cellulose before applying quantitative mass determination. Subsequent measurements of pure PEI-Mal showed no UV signal during the elution of the polymer. Therefore the UV detector was placed at the longitudinal channel outlet. This leads to the assumption, that the RB establishes a stable layer on the surface of the membrane without further influence of the detected UV signal. All subsequent measurements were performed with RB saturated membranes.

Fig. 3. UV signal peak areas of successive injections of pure RB solution for membrane modification.

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Fig. 4. Calibration curve for quantitative RB determination after polynomial fit of the detected data.

The study of the saturation of regenerated cellulose membrane with RB was essential prerequisite to generate a calibration curve for quantitative determination of non-bound RB after its separation from the mixture with RB@PEI-Mal complex during the focus step. The calibration was performed by injection of different sample loads in the range of 10–300 ␮g (Fig. 4). Five series of solutions with different concentrations were prepared by dilution of a stock solution. Each solution was injected for three times. The standard deviation of the repeated runs was below 0.2%. The calibration curve was generated by the mean values of each concentration of dilution series (Fig. 4) using a polynomial regression. The limits of the determined RB calibration lie between 16 and 80 ␮g. The lower limit is defined by the limit of detection of the UV detector. The upper limit value is caused by the dynamic range, within which Lambert–Beer law applies. Hence, higher amounts were not considered because of the decreased accuracy of the calibration curve and the related increased error of the calculated masses. The isolated amount of RB can be calculated directly from the detected peak area as follows: RB amount = 6.477 + 38.108 × (peak area) − 4.434 × (peak area)2 (1) Using this approach, the amount of bound dye can be determined at known total amount of RB in the mixture. The influence of the MWCO was studied on 5 and 10 kDa cut-off of regenerated cellulose membranes. In both cases the accuracy after the third injection shows that the deviation between the replicates is below 2%. This leads to the conclusion that no influence of MWCO on the determination of the filtered RB amount can be observed. The stability of the RB solution was the last open question before starting quantification measurements. 50 ␮L of varied concentrations of RB (0.5, 1 and 3 mg/mL) were injected over a time period of 10 days under same conditions in order to compare the UV signal intensities. The solutions were stored under ambient conditions. The results demonstrate the same values with a deviation below 1%. This indicates stability of RB solution and UV absorption behaviour, respectively, which is an important requirement for the following experiments. 3.2.3. Quantitative reproducibility tests The quantitative reproducibility of the injected and detected RB amount is the main requirement for the performance of the experiments on its complex. For this purpose RB solutions with defined concentrations were injected and measured by the described method using a modified membrane with MWCO of 5 kDa (see Section 3.2.2). The amount of the detected RB was calculated using Eq.

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Fig. 5. UV signals of reproducibility tests with RB, c = 0.33 mg/mL and various injection volumes (5, 100 and 200 ␮L).

(1). For the next experiments the injection volume was varied. A solution with a concentration of 0.33 mg/mL and different volumes is presented in Fig. 5 and the calculated amounts of detected RB are shown in Table 1. Accurate agreement between the real and the calculated masses was achieved. Subsequent reproducibility tests were carried out with solution concentrations of 0.72 and 0.9 mg/mL and with varied injection volumes (25, 50 and 100 ␮L) confirming recovery of the detected and the injected masses within an error below 1% for the first two injection volumes. However, at 100 ␮L injected volume of 0.9 mg/mL RB solution the calculated mass is about 18% lower in comparison to the injected mass. This fact can be explained with exceeding the limit of 80 ␮g within the injected RB amount defined in Section 3.2.2. This result shows that accurate UV response for high amount of RB could be obtained at low solution concentration, but increased injection volume. 3.3. Molecular weight characterization of PEI-Mal The commercially available hyperbranched poly(ethylene imine) was used as a core for the formation of the core–shell particles. The molecular weight determination of the hyperbranched PEI led to molecular weight values of Mw = 37 200 g/mol with polydispersity of Mw /Mn = 3.32. PEI derivatives with different oligosaccharide architectures (structures A–C in Fig. 6) were synthesized from hyperbranched PEI (Fig. 6; Lupasol WF possessing 29.4% of primary amino groups (T units), 40.4% of secondary amino groups (L units) and 30.3% of tertiary amino groups (D units)) through reductive amination in the presence of stoichiometric amounts of the oligosaccharide maltose (Fig. 6) (details in Appelhans et al. [11]). Structure A, formed in the presence of an excess of oligosaccharides, is characterized by a dense maltose shell (PEI-Mal A). However, most primary amino groups (T units) and secondary amino groups (L units) of PEI are converted into tertiary amino groups (D units). Therefore, only few L units formed by the monosubstitution of primary amino groups, and few non-converted secondary amino groups are present in structure A. Structure B depicts a loose shell comprising mostly L units (monosubstituTable 1 Results of reproducibility test with RB solution (c = 0.33 mg/mL). Injected volume (␮L)

