Complexes of Indomethacin with 4-Carbomethoxy-pyrrolidone

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Complexes of Indomethacin with 4-Carbomethoxy-pyrrolidone PAMAM Dendrimers Show Improved Anti-Inflammatory Properties and Temperature Dependent Binding and Release Profile Mario Ficker, Matthijs J. M. Theeuwen, Anna Janaszewska, Michal Gorzkiewicz, Søren W. Svenningsen, Barbara Klajnert-Maculewicz, and Jørn B. Christensen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00567 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Molecular Pharmaceutics

Complexes of Indomethacin with 4-Carbomethoxypyrrolidone PAMAM Dendrimers Show Improved Anti-Inflammatory Properties and Temperature Dependent Binding and Release Profile Mario Ficker1, Matthijs J. M. Theeuwen1, Anna Janaszewska2, Michał Gorzkiewicz2, Søren W. Svenningsen1, Barbara Klajnert-Maculewicz2 and Jørn B. Christensen1,* 1

Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40,

DK-1871 Frederiksberg, Denmark *Corresponding author email: [email protected] 2

Department of General Biophysics, Faculty of Biology and Environmental Protection,

University of Lodz, 90-236 Lodz, Poland

Keywords: Drug Delivery, NSAID, Thermodynamics, 4-Carbomethoxypyrrolidone, Dendrimers, Enthalpy, Entropy

ACS Paragon Plus Environment

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Molecular Pharmaceutics 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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ABSTRACT

COX-2 inhibitors such as Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common treatment for chronic inflammatory diseases like arthritis and atherosclerosis. However, they are associated with severe side effects such as cardiovascular events or stomach bleeding, due to co-inhibition of other enzymes (COX1) and off target accumulation. PAMAM-dendrimers can solubilize lipophilic drugs and increase their circulation time; furthermore PAMAM dendrimers seem to have some per se accumulation in inflammatory sides. Three different generations of 4-carbomethoxypyrrolidone (Pyr) surface modified PAMAM dendrimers were complexed with the NSAID drug Indomethacin and their in solution thermodynamic profile studied by means of NMR experiments. The binding stoichiometry was found dependent on solvent system and dendrimer generation. Larger dendrimers (G3-Pyr) were found to bind Indomethacin through entropy driven binding mode, while G1-Pyr and G2-Pyr expressed an enthalpy driven complex formation, which means that the binding constants have a generational temperature dependency. G1/2-Pyr showed reduced binding with increasing temperature, which could be important for drug release at inflammatory sites, which have in general elevated temperatures. In-Vitro studies elucidated that the Indomethacin drug remained its activity when delivered as a dendrimer-Indomethacin complex. A slight reduction in toxicity profile was noticed for G2/G3-Pyr-Indomethacin dendrimers. Both free Indomethacin and Dendrimer Indomethacin complex inhibited a variety of pro-inflammatory cytokines in LPS treated cells. However, only the Indo-Dendrimer complexes showed a significant reduction of IL-1β in LPS treated THP-1 cells, which was not present in the control with free Indomethacin.


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1. Introduction:

Inflammation is a common biological response of the innate immune system against infection or injury.1-2 However, this defensive response can in some cases, such as arthritis or atherosclerosis, turn into chronical inflammatory diseases. Here, the inflammation process does not stop naturally and a vicious circle leads to local damage and discomfort of the patient and can cause disability to work (arthritis) or even live threatening clinical symptoms such as stroke or heart attack (atherosclerosis).3-5 A common drug target, to limit or stop an inflammation process, is the inhibition of cyclooxygenases (COX) to prevent the synthesis of prostaglandin, a proinflammatory transmitter.3 COX-inhibitors, such as aspirin, ibuprofen and indomethacin belong to the most used over the counter drugs in western countries.6 The COX enzyme family consists of two important isozymes. While the COX-2 is expressed mainly at inflammation sides and its inhibition is beneficial for the treatment of inflammation, the co-inhibition of COX-1 is associated with severe side effects, especially in the gastro-intestinal track.3 COX-1 is involved in the formation of the natural mucus layer protecting the stomach tissue from the degrading acidic digestion process.7 Since most COX inhibitors are given as oral formulations, with high drug levels in the stomach, this can lead to damage of the stomach wall with symptoms such as stomach bleeding. More selective COX-2 inhibitors have found their way to the market in the last years;8 however, many of the heavily used over the counter drugs (aspirin, diclofenac, ibuprofen, indomethacin etc.) are still inhibitors of both COX isozymes. Indomethacin is a widely prescript non-steroidal anti-inflammatory (NSAID) drug for preventing chronical inflammation processes such as present in arthritis. However, its bio-availability is limited due to its poor water solubility and poor transdermal uptake as an alternative to oral formulations.9-11 Drug-delivery systems such as dendrimers could help to create transdermal drug


