Zeta Potential Changing Polyphosphate Nanoparticles: A Promising

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Zeta Potential Changing Polyphosphate Nanoparticles: A promising approach to overcome the mucus and epithelial barrier Zeynep Burcu Akkus, Imran Nazir, Aamir Jalil, Martina Tribus, and Andreas Bernkop-Schnürch Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00355 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 10, 2019

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Molecular Pharmaceutics

Zeta Potential Changing Polyphosphate Nanoparticles: A promising approach to overcome the mucus and epithelial barrier Zeynep Burcu Akkusa, Imran Nazira,b, Aamir Jalila, Martina Tribusc, Andreas BernkopSchnürcha* a Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria b Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, 22060 Abbottabad, Pakistan c Institute of Mineralogy and Petrography, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria

*Corresponding author at: University of Innsbruck, Institute of Pharmacy, Pharmaceutical Technology, Center for Chemistry and Biomedicine, Innrain 80–82/IV, A-6020 Innsbruck, Austria. Tel.: +43 512 507 58600; fax: +43 512 507 58699. E-mail address: [email protected] (A. Bernkop-Schnürch).

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Abstract Aim: The aim of the present study was to develop zeta potential changing polyphosphate nanoparticles (pp-NPs) in order to overcome the diffusion barrier of the mucus gel layer and to provide an enhanced cellular uptake. Methods: pp-NPs were obtained by in situ gelation between cationic polyethylene imine (PEI) and anionic polyphosphate. The resulting pp-NPs were characterised regarding size and zeta potential. Phosphate release studies were carried out by incubation of pp-NPs with isolated as well as cell-associated intestinal alkaline phosphatase (IAP) and quantified by malachite green (MLG) assay. Correspondingly, change in the zeta potential was measured and pp-NPs were analysed by scanning electron microscopy (SEM) studies. Mucus permeation studies were performed with porcine intestinal mucus via transwell insert method and rotating tube method. Furthermore, cell viability and cellular uptake were investigated on Caco-2 cells. Results: The resulting pp-NPs displayed a mean size of 269.16±1.12 nm and a zeta potential between -9 and -10 mV in characterisation studies. Within 4 h a remarkable amount of phosphate was released from pp-NPs incubated with isolated IAP as well as cell-associated IAP and zeta potential raised up from -9.14±0.45 mV to -1.75±0.46 mV. Compared with dephosphorylated polyphosphate nanoparticles (de-pp-NPs) a significantly enhanced mucus permeation of pp-NPs was observed. Moreover, pp-NPs did not exhibit cytotoxicity. Cellular uptake increased 2.6-fold by conversion of pp-NPs to de-pp-NPs following enzymatic cleavage. Conclusion: Taking the comparatively simple preparation method and the high mucus permeating properties of pp-NPs and high cellular uptake properties of de-pp-NPs into account, these nanocarriers might be promising novel tools for mucosal drug delivery.


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Keywords Polyphosphate, zeta potential change, polyethylene imine, mucosal drug delivery, intestinal alkaline phosphatase, cellular uptake, mucus permeation Graphical Abstract



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Introduction

Nanocarrier systems hold great promise providing various benefits for mucosal drug delivery such as protection of therapeutics towards body fluids and drug release controlling properties (1). Because of their nanometric size they can move across the mucus gel layer representing a barrier that covers mucosal membranes and provides protection against invadors such as pathogens, toxins and in a similar way drug molecules (2). After the mucus barrier, however, nanoparticles are confronted with the epithelial barrier that restricts them from cellular entry (3-5). Hence, successful mucosal delivery of nanoparticles can only be achieved when both mucus and epithelial barriers are addressed by modulating key parameters of these carrier systems including size, surface hydrophobicity and surface charge. Nanoparticles with negative or neutral surface charge can rapidly permeate the mucus gel layer, whereas positively charged nanoparticles are trapped in the mucus network due to electrostatic interactions between these nanocarriers and anionic substructures of the mucus gel layer such as sialic and sulfonic acid (1, 6). On the cell membrane, however, the situation completely changes as positively charged particles can be uptaken by epithelial cells to a much higher extent than negative ones (7-9). Generally, cationic particles do not reach the epithelium as they are either immobilized through ionic interactions in the mucus or are coated by an anionic layer on their way through the mucus to the cell membrane (10-12). In order to address this polycation dilemma, zeta potential changing carrier systems including polymeric nanoparticles and self-emulsifying drug delivery systems (SEDDS) were developed (12-15). Especially negatively charged nanocarriers bearing phosphate groups were utilised to provide high mucus permeating properties that were in the following cleaved off on the cell membrane by intestinal alkaline phosphatase (IAP) to facilitate cellular entry. Bonengel et al. have verified the charge inversion of polyelectrolyte complex nanoparticles consisting of carboxymethyl cellulose (CMC) and 6-phosphogluconic acid (6-PGA) conjugated PEI due to IAP treatment ACS Paragon Plus Environment

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(16). In another study, the surface of polyelectrolyte complex nanoparticles were enzymatically phosphorylated and the change in zeta potential was demonstrated (12). These so far established systems, however, were all based on new conjugates that would need to be toxicologically tested and upscaled to pave the way for Good Manufactruting Practices (GMP) material and clinical trials. In contrast to these new conjugates, polyphosphates are well-established and widely used in different areas as food additive, water retardant or fertilizer due to their low cost, safety and biodegradability. Usage of sodium polyphosphate (PolyP) as food additive is permitted by legal authorities in Europe (17). According to the toxicological monograph that is issued by The Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), PolyP can be used up to 70 mg/kg daily in humans (18). Moreover, cleavage of polyphosphates by IAP was already demonstrated by Yoza et al. (19). Having on the one hand the troublesome process of getting new excipients registered and on the other hand the advantages of polyphosphate in mind, it was the aim of study to apply the zeta potential change concept to polyphosphate containing nanoparticles. Hence, polyphosphate nanoparticles (pp-NPs) were prepared and characterised regarding the zeta potential change, phosphate release, mucus permeation, cell viability and cellular uptake. 2 2.1