Injected mass (␮g)

UV peak area

Calculated mass (␮g)

50 100 200

16.5 33 66

0.4918 0.9955 2.0064

17 33 64

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Fig. 6. Simplified synthetic pathway of PEI-Mal A–C, converted PEI with different ratios of maltose in the presence of borane × pyridine complex in basic solution, and their chemical structures.

tion) and only few D units (disubstitution) as peripheral groups. In other words, the peripheral secondary amino groups of structure B mostly carry one maltose unit (PEI-Mal B). Structure C possesses a mixture of T (∼50%) and L (∼50%) units as peripheral groups (PEIMal C). This leads to isolated maltose units in the periphery and the presence of unreacted primary amino groups. Table 2 summarizes the structure types and the molecular weights of the PEI derivatives employed in this study. The molecular weight determination of the non-complexed, maltose modified PEI by aqueous SEC was not possible, since the oligosaccharide shell shows strong interaction with the column packing material. However, in order to characterize the complexation behaviour of PEI-Mal, reliable molecular weight determination for PEI-Mal is essential, before investigating dye complexation. Hence, information of the influence of the concentration as well as the influence of aggregation on the separation of PEI-Mal is of significant importance. Therefore, measurements with variation in the injected masses under the same conditions were carried out. The separations were performed according to Method B described in Section 2.2, since the longer focusing time in Method A led to significant increase of the detected molecular weights due to aggregation. The injection volume was 60 ␮L. In order to provide identical separation conditions of pure and complexed PEI-Mal and to enable accurate comparison of the molecular weights, membrane saturated with RB molecules (see Section 3.2.2) was used. Nevertheless, comparison with measurements made with fresh membrane did not show any influence of the modification of the membrane on the molecular weight results of the pure PEI-Mal. Fig. 7 shows a decrease of the determined molecular weight of PEI-Mal C at higher injected mass. This effect was observed by Arfvidsson and Wahlund [37] in the

course of their studies on overloading effects in AF4. Additionally, when applying LS-RI detection it is necessary to get sufficiently high RI signal for acceptable accuracy of molecular weight calculation. The data in Fig. 7 show, that with increasing sample masses the precision of the determined molecular weight values clearly increases. This can be explained by the low concentration signal intensity of the RI detector at low sample load. The same effect was observed for PEI-Mal A and B. Therefore, sample loadings between 60 and 180 ␮g were chosen for reliable molecular weight determination of PEI-Mal. The intensities of the light scattering signals of the three PEIMal samples (Fig. 8a) confirm highest molecular weight for PEI-Mal A and lowest for PEI-Mal C. The highest molecular weight corresponds, additionally, to the broadest molecular weight distribution (Fig. 8b). Due to the high number of maltose units one can expect aggregation at different concentrations, which could influence the molecular weight results. However, the concentration dependent studies showed no significant changes even for the higher mass sample. 3.4. Complexation studies On the basis of the performed optimization of the measurement conditions, quantitative complexation studies and molecular

Table 2 Degree of decoration with maltose units and molecular weight of PEI-Mal samples determined via AF4-LS. Sample

Degree of decoration for (2 × T + L) units (%)

PEI-Mal A PEI-Mal B PEI-Mal C

90 40 20

Mn (g/mol) 215 400 200 500 114 300

Mw (g/mol)

Mw /Mn

488 900 378 500 162 900

2.27 1.89 1.43

Fig. 7. Molecular weights of PEI-Mal C as a function of injected sample mass determined by AF4-LS.

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Fig. 8. (a) Elution curves (LS angle 90◦ ) and (b) differential molecular weight distribution of PEI-Mal A–C determined via AF4-LS (injected sample mass: 120 ␮g).

weight determination of the complex can be performed. In order to check the influence of the filtration of non-bound dye in the presence of non-complexed polymer in solution, poly(ethylene glycol) (PEG) was chosen, which does not interact with RB. Mixture (1:1) of RB solution (1 mg/mL) and PEG solution (1 mg/mL) was prepared. The UV detection was set at the cross flow outlet as well as the channel exit. The UV signals at the cross flow outlet show the expected unchanged signal intensity and peak area, respectively, while at the longitudinal channel exit no UV response was detected. These experiments were repeated with a threefold higher amount of RB in the mixture confirming the full separation of the non-complexed dye from the polymer solution during the focusing step. Additionally to that, the calculated amount of RB was in a good agreement with the injected one. This leads to the conclusion that the entire amount of RB was isolated from the polymer by the cross flow and no transport effects have occurred. The aim of the present work is the investigation of the complexation behaviour of PEI-Mal with RB. Therefore, different compositions of RB and PEI-Mal mixtures were studied. The tests were carried out with the three samples of PEI-Mal. PEI-Mal A and C showed immediate precipitation after addition of the RB to the PEI-Mal solution. The explanation of this effect could be on the one hand the strong molecular weight increase due to aggregation by adding RB in case of the PEI-Mal A. However, this cannot be the only reason for this behaviour, since PEI-Mal C possesses significantly lower molecular weight. The difference between these two samples is also the degree of modification with maltose. Therefore we suppose that the pH of 6.7, which was used for all measurements, is not optimal for PEI-Mal with lower modification degrees. Further investigations with variation of pH of the solutions will shed light on this question. As a result of these observations, the following experiments were performed with PEI-Mal B, which does not show changes in the solution with the time. Kinetic studies were performed in order to elucidate the timedependence of the complexation. Therefore, both components were mixed and injected immediately after mixing followed by 15 injections over a time period of 630 min. The injected volume