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formulations of indomethacin, that are soluble in aqueous environment, can transport the drug through the skin into the circulation, and target inflammatory sites, which could minimize offtarget toxicity.12 Dendrimers are especially promising carriers for this task, since recent studies have shown that some dendrimers naturally enrich in inflammatory tissue.13-14 Furthermore, PAMAM dendrimers can release drugs using the “umbrella-effect”, where lipophilic drugs like indomethacin can be encapsulated at physiological pH in the lipophilic interior of the dendrimer. After endocytic cell entry, for instance a macrophage in an inflamed tissue, the intracellular pH change (lysosomal pH app. 5) triggers protonation of the tertiary amines in the dendrimer interior and a release of the lipophilic drug.15-18 Previous studies have shown that PAMAM dendrimers can enhance the solubility of indomethacin where especially amine terminated PAMAM-G4 helped to increase the indomethacin efficacy compared to the free drug in a rat paw model for arthritis.9-10,13 A recent study investigated the association of indomethacin on a dendrimer in solid state using STEM applying a metal ion tagging strategy.19 However, little is known about the nature of the binding interactions and binding strength between indomethacin and dendrimers in solution, which is necessary in order to design a reliable drug-carrier with defined uptake and release properties. In this study we wanted to investigate the thermodynamics of binding of a nonpolar drug, here exemplified by indomethacin in different sized (G1-G3) PAMAM dendrimers terminated with 4carbomethoxypyrrolidone groups (Figure 1). These dendrimers combine good solubility in a number of different solvents with no toxicity against various murine in vitro cell lines. Furthermore, these dendrimers did not; promote ROS generation, influence the cell membrane potential or induce hemolysis.20-21 This is in stark contrast to normal amine terminated PAMAM dendrimers which are associated with strong cellular and immune toxicity.12, 22 Additionally, our


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recent study demonstrated that 4-carbomethoxypyrrolidone dendrimers (pyrrolidone dendrimers) do not activate inflammatory signaling channels, in contrary to native amine terminated PAMAM dendrimers, which makes these surface functionalized dendrimers ideal drug delivery agents for anti-inflammatory drugs.23 The binding stoichiometry, possible binding site and binding strength were studied with means of one and two dimensional NMR techniques, with thermodynamic methods previously deployed by us.24-25 Furthermore, biological in-vitro tests were performed to study the toxicity and efficacy of the drug-formulations.

2. Materials and Methods

General Unless otherwise stated, all starting materials and indomethacin were obtained from Sigma Aldrich and used as received. Solvents were HPLC grade and used as received. 1H-NMR and 13

C-NMR spectra were performed on a 300 MHz NMR (Bruker) apparatus (300 MHz 1H-NMR,

75 MHz

13

C-NMR) or on a 500 MHz NMR (Bruker) apparatus (500 MHz 1H-NMR, 125 MHz

13

C-NMR). Chemical shifts are reported in parts per million (ppm) downfield of TMS

(tetramethylsilane) using the resonance of the deuterated solvent as internal standard. Proton couplings are described as s (singlet), d (doublet), t (triplet), q (quartet), br (broad) and m (multiplet), coupling constants are reported in Hertz.

Preparation of Dendrimers


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The PAMAM dendrimers were synthesized by published procedures,26-27 starting from 1,4diaminobutane

as

the

dendrimer-core.

The

1-(4-carbomethoxy-pyrrolidone)

surface

functionalization was achieved by reaction of the amino terminated full generation dendrimers with dimethyl itaconate as previous described.20 The reaction was monitored by Kaiser-test28 until completion. Spectroscopic data of the used dendrimers is available in the supporting information.

Indomethacin Solubility Study The solubility study of indomethacin with generation G1-3 dendrimers was carried out according to a modified procedure described by Chauhan et al.10 The samples were prepared from three stock solutions; each 1 mM dendrimer and 0 mM, 10 mM and 50 mM indomethacin respectively. The samples were prepared by mixing a total volume of 800 µl in 11 round bottom flasks, were the dendrimer concentration was kept at 1 mM and the indomethacin concentration was varied from 1 mM to 50 mM. After three hours of incubation at ambient temperatures, methanol was removed on a rotary evaporator. Deuterium oxide was added (800 µl) and stirred over night to dissolve the dendrimer-indomethacin complex, the mixtures were transferred to NMR tubes. If a precipitate of non-complexed indomethacin was observed, the samples were filtered prior to NMR measurements to remove non dissolved indomethacin.