Materials and Methods Materials

Sodium polyphosphate (PolyP, molecular mass (mm) 611 kDa, Graham´s Salt), polyethylene imine (PEI, mm 25 kDa, branched), alkaline phosphatase from bovine intestinal mucosa (7165 units/mg protein), magnesium chloride anhydrous (MgCl2) 98%, zinc chloride anhydrous (ZnCl2) 97%, potassium phosphate monobasic (KH2PO4) 99.5%, phosphatase inhibitor cocktail 2, Triton X-100, ammonium molybdate tetrahydrate 81–83%, malachite green (MLG) oxalate


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salt 90%, fluorescein diacetate (FDA), minimum essential medium eagle (MEM), glucose D (+) ≥ 99.5%, anhydrous and resazurin sodium salt were purchased from Sigma-Aldrich (Vienna, Austria). Fetal bovine serum (FBS), penicillin-streptomycin liquid and phosphate buffered saline (PBS) were purchased from Biochrom, Merck (Tutzing, Germany). D-(+)Trehalose dihydrate >98.0% (GC) was purchased from TCI Chemicals (Eschborn, Germany). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) ≥99.5% was obtained from ROTH GmbH (Karlsruhe, Germany). 2.2 2.2.1

Methods Preparation

of

Polyphosphate

Nanoparticles

and

Dephosphorylated

Polyphosphate Nanoparticles Polymeric nanoparticles were prepared by the in situ gelation method using oppositely charged hydrophilic polymers. PEI solution was prepared by dissolving the PEI in 100 mM HEPES buffer pH 7 at a concentration of 0.084 mg/ml. PolyP was dissolved in deminaralized water in concentrations as listed in Table 1 and increasing amounts of polymers were added in order to obtain uniform pp-NPs. In detail, to 6 ml of PEI solution, 1 ml of an aqueous solution of PolyP was added dropwise under constant magnetic stirring at 800 rpm and left under magnetic stirring at room temperature for 30 min. Afterwards, 2% (m/v) trehalose was added to NP suspensions in order to avoid particle aggregation prior to centrifugation. pp-NPs were purified by centrifugation at 918 g for 15 min with a Mini Spin Centrifuge (Eppendorf, Hamburg, Germany). The supernatant was removed and pp-NPs were resuspended in 100 mM HEPES buffer pH 7. de-pp-NPs were obtained by incubation of 10 ml of 0.01% (m/v), 0.05% (m/v) or 0.1% (m/v) of pp-NPs with 0.1 ml, 0.5 ml or 1 ml of IAP solution (10 U/ml), respectively, at 37˚C under constant shaking at 300 rpm by Thermomixer for 300 min (Eppendorf, Germany).


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Table 1. Size, poly dispersity index (PDI) and zeta potential of pp-NPs obtained by in situ gelation of PEI and PolyP at indicated mass ratios. Formulation PEI:PolyP PEI PolyP Size PDI Code Ratio Concentration Concentration (nm) (m/m) (mg/ml) (mg/ml) F1 1:1.5 0.084 0.75 441.87±31.78 0.298 F2 1:2 0.084 1 332.01±2.30 0.083 F3 1:2.5 0.084 1.25 269.16±1.12 0.118 F4 1:2.75 0.084 1.375 270.2±1.30 0.104 F5 1:3 0.084 1.5 267.0±1.55 0.099

2.2.2

Zeta Potential (mV) -0.29±0.74 -2.58±0.42 -9.77±1.01 -14.7±0.10 -22.3±1.68

Preparation of FDA Labelled Polyphosphate Nanoparticles

A freshly prepared 1.5% (m/v) FDA solution in acetonitrile was added to the pp-NP suspension described above in a volume ratio of 1:2. The mixture was incubated under dark conditions with continuous shaking at 600 rpm by Thermomixer (Eppendorf, Germany) at 18˚C for 2 h. Trehalose was added to the FDA labelled pp-NPs suspension in a final concentration of 1% (m/v) in in order to avoid aggregation prior to centrifugation. Thereafter, pp-NPs were purified by centrifugation at 918 g for 15 min and resuspended in 100 mM HEPES buffer pH 7. 2.2.3

Characterization of Polyphosphate Nanoparticles

Size, poly dispersity index (PDI) and zeta potential of pp-NPs were determined by photon correlation spectroscopy and laser Doppler anemometry using a Zetasizer Nano-ZS (Malvern Instruments, UK). Briefly, 0.01% (m/v) of pp-NP suspensions were prepared in demineralized water prior to analysis. Each measurement was performed in triplicate at 25 ˚C with a detection angle of 173˚. 2.2.4

Phosphate Release Studies

2.2.4.1 Malachite Green Assay Malachite green (MLG) assay was utilised to determine time dependent phosphate release from pp-NPs according to a previously described method with slight modifications (20). Briefly,


