Development of phosphorylated nanoparticles as zeta potential inverting systems

Accepted Manuscript Development of phosphorylated nanoparticles as zeta potential inverting systems Glen Perera, Maximilian Zipser, Sonja Bonengel, Willi Salvenmoser, Andreas Bernkop-Schnürch PII: DOI: Reference:

S0939-6411(15)00031-4 http://dx.doi.org/10.1016/j.ejpb.2015.01.017 EJPB 11811

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Accepted Date:

14 September 2014 19 January 2015

Please cite this article as: G. Perera, M. Zipser, S. Bonengel, W. Salvenmoser, A. Bernkop-Schnürch, Development of phosphorylated nanoparticles as zeta potential inverting systems, European Journal of Pharmaceutics and Biopharmaceutics (2015), doi: http://dx.doi.org/10.1016/j.ejpb.2015.01.017

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Development of phosphorylated nanoparticles as zeta potential inverting systems

Glen Perera1, Maximilian Zipser1, Sonja Bonengel1, Willi Salvenmoser2 and Andreas Bernkop-Schnürch1,*

1

Department of Pharmaceutical Technology, Institute of Pharmacy, University of

Innsbruck, Innrain 80/82, Center for Chemistry and Biomedicine, 6020 Innsbruck, AUSTRIA 2

Institute of Zoology, University of Innsbruck, Technikerstr. 25, 6020 Innsbruck,

AUSTRIA

*

Corresponding author:

Department of Pharmaceutical Technology Institute of Pharmacy, University of Innsbruck Innrain 80/82, Center for Chemistry and Biomedicine 6020 Innsbruck, AUSTRIA Tel. : +43-512-507-58600 Fax. : +43-512-507-58699 e-mail : [email protected] 1

ABSTRACT The objective of this study was to generate nanoparticles with a slightly negative zetapotential which switches to positive values under the influence of intestinal alkaline phosphatase in order to address two major physiological barriers (mucus and membrane barrier). Carboxymethyl cellulose and chitosan were modified with phosphotyrosine by means of a water-soluble carbodiimide and polyelectrolyte complexes were formed by mixing two polymer solutions in an appropriate ratio. Due to this modification, phosphate ions could potentially be released which would lead to a change in zeta potential. Their sizes were found to be between 200 and 300 nm while their zeta potentials ranged from - 8 mV to - 5 mV prior to incubation with the enzyme. It could be shown that phosphate ions are released from the modified polymers and nanoparticles by isolated phosphatase and in a Caco-2 cell model. Incubation with phosphatase led to a change in zeta potential of the nanoparticles up to + 8 mV. As neither polymers nor particles display toxic properties within the resazurin assay, these nanoparticles appear to be useful tools in future drug delivery systems as they have appropriate properties regarding particle size and surface charge in order to overcome the mucus and the membrane barrier.

Keywords: mucus, nanoparticles, zeta potential, intestinal alkaline phosphatase, phosphotyrosine, intestinal barriers

2

INTRODUCTION As of this writing, oral drug administration is still unrivaled regarding its patient compliance. However, the number of drugs that have to be administered on parenteral routes is consistently increasing as peptide and nucleic acid drugs become ever-increasingly attractive due to their numerous therapeutic options. When administered orally, such drugs are confronted with different physiological barrier functions that minimize their oral bioavailability often below 1 %. Among these, the enzymatic barrier [1], the membrane barrier [2] and the mucosal barrier [3] play key roles in limiting bioavailability of hydrophilic macromolecular drugs. While there are numerous enzyme inhibitors and encapsulation methods available that can help to prevent premature enzymatic degradation during the passage of the gastrointestinal tract [1], addressing the mucosal barrier and the membrane barrier at the same time appears to be a more challenging task within oral drug delivery. In recent time, several nanoparticle strategies have been developed in order to pass the mucus barrier. A major strategy depends on PEG-coating of nanoparticles aiming at particles with a slightly negative to neutral zeta potential [4]. These nanoparticles display a much better mucus penetration compared to positively charged particles which are decelerated within mucus due to ionic interactions with sialic acid and sulfonic acid moieties of the mucus [5]. Particle size may be another crucial parameter. However, particles as large as 500 nm can traverse mucus as soon as the surface properties are chosen soundly [6]. Unfortunately, different properties are necessary for an uptake by intestinal epithelial cells. When nanoparticles enter the intestine after oral administration, an uptake by lymphoid tissues of Peyer’s patches as well as non-lymphoid epithelial cells could be demonstrated even though uptake by lymphoid tissue seems to play the more important role [7]. However, after passage of the mucus, more interactions with negatively charged cell membranes can be 3