was 100 ␮L in all experiments. Time-dependent free RB determination and molecular weight of the dye@PEI-Mal complex within one experiment (Method A, see Section 2.2) was performed. A mixture of 75 ␮g RB and 87.5 ␮g PEI-Mal B (Table 3) was prepared and measured by Method A (see Section 2.2) combining the filtration of the free RB and the molecular weight determination of the dye@PEIMal B complex. The molecular weight increase in the mixture can be detected immediately after mixing indicating fast formation of the dye@PEI-Mal complex without significant changes with the time (Fig. 9a). Despite the strong focussing conditions the stability is evident. The molecular weights are constant over a time period of 630 min with very low deviation between the individual injections. The determined amounts of free RB show standard deviation below 1.7%. In order to calculate the amount of complexed dye out of the known, total injection amount of RB, we used the calibration according to Eq. (1) (Fig. 9b). As a result for this specific mixture approximately 96 RB molecules per polymer molecule in a complex were calculated. However, the detected molecular weight of Mn = 464 000 g/mol exceeds significantly the molecular weight expected from the calculation corresponding to Mn = 200 500 g/mol of the polymer and to 96 RB molecules with Mw = 1017.6 g/mol. In order to check the reliability of the molecular weight of the complex determined by a single measurement (Method A), the influence of the long focusing time for filtration of the free dye should be carefully taken into account. Therefore, subsequent studies with gentle focussing conditions applying Method B (see Section 2.2) were performed. The determined molecular weights of the complex are lower than the values determined with longer focusing time. The molecular weight values determined by Method B were stable over the same time period of 630 min. This shows us clearly that not in the solution mixture, but during the long focusing time of 20 min, aggregation of the complex occurs which leads to increased values of the molecular weight. However, this aggregation does not affect the complexation of dye molecules. This can be concluded from the fact that the calculated number of complexed RB molecules based on the detected free RB is in a very good agreement with the increase of the molecular weight of the

Table 3 Quantitative complexation studies (pure water) of RB and PEI-Mal B. Injected amount RB

Injected amount PEI-Mala

Molar ratio RB:PEI-Mal injected

Isolated RB (mol)

Bound RB (mol)

Mn b RB@PEI-Mal (g/mol)

Molar ratio RB:PEI-Mal in the complex

50 ␮g (4.91 × 10−8 mol) 40 ␮g (3.93 × 10−8 mol) 75 ␮g (7.37 × 10−8 mol) 50 ␮g (4.91 × 10−8 mol) 200 ␮g (1.96 × 10−7 mol)

240 ␮g (1.20 × 10−9 mol) 60 ␮g (2.99 × 10−10 mol) 87.5 ␮g (4.36 × 10−10 mol) 40 ␮g (2.00 × 10−10 mol) 60 ␮g (2.99 × 10−10 mol)

41 131 169 246 657

0 1.73 × 10−8 3.17 × 10−8 2.05 × 10−8 n.d.c

4.91 × 10−8 2.20 × 10−8 4.20 × 10−8 2.86 × 10−8 –

234 400 272 300 291 800 342 800 476 300

41 74 96 144 271d

a b c d

In this study PEI-Mal B was used (Mn = 200 500 g/mol). Determined by AF4, Method B (see Section 2.2). RB amount is higher than the maximum detectable amount of 80 ␮g. Calculated from Mn of the complex, the molecular weight of RB and Mn of pure PEI-Mal B.