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MLG salt was dissolved in 3.6 M H2SO4 at a concentration of 0.15% (m/v). 100 μl of 11% (m/v) Triton X-100 solution was added to 2.5 ml of MLG solution and incubated at room temperature for 5 min. Thereafter, 1.5 ml of 8% (m/v) ammonium molybdate solution was added to this mixture dropwise under constant mixing by vortex. 100 μl of this MLG assay solution was added to 50 μl of test samples in the 96 well plate and absorbance was measured at 630 nm with a microplate reader (Tecan inﬁnite M 200; Gröding, Austria). The amount of released phosphate was calculated using a standard curve that was obtained by measuring the absorbances of increasing amounts of KH2PO4. 2.2.4.2 Enzymatic Phosphate Cleavage by Isolated Intestinal Alkaline Phosphatase Time dependent phosphate cleavage from pp-NPs was investigated using isolated IAP and phosphate release was quantified by MLG assay as described above. In brief 0.01% (m/v) of pp-NPs suspension was prepared in 100 mM HEPES buffer pH 7.2 containing 5 mM MgCl2 and 0.2 mM ZnCl2. Afterwards, 100 μl of IAP solution (10 U/ml) was added to 10 ml of pp-NP suspension and incubated at 37 ˚C under continuous shaking at 300 rpm for 240 min. Aliquots of 50 μl were withdrawn at predetermined time points (0, 15, 30, 90, 180 and 240 min) and placed on a 96 well plate. pp-NP suspensions omitting IAP served as control and were incubated under the same conditions. Enzymatic activity was terminated by the addition of 5 μl of 3.6 M H2SO4 to the 50 μl aliquots and phosphate release was determined by MLG assay. 2.2.4.3 Enzymatic Phosphate Cleavage by Caco-2 Cell Monolayer Caco-2 cells were purchased from European collection of cell cultures (ECACC, health protection agency, UK). Cells were seeded in 24 well plates (25,000 cells/well) and cultured with minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ˚C and 5% CO2 atmosphere. Old culture medium was changed with fresh medium in alternate days until cell monolayer was observed. Prior to experiment, cells were washed twice with glucose-HEPES buffer (268 mM glucose and 25 mM ACS Paragon Plus Environment

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HEPES at pH 7.4). Cells were incubated with 500 μl of 0.01% (m/v) of pp-NP suspensions prepared in the glucose-HEPES buffer. 50 μl aliquots were transferred into a 96-well plate at predetermined time points (0, 5, 15, 30, 60, 120, 180 and 240 min). Furthermore, the test was carried out in the presence of the phosphatase inhibitor cocktail diluted at a ratio of 1:100 in the same buffer as control. Enzymatic activity was terminated by addition of 5 μl of 3.6 M H2SO4 to the 50 μl aliquots and phosphate release was determined by MLG assay as described above. 2.2.5

Zeta Potential Change

Time dependent change in zeta potential was determined in the presence of isolated IAP utilising photon correlation spectroscopy as mentioned above (Zetasizer Nano-ZS, Malvern nstruments, UK). 0.01% (m/v) of pp-NP suspensions were prepared in 100 mM HEPES buffer pH 7.2 containing 5 mM MgCl2 and 0.2 mM ZnCl2. To 10 ml of pp-NP suspension, 100 μl of IAP solution (10 U/ml) was added and incubated for 300 min. Nanoparticle suspensions omitting IAP served as control and were incubated under the same test conditions. Enzyme induced zeta potential change of pp-NPs was measured at predetermined time points (0, 5, 15, 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min). 2.2.6

Scanning Electron Microscopy Studies

In order to evaluate the effect of IAP on the pp-NPs visually, SEM studies were performed. Accordingly, 0.1% (m/v) of pp-NPs and de-pp-NPs suspensions were prepared in 100 mM HEPES buffer pH 7.2. The 50 μl of suspensions of pp-NPs and de-pp-NPs were placed on carbon tapes as small droplets and were left at 37˚C in an incubator (Memmert Typ U15, Germany) for 24 h. Thereafter, pp-NPs and de-pp-NPs were coated with a thin layer of gold, 10 nm in thickness using a Polaron SEM coating unit E5100. Microstructural images with a magnification of 15,000 x at a working distance of 14 mm were obtained on a JEOL JSM-6010 LV scanning electron microscope. The SEM was operated under high vacuum conditions at 15 kV acceleration voltage.


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2.2.7

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Mucus Permeation Studies

2.2.7.1 Intestinal Mucus Purification Porcine intestine was obtained freshly from a local slaughterhouse. The intestine was divided into small fragments and incised longitudinally. Thereafter, mucus was collected from the walls of the fragments with the aid of a spatula to the falcon tubes. Collected mucus was weighed and 5 ml of 0.1 M sodium chloride was added to each 1 g of mucus and incubated under gentle stirring at 4 ˚C for 1 h. In order to purify mucus from the remaining debris, mixture was centrifuged at 11,246 g at 4 ˚C for 1 h and the supernatant was removed. Clean parts were collected, weighed and 2.5 ml of 0.1 M sodium chloride was added to each 1 g of mucus and incubated under gentle stirring at 4 ˚C for 1 h. Afterwards, centrifuge step was repeated once at 11,246 g at 4 ˚C for 1 h. Clean mucus was collected and stored at -20 ˚C until further use. 2.2.7.2 Transwell Insert Method In order to determine the mucus permeation ability of pp-NPs transwell insert method was applied as described previously with some modifications (21). For mucus permeation studies, transwell inserts with 33.6 mm2 surface area and 3 μm pore size (Greiner-Bio One, Austria) were placed on 24 well plate and covered with 60 mg of porcine mucus. The acceptor chamber of all wells was filled with 500 μl of 100 mM HEPES buffer pH 7. The donor chambers were filled with 250 μl of FDA labelled 0.05% (m/v) of pp-NPs or de-pp-NPs suspensions in 100 mM HEPES buffer pH 7 or 100 mM HEPES buffer pH 7 only. Afterwards, aluminium foil covered plate was incubated on a shaking board at 37 ˚C and 300 rpm (Vibramax 100, Heidolph Instruments, Schwabach, Germany). Aliquots of 100 μl were withdrawn from each well and replaced with the same amount of pre-warmed buffer solution at predetermined time points (0, 1, 2, 3 and 4 h). After 4 h, samples were incubated with 10 μl of 5 M NaOH for 30 min, to hydrolyse FDA to its fluorescently measurable analogue, sodium fluorescein. Fluorescent intensities were measured at an excitation wavelength of 480 nm and an emission wavelength ACS Paragon Plus Environment