expected from positively charged nanoparticles and it has been observed that positively charged particles offer the advantage of being endocytosed more efficiently than others. Hence, a positive zeta potential may be highly advantageous for both possible ways of membrane passage as both ways rely on interactions between nanoparticles and cell membrane [8,9]. Consequently, a drug delivery system with a neutral to slightly negative zeta potential during mucus passage and a positive zeta potential at intestinal membranes would be an appealing tool for oral delivery of drugs that need to be delivered to intestinal cells and cannot pass the gut unhindered by themselves. Quite recently, it was found that Peyer’s patches are also producing mucus, which make the mucus passage also necessary for this route of particle uptake [10]. Hence, such drug delivery systems can be of particular interest for nucleic acid drugs and vaccines. In order to achieve this aim, a strategy based on enzymatic cleavage by brush border membrane-bound digestive enzymes has been followed within this study. As enzymatic cleavage should either induce positive charges or reduce negative charges on the particle surface, a substrate of intestinal alkaline phosphatase (IAP), namely phosphotyrosine (PTyr), was coupled to chitosan (CS) as well as carboxymethyl cellulose (CMC) via amide formation and particles were produced via polyelectrolyte complexation of the two novel polymers. It is known from the literature that CMC and CS spontaneously form nanoparticles whose zeta potential highly depends of the utilized ratio of the two polymers [11]. Particles with a slightly negative zeta potential have been generated. Hence, IAP can cleave the phosphate ester and release negatively charged phosphate ions, reduce the negative net charge of the nanoparticles and generate positively charged nanoparticles in close proximity to the intestinal membrane.

4

MATERIAL AND METHODS

Materials Phosphotyrosine was purchased from Bachem AG (Bubendorf, Switzerland). NHydroxysuccinimid was obtained from Acros Organics (Geel, Belgium). 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide

(EDAC)

was

purchased

from

Carbosynth

(Compton, Berkshire, UK). Minimum essential medium, fetal calf serum and penicillin-streptomycin liquid (100x) were purchased from PAA (Pasching, Austria) All other chemicals were purchased from Sigma-Aldrich, Steinheim, Germany.

Methods

Depolymerization of chitosan Low molecular weight chitosan was prepared by oxidative degradation with NaNO2 at room temperature. Briefly, 2 g chitosan (medium molecular weight) was dissolved in 100 mL of 6 % acetic acid solution under magnetic stirring. When chitosan was completely dissolved, 80 mg of NaNO2 was added and the reaction was conducted at room temperature for 1 h. Subsequently, the reaction mixture was neutralized with 5 M NaOH and adjusted to pH 9.0 to precipitate chitosan. The precipitated chitosan was re-dissolved in 0.1 % acetic acid and purified by multiple dialysis steps against purified water. The dialyzed product was then frozen at -80 °C and subsequently lyophilized under reduced pressure (Benchtop 2K, VirTis, NY, USA) [12,13]. It is known from the literature that this method delivers chitosan with a molecular weight of approximately 20,000 Da [14].

Synthesis and characterization of phosphotyrosine-modified polymers

5

Phosphotyrosine-modified CMC (CMC-PTyr) was synthesized by a straightforward carbodiimide method which has been adopted from previous syntheses of our research group [15]. For this purpose, 1 g of CMC (90 kDa) was dissolved in deionized water, 2 g of EDAC was added after complete dissolution and pH was adjusted to 5. After 15 minutes of incubation, 500 mg of PTyr was added and pH was adjusted to 5 again in order to minimize potential hydrolysis of the phosphate ester. The reaction mixture was heated to 40 °C and stirred overnight. Finally, this reaction mixture was dialyzed against water six times for 12 hours, frozen and lyophilized (Benchtop 2K, VirTis, NY, USA). Phosphotyrosine-modified chitosan (CS-PTyr) was synthesized by a similar method [16]. First, 250 mg phsophotyrosine and 500 mg of depolymerized chitosan were dissolved in 50 mL water and 1 g of EDAC was added. Finally, pH was slowly adjusted to 5 and heated to 40 °C overnight. The reaction mixture was then treated as described for CMC-PTyr. The syntheses of both polymers are schematically depicted in Fig. 1. These novel polymers were quantified by means of a spectrophotometric method. CMC-PTyr was dissolved in TRIS buffer (20 mM, pH 8.5) and CS-PTyr was dissolved in 0.5 M HCl. PTyr standards were dissolved in the corresponding medium for analysis. The absorbance of polymer solutions (1 mg/mL) was measured at λ = 265 nm and correlated to a calibration curve of PTyr standards with decreasing concentrations.