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Fig. 9. Kinetics of the complexation of RB and PEI-Mal B: (a) molecular weight of the complex determined by AF4-LS and Method A (see Section 2.2) and (b) amount of RB (injected total amount 7.37 × 10−8 mol).

polymer complex. Fig. 10 shows the comparison of the molecular weight distribution between the pure PEI-Mal B and RB@PEI-Mal B complex. The following experiments show that the content of RB molecules in the complexes is strongly dependent on the molar ratio RB:PEI-Mal B. The values from the analysis of different mixtures are collected in Table 3. Evidently, with higher amount of RB in relation to PEI-Mal B the number of bound molecules tends to increase. The highest detected number of bound dye molecules (144) was reached at a molar ratio 246:1 RB to PEI-Mal B. The bound RB in the complex was calculated on the basis of the filtered free RB amount. While at molar ratio of 41:1 the complete RB is introduced in the complex, the amount of bound RB in the residual mixtures approximates 58% of the whole RB amount. This corresponds to a linear increase of the bound RB with the RB amount in the mixture (Fig. 11). This fact gives a clear indication that equilibrium conditions are reached within injected molar ratios RB:PEI-Mal B between 131 and 246. As expected, the number averaged molecular weights are continuously increasing at higher amount of RB in the complex. Their values correspond very well to the expected increase according to sum of the molecular weights of PEI-Mal B and RB. The injection of a very high excess of RB (657:1) leads to further increase of the molecular weight of the complex. However, the isolated amount of free RB exceeded the determined detection limit (see Section 3.2.2) and hence, no information about the introduced number of RB molecules in the complex was available. The supposed value calculated from the increase of Mn of the complex compared to the pure polymer would be 271 RB molecules per one polymer

Fig. 11. Molar ratio RB:PEI-Mal B in the complex and number average molecular weight of the complex in dependence of the molar ratio RB:PEI-Mal B in the mixture.

molecule. Fig. 11 shows deviation from the linearity in this molecular weight region. This is an indication that the RB in this complex approximates a state of saturation. The complexation study shows that the RB@PEI-Mal B complexes exist preferably as isolated particles in solution of pure water. The main driving force to complex RB molecules in PEI-Mal B is the electrostatic interaction between the anionic RB and the cationic PEI-Mal B [11]. One can assume that the most of the RB molecules are attached in the outer sphere of the PEI-Mal B macromolecules and few within the dendritic scaffold. The high molar amount of RB complexed by PEI-Mal B here in this study, and preferred presence of isolated complex particles in solution were confirmed by another study in our working group, using ultra-filtration method combined with UV–vis measurements and dynamic light scattering [38]. 4. Conclusions

Fig. 10. Differential molecular weight distribution of pure PEI-Mal B and RB@PEIMal B determined by AF4-LS, Method B (see Section 2.2).

The present work describes the development of an alternative route for the quantitative determination of RB with poly(ethylene imine) decorated with maltose units via AF4 for the application in encapsulation or complexation studies. Comprehensive molecular weight determination of the nonmodified and maltose modified PEI via SEC and AF4-LS was performed. In respect to different degrees of modification of PEI the molecular weights of three types of PEI-Mal were studied by AF4LS. Rose Bengal complex with PEI with 40% modification degree with maltose was stable over long period of time. With the subsequently developed AF4 method it was possible to filtrate the free RB from mixture of complexed and non-bound RB. During the focus step the eluent flow transports the small free RB molecules through the ultra-filtration membrane out of the channel while the

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polymer bound dye molecules remain inside. Different parameters like membrane material and molecular weight cut-off were studied. Regenerated cellulose membranes with a MWCO of 5 and 10 kDa were found to be suited for this separation. One important prerequisite for the quantitative determination of bound RB in RB@PEI-Mal complexes is the modification of the regenerated cellulose membrane, whereas the surface of the membrane has to be saturated with RB molecules. In this context, reproducible calibration for the isolated RB amount was established for further evaluation of bound RB in RB@PEI-Mal B complex. The determination of the molecular weight of the complex and the quantification of the complexed RB amount within one AF4 measurement was successful. However, aggregation of the complex during the filtration of the dye in the focusing step was observed. Reliable molecular weight determination of the dye@polymer complex was possible separately, after gentle focusing conditions preventing aggregation. The complexation studies on different RB:PEI-Mal mixtures were performed after (i) RB saturation of the membrane; (ii) filtration and detection of the amount of free dye during one measurement; and (iii) fractionation and molecular weight determination of the complex during a second measurement. These studies show stable equilibrium of bound and free RB within 131 and 246 molar ratio of RB:PEI-Mal, whereas for lower and higher ratios no equilibrium values were obtained. Maximum amount of 144 RB in the complex with PEI-Mal B with Mn of 200 500 g/mol was detected. Further applications of the developed AF4-LS method will be the determination of the maximum dye content, which can be introduced in the complex; pH dependent studies as well as variation of the modification degree for the optimization of the complex formation will be performed. In this context, this approach can be used as a potential analytical tool to study encapsulation/complexation of drug with carrier systems, including the size and molecular weight of drug@polymer complexes in the (bio-)medical and pharmaceutical field. References [1] B.I. Voit, A. Lederer, Chem. Rev. 109 (2009) 5924. [2] A.W. Bosman, H.M. Janssen, E.W. Meijer, Chem. Rev. 99 (1999) 1665.

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