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of 520 nm with microplate reader (TECAN infinite M200, Austria). The transwell inserts without mucus gel layer and the transwell inserts without pp-NP suspension represented the positive and negative control, respectively. The percentages of nanoparticle permeation were calculated based on the values of positive and negative controls including the cumulative corrections. 2.2.7.3 Rotating Tube Method To further investigate the diffusion of pp-NPs across purified porcine intestinal mucus, rotating tube method was applied as described previously (22). Briefly, silicon tubes were divided into 60 mm length pieces and their one end closed with end cap. Afterwards, silicon tubes were filled with 300 μl of mucus. FDA labelled 0.05% (m/v) pp-NP or de-pp-NPs suspensions were dispersed in 100 mM HEPES buffer pH 7 and 50 μl of of each suspension were added to open end of mucus filled tubes and closed with an end cap. After 24 h incubation time the tubes were frozen at -80 ˚C and then cut into 2 mm slices. Slices were treated with 5 M NaOH for 30 min in order to obtain sodium fluorescein from FDA. Thereafter, samples were sonicated in an ultrasonic bath for 1 h and centrifuged for 5 min at 12045 g with a Mini Spin Centrifuge (Eppendorf, Hamburg, Germany). 100 μl of supernatant from each sample was added to 96 well black plates and fluorescence intensities were measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm with microplate reader (TECAN infinite M200, Austria). Silicon tubes containing mucus without nanoparticle suspensions and silicon tubes containing nanoparticle suspensions without mucus served as negative and positive controls, respectively. The percentages of nanoparticle permeation were calculated based on the values of positive and negative controls. 2.2.8

Cellular Viability

Viability studies were performed on Caco-2 cell monolayer and cellular viability was assessed by resazurin assay as described by Perera et al. with some modifications (13). Cells were ACS Paragon Plus Environment

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incubated at a density of 25,000 cells/well in a 24 well plate with serum-supplemented MEM at 37 ˚C in an atmosphere with 5% CO2 and 95% humidity. The MEM was replaced with fresh medium on alternate days until a cell monolayer was obtained and one day before the experiment MEM was replaced with MEM omitting the FBS. pp-NP suspensions were diluted properly in 10 mM PBS pH 7.4 to obtain following concentrations of pp-NPs: 0.005 (m/v), 0.01 (m/v) and 0.02 (m/v). Afterwards, cells were washed twice with phosphate buffered saline (PBS) and NP suspensions were added to the wells. PBS and 2% (m/v) Triton X-100 solution were utilised as the positive control and negative control, respectively. At predetermined time points (3 h and 24 h) cells were washed twice with PBS and 2.2 mM resazurin solution was added to each well and incubated at 37˚C in dark conditions for 3 h. Subsequently, 100 µl of aliquots from each well was transferred into a 96 well black titer plate and fluorescence intensities were measured at excitation wavelength 540 nm and emission wavelength 590 nm (Tecan infinite M200; Gröding, Austria). Cell viability was assessed according to following equation:

Cell viability (%) =

2.2.9

Sample Fluorescence x100 Control Fluorescence

In Vitro Cellular Uptake

In vitro cellular uptake studies of FDA labelled pp-NPs were performed in Caco-2 cells. Accordingly, Caco-2 cells were seeded in 24 well plates at a density of 25,000 cells/well in the presence of MEM supplemented with 10% FBS. Medium was changed in alternate days until a cell monolayer was observed. Prior to experiment cells were washed twice with glucoseHEPES buffer (268 mM glucose and 25 mM HEPES at pH 7.4). Afterwards, cells were incubated with glucose-HEPES buffer with or without 1% v/v phosphatase inhibitor for 1 h. Cells were washed twice with glucose-HEPES buffer and pp-NP suspensions were applied


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(0.01% m/v in glucose-HEPES buffer) to the cells and incubated for 1 h and 3 h. Then, cells were washed with glucose-HEPES buffer and 500 μl of glucose-HEPES buffer were added to each well except positive control wells (100%) at the end of incubation times (1 h and 3 h). Thereafter cells were incubated with 200 μl of 2% Triton-X 100 solution in 5 M NaOH for 30 min in order to obtain sodium fluorescein following cell lysis. Aliquots were transferred to 96 well plates and fluorescence intensities were measured with microplate reader at λext=485 nm and λem=535 nm. Cells which are incubated only with buffer represented the negative control (0%). Uptake efficiency was measured utilising the following equation,

Uptake Efficiency (%) =

Ft – F0 F100 – F0

where the F100 is 100% value, F0 is 0% value and Ft is intensity of the sample. 2.2.10 Statistical Data Analysis In this study, GraphPad Prism 5.01 software was utilised for statistical data analysis. Unpaired student´s t-test was used in order to investigate the difference between two independent groups. *p≤0.05 and **p˂0.01 were set as levels of significant, very significant and highly significant, respectively. Mean values were calculated from at least three independent measurements and data expressed as mean ± standart deviation (SD). 3 3.1

Results and Discussion Preparation and Characterisation of Polyphosphate Nanoparticles

pp-NPs were obtained via in situ gelation utilising the electrostatic interactions between the oppositely charged groups of PEI and PolyP as illustrated in Figure 1.


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NH2 NH2

N

NH2

NH N

N H

H N

N

NH2

N NH2

H2N Branched polyethylenimine, 25 kDa

n

Sodium polyphosphate

OH

OH

O

O HO

O

O HO

NH2

O NH3

NH N

N H

O

P

HO

O

P O NH3

N

O

P

HO

O

P

O

P

HO

O

P

n

HO

n

H N

N

NH2

N O O

H3N

P

OH

O

O

NH3

P

OH

O

P

OH

n

O

P

OH

O

O O

n

O

O n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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P

OH

O

OH

P

OH

OH

Polyphosphate Nanoparticles

Figure 1. Formation of pp-NPs by in situ gelation between branched chain PEI (25 kDa) and PolyP.