Nanoparticle preparation and characterization Nanoparticles were prepared by interpolymer complexation between PTyr-modified CS and PTyr-modified CMC exploiting the opposite charge of the two polymers. For this purpose, 1 % stock solutions were prepared of both polymers and pH was 6

adjusted to 6.8. Then, 0.5 to 1.0 mL of CMC stock solution was mixed with 7 mL of water and different amounts of CS stock solution were added drop-wise under magnetic stirring. Different ratios of CMC-PTyr:CS-PTyr have been tried in order to investigate the correlation between polymer ratio and zeta potential. Subsequently, the zeta potentials of all investigated nanoparticles within this study were controlled by the ratio of the two polymers while the main purpose of the modification lies within the zeta potential inversion by enzymatic cleavage. During nanoparticle preparation, zeta potential was measured in order to allow the formation of nanoparticles with a negative, monadic zeta potential. Particle sizes and zeta potential were measured with a Nicomp 380 DLS/ZLS (Santa Barbara, CA, USA). Zeta potential was measured for 180 s at a field strength of E = 5 V/cm. Moreover, nanoparticles were investigated with a transmission electron microscope. Specimens were examined with a ZEISS LIBRA 120 digital energy filter transmission electron microscope (EFTEM). Elastic imaging with the in-column omega filter (0 eV) and inelastic imaging with a selected energy loss of 50–130 eV were applied. Furthermore, electron energy loss spectroscopy (EELS) was performed in order to detect nitrogen atom distribution within the particles in order to confirm that chitosan is present within these.

Cell viability assays Cell viability was investigated by means of the resazurin assay which is a routine test at the authors’ laboratory. This assay is an easy and inexpensive alternative to other more common tests. In comparison to MTT assay it also investigates cell metabolism but no lysis of cells is necessary. Compared to assays which investigate membrane integrity, such as LDH assay, it allows a more distinct conclusion regarding cell health. 7

Cell viability was investigated on Caco-2 cells and the medium was composed of 79 % minimum essential medium (MEM) and 20 % FCS and 1% penicillinstreptomycin (100x). The cells were plated in 24-well plates at a density of 1 × 105 cells/well in a final volume of 0.5 mL cell culture medium and incubated at 37 °C in an atmosphere of 5 % CO2, 95% relative humidity. The medium was changed every other day. Caco-2 cell cultures were used for resazurin assays at day 14 after plating. One day before the actual assay, medium was changed to the same composition omitting FCS in order to minimize interferences with FCS. For the assay, the medium was replaced by 0.5 % solution of both polymers in HEPES-buffered saline (HBS) (0.5 mL) after washing the cells with HBS (500 μL) two times. A nanoparticle suspension was prepared in HBS as mentioned above but under aseptic conditions and 500 µL were used for the resazurin assay. HBS served as positive control. Experiments were performed in triplicate. The plate was incubated at 37 °C, 5% CO2, 95% relative humidity for 24 h. After washing the cells with HBS solution two times, 500 μL of a 2.2 µM resazurin solution in MEM were transferred into each well of the plate and incubated for 3 h at 37 °C. Fluorescence measurements with an excitation wavelength λ = 540 nm and an emission wavelength λ = 590 nm were performed in order to determine the degree of resazurin metabolism within Caco-2 cells (Tecan Infinite M200 (Grödig, Austria) [17]. The cell viability was calculated by the following equation:

cell viability % 

sample luorescence  100 control luorescence

Enzymatic phosphate-elimination by isolated IAP 8

In order to investigate whether such polymers and their corresponding nanoparticles are still susceptible to enzymatic cleavage by IAP, polymer solutions (0.7 mg/mL) were prepared. To 10 mL of these solutions 3 mU of IAP were added. Samples (200 µL) were taken from this solution after pre-determined time points (0, 5, 15, 30, 60, 90, 120, 180 and 240 minutes). Further, nanoparticle suspensions were investigated, prepared as mentioned above. IAP was added to 10 mL of the suspensions (3 mU). This mixture was kept at 37 °C and samples were withdrawn at mentioned time points. Samples were centrifuged with a MiniSpin® table centrifuge (Eppendorf, Hamburg, Germany) at 13,000 rpm and the supernatants were analyzed for their phosphate content. Eliminated phosphate was quantified by means of the well-established malachite green (MLG) assay in a slightly modified way [18]. The MLG reagent solution was prepared by dropwise addition of 5 mL of a 7.5 % ammonium molybdate solution to 10 mL of a 0.15 % solution of MLG in 3.6 M H2SO4. In order to stabilize the coloured products during the assay 0.4 mL of a 11 % Triton X-100 solution was added. Samples were analyzed by adding 100 µL sample solution to 300 µL MLG reagent solution and the absorbance was measured at λ = 630 nm. The amount of eliminated phosphate was concluded from a calibration curve of phosphate standards with decreasing phosphate concentration.

Enzymatic phosphate elimination by Caco-2 monolayers In addition to the phosphate elimination studies with isolated IAP, it was investigated whether CMC-PTyr and CS-PTyr are subject to phosphate elimination by Caco-2 cells. For this purpose, Caco-2 cells were grown in 24-well plates at an initial seeding density of 104 cells per well in 1 mL of supplemented MEM medium. Cells were 9

allowed to grow and differentiate for 14 days at 37 °C/5 % CO2 while being fed every other day with supplemented MEM. One day prior to the experiment, cells were fed with MEM without FCS in order to improve reproducibility of the results. Prior to the experiment, cells were washed thrice with HBS in order to reduce phosphate levels to a minimum. For the elimination studies, CMC-PTyr and CS-PTyr were dissolved in HBS at a final concentration of 0.7 mg/mL and 1.5 mL of this solution were added to each well. Samples of 200 µL were taken after pre-determined time-points (0, 5, 15, 30, 45, 60, 90 and 120 minutes). Samples were then analyzed for their phosphate content with the MLG assay as described above. For these experiments, nanoparticles were prepared in HBS (pH 6.8), as well, and 1.5 mL of these suspension were added to the cells. Samples were withdrawn at mentioned time points and treated as mentioned above in order to analyze them with the MLG assay.

Enzyme-induced zeta potential switch In order to demonstrate that the zeta potential of nanoparticles can be changed under the influence of brush border membrane-bound enzymes, nanoparticle suspensions were prepared as mentioned above. The zeta potential was measured prior to the addition of IAP (E = 5 V/cm, 180 s measurement). Subsequently, two units of IAP were added to the suspension and zeta potential was measured again under the same conditions after five minutes. Zeta potential measurements were carried out in demineralized water after slight pH correction to pH 6.8 in order to minimize the influence of buffer salts. Particles were stable in this medium during all investigations. An excess of IAP was utilized to allow a rapid measurement.

RESULTS

Synthesis and characterization of phosphotyrosine-modified polymers 10

Covalent attachment of PTyr to CMC and CS was achieved by amide formation via a simple carbodiimide technique. A schematic depiction of the novel polymers is provided in Figure 1. Spectrophotometric analysis was performed in order to quantify the amount of attached PTyr. It was calculated from a calibration curve that 270.87 ± 6.59 µmol PTyr was attached per gram CMC whereas 698.24 ± 11.06 µmol/g PTyr was attached to CS. This means, approximately 6 % of the anhydroglucose units of CMC and 12 % of the glucosamine units of CS are grafted with PTyr. All purified and lyophilized conjugates were white, fibrous, odorless and easily water soluble.