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In case of 1 mg/ml solution of PEI, formation of white precipitates were observed by the dropwise addition of PolyP within minutes. This aggregation is caused by the strong inter-chain electrostatic interactions between PEI and PolyP due to the high concentration of branched PEI. Moreover, the intra-chain repulsion of the amine groups of branched PEI decreases in the concentrated solutions and therefore a collapse of the hydrophobic backbone of the polymer occurs (23). Similarly, Suschaoin et al. observed at higher concentrations a more pronounced precipitation of branched PEI than linear PEI with CMC (24). Therefore, more diluted stock solutions of PEI (0.083 mg/ml) were used for the subsequent studies. Furthermore, mixing ratio and addition order of the polymers affect aggregation and precipitation. Milky suspensions without precipitates occurred by the dropwise addition of PolyP solution to PEI solution under continuous stirring. Thereby pp-NPs in the range of 260-340 nm with a narrow size distribution were obtained. As illustrated in Table 1, particle size decreased with the addition of higher amounts of PolyP due to increased electrostatic interactions between oppositely charged polymers resulting in more compact nanoparticles. Resulting pp-NPs displayed zeta potential values from -0.286±0.74 mV to -22.3±1.68 mV depending on the added amount of PolyP as listed in Table 1. These results are in good agreement with the study of Kiilll et al. that indicated a decrease in positive zeta potential values of chitosan-PolyP nanoparticles with increasing amounts of PolyP due to compensation of positive charges (25). Accordingly formulation F3 displaying a mean size of 269.16±1.12 nm and a zeta potential of -9.77±1.01 mV was chosen for further experiments. 3.2 3.2.1

Phosphate Release Studies Enzymatic Phosphate Cleavage by Isolated Intestinal Alkaline Phosphatase

IAP is a membrane bound metalloenzyme containing bound Zn2+ and Mg2+ ions in its active sites which are required for phosphate cleavage activity (26, 27). Moreover, Lorenz and Schröder have observed that polyphosphate degradation activity of intestinal alkaline ACS Paragon Plus Environment

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phosphatase was inhibited in the presence of EDTA confirming that Zn2+ and Mg2+ ions might be required for enzymatic activity (28). Therefore MgCl2 and ZnCl2 were utilised in order to guarantee the enzymatic activity of isolated intestinal alkaline phosphatase. On the other hand, IAP is a brush border enzyme which hydrolyses a variety of moieties such as phosphate esters of alcohols, amines and phenols (29). Moreover, Yoza et al. indicated the catalytic activity of calf IAP on the hydrolyses of pyrophosphates and triphosphates due to the cleavage of the PO-P bonds, in addition to the catalytic effect of IAP on the cleavage of P-O-C bonds of monophosphate esters (19). It has also been shown that IAP has an ability to cleave polyphosphates (30, 31). Within this study, pp-NPs were incubated with isolated IAP and timedependent release of phosphate groups was determined by MLG assay. As shown in Figure 2, 1.12±0.06 µmol of phosphate were released per mg of pp-NPs within the first 90 min, followed by a plateau phase thereafter. In case of the control group without addition of IAP, no phosphate release was observed confirming that the cleavage of polyphosphate groups from the surface of pp-NPs is exclusively related to the enzymatic activity of IAP. This behaviour was also in good agreement with previous studies of Lorenz and Schroder investigating the catalytic activity of IAP on polyphosphates of different chain lengths. Their results revealed a time-dependent increase of free phosphate being released from polyphosphates with a chain length of 12-23 phosphate residues within 90 minutes (28).


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Figure 2. Phosphate release from pp-NPs in 100 mM HEPES buffer pH 7.2 containing 5 mM MgCl2 and 0.2 mM ZnCl2 by enzymatic cleavage with isolated IAP at 37˚C over time. Data is presented as mean ± SD (n = 3). 3.2.2

Enzymatic Phosphate Cleavage by Caco-2 Cells

Caco-2 cell line has been used as an effective in vitro model of intestinal epithelium for drug permeability studies due to its morphological and biochemical features which resemble intestinal brush border membrane (32). Prüfert et al. verified the presence of IAP in Caco-2 cell line by immunocytochemistry images (33). Moreover, Wu et al. have analysed the activity of IAP via a commercial alkaline phosphatase assay kit and results indicate that Caco-2 cell line expresses higher amounts of IAP compared with HT29-MTX-E12 cell line which is also used as an in vitro epithelium model (34). Therefore Caco-2 cell line was utilised for time dependent phosphate release studies in order to evaluate the phosphate release from pp-NPs under in vitro conditions and results are shown in Figure 3. Within 90 min 0.92 ± 0.29 µmol of phosphate was released per mg of pp-NPs being in accordance with isolated IAP studies. Similar to the studies carried out with isolated IAP, in case of control group containing an inhibitor cocktail to supress IAP, no phosphate release was observed. These results were in good agreement with previous studies of Bonengel et al. where phosphate release was observed with phosphate group (6-PGA) bearing branched PEI nanoparticles changing their zeta potential on Caco-2 cell line (15).


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Figure 3. Phosphate release from pp-NPs in 268 mM glucose 25 mM HEPES buffer pH 7.4 by enzymatic cleavage with IAP expressed on Caco-2 cell line at 37˚C over time. Data is presented as mean ± SD (n = 3). 3.3 Zeta Potential Change Phosphate release studies were supported by time dependent zeta potential measurements of pp-NPs. A considerable change in zeta potential from -9.14±0.45 mV to -1.75±0.46 mV was observed within the first 90 min similar to time dependent phosphate release studies. Conformably, a plateau was observed after 90 min and zeta potential raised up to -1.18±0.12 mV within 5 h as indicated in Figure 4. Within this study, anionic charges were placed on the surface of nanoparticles with the aid of polyphosphates in order to obtain positive zeta potential following IAP treatment. After 90 min, however, velocity of zeta potential change decreased and within 5 h just a slightly negative charge could be observed. This downtrend could be explained by the inhibitory effect of free phosphate ions being released by enzymatic cleavage with IAP. It has been demonstrated that bound phosphate stimulates IAP activity in a dose dependent manner. On contrary, free phosphate ions are substrates and competitive inhibitors of IAP (26, 35, 36). Accordingly, time dependent phosphate release and thus change in zeta potential can be attributed to the activation of IAP by bound PolyP. As soon as the concentration of released free phosphate groups reaches a sufficiently high level a further change in the zeta


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potential is restricted by inhibition of IAP. Similar results were obtained in a previous study by Nazir et al. showing a zeta potential change of phosphorylated chitosan-chondroitin sulphate nanoparticles (12).