Nanoparticle preparation and characterization Polyelectrolyte complexes were formed between CMC-PTyr and CS-PTyr at pH 6.8 in the nano-size range. Particle sizes were measured by dynamic light scattering. In Figure 2, a characteristic size distribution for such particles is shown. Very different ratios can be used in order to prepare particles from CMS and CS. During this present study it was found that the ratio of the two polymers did not have a distinct effect on particle size distributions. However, the desired zeta potential can only be achieved within quite a narrow range of polymer ratios. Outside of this range, the zeta potential gets dominated by one of the polymers and leads to higher positive or negative values. Mean diameters of these nanoparticles and zeta potentials are provided in Table 1. For all prepared nanoparticles, polydispersity indices were between 0.125 and. 0.140. Particle sizes have not been changed significantly due to polymer modification as unmodified polymers showed similar distributions and mean diameters ranging from 200 to 300 nm in preliminary experiments depending on the polymer ratio and the resulting zeta potential. Similar particle size could be observed during electron microscopy as can be seen in Figure 3. Furthermore, EELS showed a rather dense nitrogen distribution within the recorded particles (Figure 4) which 11

justifies the assumption that chitosan is a major component of these particles as no other utilized molecule has that many nitrogen atoms within its structure. Therefore, it can be regarded as certain that the observed structures are the synthesized particles. Smaller and less dense structure may be artefacts of polymer remnants that have not formed particles or could not be removed by centrifugation.

Cell viability assay Cell viability of Caco-2 cells was investigated by means of the resazurin assay after treatment with 0.5% solutions of both polymer conjugates and nanoparticles prepared as stated above. Cell viability after treatment with conjugates and nanoparticles does not drop below 80% within the time of investigation as shown in Figure 5. The observed cell viabilities of polymer conjugates and nanoparticles are in good accordance with the fact that all utilized materials can be regarded as safe. During the experiments, no detachment of Caco-2 cells from the plates was observed.

Enzymatic phosphate-elimination by isolated IAP In preliminary experiments, it was found that phosphotyrosine is subject to enzymatic cleavage by IAP (data not shown). As the enzymatic cleavage is supposed to be crucial to a potential zeta potential switch, it was investigated whether PTyr-modified polymers and their corresponding nanoparticles are still prone to phosphate elimination by IAP. Figure 6 shows the time-dependent phosphate elimination of polymers and nanoparticles. It can be concluded that phosphate can still be eliminated even though it needs to be mentioned that the rate of elimination is significantly reduced compared to free PTyr.

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Enzymatic phosphate elimination by Caco-2 monolayers In orientating studies, it was investigated that phosphate can be eliminated from PTyr by Caco-2 cells which serve as a model for the membrane underneath the mucosa. It is assumed, that enzymatic cleavage by membrane-bound enzymes is utterly important to zeta potential inversion at the membrane. Hence, PTyr-modified polymers and corresponding nanoparticles were added to Caco-2 cells. Phosphate elimination was quantified via the malachite green assay. Figure 7 shows that polymers and nanoparticles are cleaved by enzymes secreted by Caco-2 cells. The obtained data correspond well with the results from experiments with isolated IAP as the rank order is pretty similar.

Enzyme-induced zeta potential switch Zeta potential inversion was finally investigated by measuring the zeta potential prior to and after addition of isolated IAP of differently composed nanoparticles. These nanoparticles displayed the desired properties (low monadic negative to neutral zeta potential). Additionally, mean diameters were measured. Results from these experiments are put together in Table 1. As can be seen, the zeta potentials of these nanoparticles changed from approximately -8 mV to + 8 mV in best case. These results are in good accordance with unmodified nanoparticles which were produced from corresponding unmodified polymers. When produced with comparable polymer ratios, zeta potential of unmodified particles ranged from – 2 mV to + 10 mV. All prepared particles changed their potential from the negative to the positive range. These results show that the zeta potential can be changed from negative to positive under the influence of IAP.

DISCUSSION 13

Oral delivery of macromolecules is still one of the major challenges in pharmaceutical sciences due to several physiological barrier functions that drastically reduce the oral bioavailability of such compounds. The most important barriers are the enzyme barrier, the mucus barrier and the membrane barrier. It is considered to be rather easy to master the enzyme barrier by enzyme inhibitory additives or encapsulation, preferably with carrier materials which have enzyme inhibitory properties themselves. However, the membrane barrier and mucus barrier still represent important obstacles to oral administration of therapeutic macromolecules. Some authors suggested either mucus disruption or mucus penetration as possible strategies to overcome the mucus barrier [4,5,19]. Olmsted and co-workers defined a variety of properties that are favorable when mucus should be penetrated: (i) size that allows unrestricted passage of the mucin mesh, (ii) surface without mucoadhesive hydrophobic areas and (iii) high charge density with a net-neutral, highly hydrophilic surface [20]. Rather recently, it was found that under certain circumstances, it may be even possible to traverse mucus layers with nanoparticles as big as 500 nm in diameter [6]. Herein, it was found that particles with a similar size as the ones of the present study diffuse only 6fold slower in mucus than in water