Figure 4. Time dependent zeta potential change of pp-NPs by isolated IAP in the presence ( 3.4

) and absence (

) of isolated IAP. Data is presented as mean ± SD (n = 3).

Scanning Electron Microscopy Studies

To evaluate the effect of IAP application on the morphology and integrity of pp-NPs, SEM studies were performed. Prior to IAP application, spherically shaped pp-NPs with surface phosphate groups as light apparent rings were observed as indicated in Figure 5 (A). Incubation of particles in the presence of IAP for 5 h, however, caused an increase of particle size, alteration of particle morphology and aggregation as illustrated in Figure 5 (B). These changes are explained by the cleavage of phosphate groups from the surface of pp-NPs by IAP. Aggregation likely occurred due to the partial disintegration of the nanoparticle structure triggered by the enzymatic cleavage of ionically crosslinking PolyP on pp-NPs, in a similar way to the study of Baier et al.. Within SEM studies, they observed change of particle morphology and increase of particle size due to disruption of polymer chains of poly (L-lactide) (PLLA) nanoparticles by protein kinase K activity resulting in a targeted drug release (37). In addition, the decrease in zeta potential of pp-NPs due to the removal of phosphate groups leads to a ACS Paragon Plus Environment

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reduction of electrostatic repulsion between the de-pp-NPs causing aggregation and increase of particle size of the de-pp-NPs.

Figure 5. SEM images of pp-NPs (A) and de-pp-NPs (B). Scale bar is equal to 1 μm. 3.5

Mucus Permeation Studies

Permeation behaviour of pp-NPs in the purified intestinal porcine mucus was determined by two different procedures. In case of transwell insert method nanoparticles move vertically whereas in the rotating tube method they move horizontally without a membrane barrier directly contacting with the mucus (38). 3.5.1

Intestinal Mucus Purification

Porcine intestinal mucus is the closest model to human intestinal mucus compared with other animal models, in terms of certain features including intestinal mucus thickness, rheological properties and mucin molecular weight (39-41). Nevertheless, it has been stated that native mucus samples might exhibit significant variations of rheological properties and mucin content depending on the porcine intestines obtained from different animals. Hence, the usage of commercial mucin products was addressed (42, 43). However, these products are inadequate to resemble human mucus even in the higher concentrations due to absence of gel forming properties caused by alteration of mucin structure during purification process (38). Moreover,


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high degree of variations were also reported in case of human mucus between individuals as well as in case of each single individual (40). Therefore, native mucus from porcine intestine was purified and afterwards utilised for following mucus permeation studies. 0.1 M NaCl solution was utilised in order to wash the scraped mucus from intestine to remove the food particles and debris prior to the centrifugation step (38). Washing process is essential to obtain visually clean mucus which is required for mucus permeation studies in order to evaluate permeation behaviour of the particles in nanosize range. 3.5.2

Transwell Insert Method

In order to evaluate the permeation behaviour of pp-NPs across the mucus-covered membrane, transwell insert method was used and results are illustrated in Figure 6. As control group, ppNPs were incubated with IAP prior to experiment in order to cleave the phosphate groups and to obtain de-pp-NPs. Within this experiment, pp-NPs displayed 2-fold higher mucus permeating properties than de-pp-NPs. This result can be explained by the electrostatic repulsion between the negatively charged sialic and sulfonic moieties of the mucus gel layer and negatively charged pp-NPs facilitating the movement through mucus gel layer. Likewise, similar data were obtained in a previous study by Suchaoin et al. where a comparatively higher mucus diffusion behaviour of negatively charged phosphorylated self-emulsifying drug delivery systems (SEDDS) versus dephosphorylated SEDDS was observed (6). Apart from electrostatic interactions, hydrophobic interactions between the nanoparticles and mucus gel layer limiting effective mucus permeation have to be taken into consideration (1). Mahlert et al. compared mucus permeation of poly(ethylene)glycol (PEG) and chitosan coated hydrophobic poly(dllactide-co-glycolide) (PLGA) nanoparticles on transwell inserts containing mucus generating HT-29-MTX cells. According to their results PEG coated PLGA nanoparticles show higher mucus permeation than chitosan coated ones due to the muco-inert properties of PEG (44).


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Likewise, the hydrophilic nature of PEI and polyphosphate contribute to the high mucus permeation of pp-NPs avoiding hydrophobic interactions with lipophilic mucus substructures.

Figure 6. Permeation studies of FDA labelled pp-NPs through native porcine intestinal mucus using transwelI chambers at 37 ˚C for 4 h. Black bars indicate de-pp-NPs while white bars indicate pp-NPs. Data is presented as mean ± SD (n = 3), (*p≤0.05, **p˂0.01). 3.5.3

Rotating Tube Method

Following the transwell insert studies, pp-NPs were applied to the mucus containing silicon tubes as elongated mucus barrier model, in order to investigate the permeation ability of ppNPs into deeper regions of the mucus gel layer. Results of this study are illustrated in Figure 7. In case of pp-NPs, a 2.5-fold increase in diffusion was observed in segment 3 and 4 compared to de-pp-NPs. These results were in good agreement with data of transwell insert experiments confirming electrostatic repulsion phenomena between negatively charged mucus substructures and negatively charged pp-NPs. Similarly, Dünnhaupt et al. demonstrated a higher diffusion ability of anionic poly (acrylic acid) particles (PAA) compared to cationic chitosan particles by rotating silicone tube method (22). In another study, chitosan-chondroitin sulphate nanoparticles with highly densely surface charge consisting of both negative and positive charges on the surface, were investigated in terms of mucus permeation behaviour by de Sousa et al. Compared with highly negatively charged 50/50 DL-lactide/glycolide copolymer NPs, ACS Paragon Plus Environment

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chitosan-chondroitin sulphate nanoparticles mimicking the dual surface charge characteristics of viruses displayed an even higher mucus permeation behaviour (45).

Figure 7. Permeation studies of FDA labelled pp-NPs through native porcine intestinal mucus. Black bars indicate de-pp-NPs while white bars indicate pp-NPs. Data is presented as mean ± SD (n = 3) (*p≤0.05). 3.6

Cellular Viability

According to resazurin assay, a pink colour was observed and high fluorescent intensity was measured due to the reduction of resazurin to its highly fluorescent form, resorufin, by living cells (46). PEI has favourable properties as polymeric excipient especially for non-viral gene delivery as stated by Huang et al. (47). As a polycationic delivery system PEI nanoparticles can enhance the cellular uptake by interfering with negatively charged cell membrane components making membranes more vulnerable (10). This cytotoxic effect of PEI nanoparticles is caused by abounding amine groups and cationic nature of PEI damaging cell membranes (48, 49). In order to reduce this toxic effect, the cationic charge of PEI was shielded by anionic PolyP through ionic interactions. Correspondingly, within this study, de-pp-NPs did not display a cytotoxic potential. As illustrated in Figure 8 no significant reduction in cell viability was observed in case of de-pp-NPs in a concentration of 0.005% (m/v) and 0.01% (m/v) for 24 h and 0.02% (m/v) for 3 h. However, in case of 0.02% (m/v) pp-NPs increased reduction in cell


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viability was observed after 24 h which could be caused by complete degradation of pp-NPs over time releasing the toxic PEI subunits.

Figure 8. Cell viability on Caco-2 cells after 3 h (white bars) and 24 h (black bars) incubation with various concentrations of pp-NPs measured by resazurin assay. Data is presented as mean ± SD (n = 3). 3.7

In Vitro Cellular Uptake

In the present study Caco-2 cell line was used in order to test the extent of cellular uptake of the pp-NPs in the presence or absence of alkaline phosphatase inhibitor cocktail. In case of cells that were not incubated with the inhibitor cocktail, a 2.6-fold increase in the cellular uptake was observed compared to cells that were incubated with inhibitor (Figure 9). Moreover, a 3.1-fold increase was observed at the end of 3 h compared with the cellular uptake at the end of 1 h. This time dependent increase in cellular uptake is in line with the time dependent zeta potential change and phosphate release data as mentioned above confirming that via the cleavage of phosphate by IAP over time, pp-NPs become less negative and are uptaken by Caco-2 cells in a surface charge dependent manner. Shan et al. prepared zwitterionic nanoparticles consisting of polylactic acid (PLA) and dilauroylphosphatidylcholine (DLPC) and investigated their cellular uptake efficacy. Similar to our results, they indicated an enhanced cellular uptake of ACS Paragon Plus Environment

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hydrophilic and slightly negatively charged zwitterionic particles by HT29-MTX-E12 human colon cells (50).

Figure 9. Cellular uptake studies of pp-NPs with Caco-2 cell line. Black bars represent the treated cells with IAP inhibitor cocktail while white bars represent untreated cells. Data is presented as mean ± SD (n = 3), (*p≤0.05, **p˂0.01). 4. Conclusion Within this study, a polyphosphate containing zeta potential changing system was developed for the first time without challenging chemical or enzymatic phosphorylation steps. Particle size and surface charge were arranged to preferred values by adjusting the ratios of cationic PEI and anionic polyphosphate. Hydrophilic, negatively charged pp-NPs were able to cross the mucus barrier to a higher extent than de-pp-NPs. PEI nanoparticles are reported to be toxic due to their cationic nature (48). Combining the anionic polyphosphate with PEI, toxic effects derived from the cationic charge of PEI could be eliminated in a concentration dependent manner and improved cellular uptake achieved in Caco-2 cell line. Furthermore, polyphosphate is a wellestablished safe compound according to FAO/WHO reports. Consequently, zeta potential change concept was successfully applied to the non-toxic pp-NPs crossing the mucus and epithelial barriers readily. This system could be utilised as a promising mucosal drug and gene delivery system in order to overcome intestinal absorption membrane barrier based on the cleavage of polyphosphate structures in the future.