depending on the surface properties of the

nanoparticles. Particles with a distinct positive zeta potentials caused by tertiary amines on their surface were found to be strongly mucoadhesive [5]. Strong mucoadhesion may consequently lead to immobilization in the mucus layer. All these requirements should be addressed by the nanoparticle system that has been developed within this study as polyelectrolyte complexes will provide the high charge density with a hydrophilic surface. As very hydrophilic polymers have been utilized, the presence of hydrophobic areas on the surface is rather unlikely. The required particle size can be ensured by an appropriate manufacturing process. In this present

14

study, particles display mean diameters around 200-300 nm which would be capable of mucus passage. These particles were prepared with a polyelectrolyte complexation method. Similar methods are very common in current literature and usually display yields between 50 and 70 % of the utilized polymers [21,22]. If it is assumed that it is possible to traverse the mucus layer, it is still challenging to get therapeutic macromolecules across intestinal membranes. According to contemporary literature, it is assumed that positively charged nanoparticles are more susceptible to endocytosis than negatively charged ones. This is explained by ionic interactions between negatively charged proteoglycans on cell membranes and positive charges on the particle surface [23]. Hence, an inversion of the zeta potential would be favorable for a drug delivery system. In order to investigate if this is feasible, different experiments have been performed. Phosphate elimination assays with isolated IAP and on Caco-2-monolayers have shown that phosphotyrosine which is bound to polymers as well as corresponding polymer nanoparticles is still a substrate to the enzyme. Consequently, it must be possible to release negatively charged phosphate ions from PTyr and leave back the less charged phenolic hydroxyl group. With an approximate pKa of 7 for the phosphate structure, the ratio between charged and uncharged molecules will be close to 1:1 at physiological pH values according to the Henderson-Hasselbalch equation. In comparison, the remaining phenol structure will be present with an approximate ratio of 1:1000 (charged:uncharged) due to its pKa of about 10. This difference in charge distribution on the backbone of both polymers can have significant impact on the net surface charge of corresponding polymer nanoparticles. Phosphate elimination assays have been performed with isolated enzyme as well as on cell cultures in order to show that a cell model close to human intestine is capable of cleaving the phosphate structure 15

utilized within this study [24]. The results in this study show that Caco-2 cells are able to do so, even though to a significantly reduced extent compared to isolated enzyme. However, this may be a result of several washing steps prior to the experiment. One the one hand, these washing steps prevent falsification of the results by phosphate ions on the cell surface but on the other hand phosphatase which is secreted by Caco-2 cells is also washed of. The fact that no saturation can be observed also hints at rather low enzyme concentrations on the surface of Caco-2 cells within these experiments. Nevertheless, the results of these two experiments suggest, that an in vivo cleavage of such structure may be possible. However, for the overall aim this cleavage alone will not do the job. In order to be an appropriate delivery system for mentioned drugs, it is vital for them to change their zeta potential under the influence of such enzymes in order to facilitate endocytosis of the prepared nanoparticles. Within this study, it was found that zeta potential can be adjusted by using the right ratio of the two oppositely charged polymers within a certain range. Hence, it was possible to prepare different formulations with different zeta potentials between 0 and – 10 mV. These different formulations have been incubated with IAP and zeta potential

was

measured

once

again

after

incubation.

These

experiments

demonstrated that these particles can change their zeta potential when they are incubated with IAP. These results of these experiments show that it is possible to create nanoparticles with an appropriate zeta potential in order to pass the mucus barrier. When they do so, it also appears possible for them to change their zeta potential due to enzymatic cleavage in a manner that allows endocytosis by endothelial cells where potential drugs can be released and take their full effect. This means, within this study the basis to master two major barriers for oral nucleotide delivery is provided. However, these nanoparticles will not be stable at gastric pH due to the dramatically reduced charge of CMC. Therefore, it will be necessary to use 16