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5. Acknowledgements This research was funded by the FWF (Fonds zur Förderung der wissenschaftlichen Forschung), Austria, under project number P 30268-B30. References 1. Liu M, Zhang J, Shan W, Huang Y. Developments of mucus penetrating nanoparticles. Asian Journal of Pharmaceutical Sciences. 2015;10(4):275-82. 2. Wu L, Shan W, Zhang Z, Huang Y. Engineering nanomaterials to overcome the mucosal barrier by modulating surface properties. Adv Drug Deliv Rev. 2018;124:150-63. 3. Lundquist P, Artursson P. Oral absorption of peptides and nanoparticles across the human intestine: Opportunities, limitations and studies in human tissues. Advanced Drug Delivery Reviews. 2016;106:256-76. 4. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev. 2012;64(6):557-70. 5. Barua S, Mitragotri S. Challenges associated with Penetration of Nanoparticles across Cell and Tissue Barriers: A Review of Current Status and Future Prospects. Nano Today. 2014;9(2):223-43. 6. Suchaoin W, Pereira de Sousa I, Netsomboon K, Lam HT, Laffleur F, BernkopSchnurch A. Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation. Int J Pharm. 2016;510(1):255-62. 7. Pepic I, Lovric J, Filipovic-Grcic J. How do polymeric micelles cross epithelial barriers? Eur J Pharm Sci. 2013;50(1):42-55. 8. Mislick KA, John D. Evidence for the role of proteoglycans in cation mediated gene transfer. Proceedings of the National Academy of Sciences. 1996;93(22):12349-54. 9. Zhi D, Bai Y, Yang J, Cui S, Zhao Y, Chen H, et al. A review on cationic lipids with different linkers for gene delivery. Adv Colloid Interface Sci. 2018;253:117-40. 10. Bernkop-Schnürch A. Strategies to overcome the polycation dilemma in drug delivery. Advanced Drug Delivery Reviews. 2018. 11. Wang T, Upponi JR, Torchilin VP. Design of multifunctional non-viral gene vectors to overcome physiological barriers: Dilemmas and strategies. International Journal of Pharmaceutics. 2012;427(1):3-20. 12. Nazir I, Leichner C, Le-Vinh B, Bernkop-Schnurch A. Surface phosphorylation of nanoparticles by hexokinase: A powerful tool for cellular uptake improvement. J Colloid Interface Sci. 2018;516:384-91. 13. Perera G, Zipser M, Bonengel S, Salvenmoser W, Bernkop-Schnurch A. Development of phosphorylated nanoparticles as zeta potential inverting systems. Eur J Pharm Biopharm. 2015;97(Pt A):250-6. 14. Salimi E, Le-Vinh B, Zahir-Jouzdani F, Matuszczak B, Ghaee A, Bernkop-Schnurch A. Self-emulsifying drug delivery systems changing their zeta potential via a flip-flop mechanism. Int J Pharm. 2018;550(1-2):200-6. 15. Bonengel S, Prufert F, Jelkmann M, Bernkop-Schnurch A. Zeta potential changing phosphorylated nanocomplexes for pDNA delivery. Int J Pharm. 2016;504(1-2):117-24. 16. Bonengel S, Prufert F, Perera G, Schauer J, Bernkop-Schnurch A. Polyethylene imine6-phosphogluconic acid nanoparticles--a novel zeta potential changing system. Int J Pharm. 2015;483(1-2):19-25. 17. Kulakovskaya TV, Vagabov VM, Kulaev IS. Inorganic polyphosphate in industry, agriculture and medicine: Modern state and outlook. Process Biochemistry. 2012;47(1):1-10.


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38. Grießinger J, Dünnhaupt S, Cattoz B, Griffiths P, Oh S, Gómez SBi, et al. Methods to determine the interactions of micro- and nanoparticles with mucus. European Journal of Pharmaceutics and Biopharmaceutics. 2015;96:464-76. 39. Taherali F, Varum F, Basit AW. A slippery slope: On the origin, role and physiology of mucus. Advanced Drug Delivery Reviews. 2018;124:16-33. 40. Lai SK, Wang YY, Wirtz D, Hanes J. Micro- and macrorheology of mucus. Advanced Drug Delivery Reviews. 2009;61(2):86-100. 41. Varum FJO, Veiga F, Sousa JS, Basit AW. Mucus thickness in the gastrointestinal tract of laboratory animals. Journal of Pharmacy and Pharmacology. 2012;64(2):218-27. 42. Boegh M, Nielsen HM. Mucus as a Barrier to Drug Delivery - Understanding and Mimicking the Barrier Properties. Basic Clin Pharmacol. 2015;116(3):179-86. 43. Groo AC, Lagarce F. Mucus models to evaluate nanomedicines for diffusion. Drug Discov Today. 2014;19(8):1097-108. 44. Mahlert L, Anderski J, Mulac D, Langer K. The impact of gastrointestinal mucus on nanoparticle penetration –in vitro evaluation of mucus-penetrating nanoparticles for photodynamic therapy. European Journal of Pharmaceutical Sciences. 2019. 45. de Sousa IP, Steiner C, Schmutzler M, Wilcox MD, Veldhuis GJ, Pearson JP, et al. Mucus permeating carriers: formulation and characterization of highly densely charged nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics. 2015;97:273-9. 46. Csepregi R, Lemli B, Kunsagi-Mate S, Szente L, Koszegi T, Nemeti B, et al. Complex Formation of Resorufin and Resazurin with Beta-Cyclodextrins: Can Cyclodextrins Interfere with a Resazurin Cell Viability Assay? Molecules. 2018;23(2). 47. Huang X, Shen S, Zhang Z, Zhuang J. Cross-linked polyethylenimine-tripolyphosphate nanoparticles for gene delivery. Int J Nanomedicine. 2014;9:4785-94. 48. Patnaik S, Gupta KC. Novel polyethylenimine-derived nanoparticles for in vivo gene delivery. Expert Opin Drug Del. 2013;10(2):215-28. 49. Wang FZ, Xing L, Tang ZH, Lu JJ, Cui PF, Qiao JB, et al. Codelivery of Doxorubicin and shAkt1 by Poly(ethylenimine)-Glycyrrhetinic Acid Nanoparticles To Induce AutophagyMediated Liver Cancer Combination Therapy. Mol Pharm. 2016;13(4):1298-307. 50. Shan W, Zhu X, Tao W, Cui Y, Liu M, Wu L, et al. Enhanced Oral Delivery of Protein Drugs Using Zwitterion-Functionalized Nanoparticles to Overcome both the Diffusion and Absorption Barriers. ACS Appl Mater Interfaces. 2016;8(38):25444-53.


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pp-NPs

Mucus Permeation Sodium polyphosphate

Mucus Barrier

de-pp-NPs

Cellular Uptake

Branched polyethylenimine ACS Paragon Plus Environment

Enterocytes

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Zeta Potential Changing Polyphosphate Nanoparticles: A Promising

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