gastro-resistant delivery devices in order to deliver these nanoparticles to the intestine. In order to allow the release of phosphate ions by IAP it is necessary that phosphate esters are easily accessible to the enzyme. From a toxicology point of view, it is of course favorable to use as many well-known, unmodified substances as possible. However, both polymers were modified herein as the structure of the polymers within the particles after their entanglement is not known. By this approach the probability to have phosphate ester on the surface of the nanoparticles was maximized. In order to be a valuable tool for oral delivery of the mentioned drugs, it is of course necessary that the prepared polymers and nanoparticles do not show toxic effects. As CMC is categorized as GRAS (generally regarded as safe) by the FDA and several CS-products have been granted GRAS status and PTyr even shows cytoprotective effects in some studies [25], it is not expected that the generated polymers display toxic effects. However, a cell viability assay has been performed with the polymers and corresponding nanoparticles within the scope of this study. This assay did not show a significant toxic effect on Caco-2 cells. Hence, it can be assumed that the polymers and nanoparticles of this study do not show significant toxic properties.

CONCLUSION Within this study, two novel polymer modifications have been generated and nanoparticles have been prepared from these. These nanoparticles are hydrophilic in nature and probably show a dense charge distribution on their surface as they are polyelectrolyte complexes. However, their surface charge is rather low. Hence, they might show some similarity to viruses that are capable of mucus passage [20]. These particles are prone to enzymatic cleavage of their phosphate ester which obviously 17

results in a zeta potential change to the positive range. Positive nanoparticles can finally be internalized by endothelial cells which can be the target for certain drug actions. Hence, a novel enzymatically induced zeta potential inverting system has been generated. This innovative development may set novel and fascinating options for future drug delivery systems of hydrophilic macromolecules.

ACKNOWLEDGEMENTS The research leading to these results has received funding from the European Community´s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 280761.

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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

FIGURE CAPTIONS

Figure 1. Schematic depiction of the two novel polymer modifications CMC-PTyr (A) and CS-PTyr (B)

Figure 2. Exemplary size distribution of interpolymer complex nanoparticles generated from CMC-PTyr and CS-PTyr

Figure 3. Transmission electron microscopy photographs of prepared nanoparticles

Figure 4. Electron energy loss spectroscopy photographs of prepared nanoparticles. Green dots display nitrogen distribution within the particles.

Figure 5. Cell viability according to intracellular resazurin metabolism after 3 h incubation Indicated values are means of at least three experiments ± standard deviation

Figure 6. Time-dependent phosphate elimination of CS-PTyr (♦), CMC-PTyr (■) and nanoparticles (▲) by isolated intestinal alkaline phosphatase. Indicated values are means of at least three experiments ± standard deviation.

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Figure 7. Time-dependent phosphate elimination of of CS-PTyr (♦), CMC-PTyr (■) and nanoparticles (▲) by secretions of cultured Caco-2 cells. Indicated values are means of at least three experiments ± standard deviation.

Table 1. Characterization of different nanoparticle formulations regarding mean diameter and zeta potential prior (ZP0) and after (ZPIAP) incubation with intestinal alkaline phosphatase. Indicated values are means of at least three experiments ± standard deviation. CMC-PTyr [%]

CS-Ptyr [%]

Mean diameter [nm]

ZP0 [mV]

ZPIAP [mV]

0.05

0.025

301.50 ± 101.91

-7.77 ± 0.24

8.10 ± 0.22

0.05

0.03

271.80 ± 80.90

-5.96 ± 0.44

6.68 ± 0.27

0.05

0.0325

245.60 ± 84.01

-6.56 ± 0.23

5.36 ± 0.30

0.05

0.035

208.30 ± 49.56

-5.07 ± 0.49

5.37 ± 0.21

0.05

0.0375

198.30 ± 66.24

-5.12 ± 0.19

4.64 ± 0.48

22

23

24 4

25

26

Gra aph hica al abs a stra act

Hig ghlligh hts s • • • • •

Tw wo novel ph hosp pho orylate ed poly p yme ers were syn s nthe esiz zed Nano opa articcless we ere e prepa ared d by poly p yele ectro olytte com c mple exa ation n off the tw wo pollym merss Thesse nan n nopartiicle es disp d playy ap ppro opriiate e ch hara acte eris sticss fo or m muccus perme eatiion Enzyyma aticc de epho osp pho oryla atio on of o th he nan n nop partiicle es le ead ds to o zeta a po oten ntial inverrsio on Nove el poly p yme ers and d co orre esp pond ding g nano opa articcless do o no ot sshow toxicc effeccts

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