NMR Job Plots The binding stoichiometry of the drug dendrimer complex was evaluated by performing NMR Job plots as earlier described.24-25

Two stock solutions were made for each experiment,


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consisting of A) 10 mM indomethacin and B) 10 mM dendrimer in each 5 mL deuterated NMR solvent (methanol or chloroform). The samples were prepared by pipetting a total volume of 500 µL in 17 NMR tubes for each Job plot, keeping a constant total concentration of 10 mM ([den]+[ind]), where the ratio [den]/[ind] was varied. NMR Binding study The dissociation constants were determined by NMR binding studies, employing a previously described binding model.24-25 A series of NMR samples was prepared where the total dendrimer concentration was kept constant at 1mM, while the concentration of indomethacin was varied between 0 and 100 mM with a constant volume of 500 µL within the NMR tube. Experiments were conducted using dendrimers of generation G1-G3 and deuterated methanol or chloroform as solvent. After sample preparation, the vials were incubated for three hours at ambient temperature before measurement. Furthermore, temperature dependent experiments were conducted measuring the 1H-NMR samples in an NMR machine thermostated at 300 K, 310 K, 320 K and 330K. Binding saturation curves were fitted employing Origin 9.0.

Cell culture The THP-1 (acute monocytic leukemia) and U937 (histiocytic lymphoma) human cell lines were purchased from ATCC (USA) and were maintained under standard conditions in RPMI-1640 Medium (Gibco) containing 10% fetal bovine serum (Sigma Aldrich) at 37oC in an atmosphere of 5% CO2. Cells were sub-cultured three times per week.

Cytotoxicity Assay


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To estimate the cytotoxicity of indomethacin and dendrimers, and to choose appropriate concentrations for further analysis, resazurin viability assay was performed. Cells were seeded into 96-well black plates at a density of 1.5 x 104 cells per well and treated with increasing concentrations of selected compounds, ranging from 0.1-400 µM, for 24h. Following the incubation, 10 µg/ml resazurin in culture medium solution was added and the plates were incubated for 90 minutes to allow conversion of resazurin to resorufin. Fluorescence measurement was performed at 530-nm excitation and 590-nm emission using an EnVision luminometer plate reader (PerkinElmer, Waltham, USA).

Gene Expression Assay The expression level of cytokine genes (IL1B, IL6, IL10) was determined by quantitative realtime RT-PCR. A detailed description and table showing the primer sequences is presented in the Supporting Information.

Statistics For statistical significance testing we used one-way ANOVA for concentration series followed by post-hoc Tukey’s test for pairwise difference testing. For single pairwise comparisons, Student’s t test was applied. In all tests, p values < 0.05 were considered to be statistically significant. Data was presented as arithmetic mean ± S.E.M.

3. Results and Discussion:

The molecular interactions between indomethacin with different sized 4-carbomethoxy pyrrolidone (Pyr) terminated PAMAM dendrimers (G1-G3) were elucidated applying various


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1

H-NMR methods in three different NMR solvent, ranging from polar protic solvents like water

and methanol to chloroform as more lipophilic solvent. The obtained dendrimer-indomethacin complexes were afterwards investigated for their in-vitro behavior in two cell-lines concerning cellular toxicity and anti-inflammatory response. First, the possible uptake and solubilization of indomethacin within Pyr-terminated dendrimers was investigated. The PAMAM-Pyr dendrimers of generation G1 to G3 were applied in this study. It has to be mentioned that the pyrrolidone surface functionalization approximately doubles the molecular weight of the dendrimer. A G3-Pyr dendrimer is thus similar in size and molecular weight to a native amine terminated PAMAM-G4 (Supporting Information). Since indomethacin is not soluble in D2O in the concentration range of NMR (Supporting Information), the solubilization of indomethacin in water can be directly associated with a dendrimer interaction. A NMR series of different concentrations of indomethacin with the corresponding G1 to G3 dendrimer was prepared and the solubilized indomethacin fraction quantified (Table 1).


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Figure 1. G3-PAMAM dendrimer with 4-carbomethoxy pyrrolidone surface groups.

Table 1. Indomethacin solubility enhancement by dendrimer, loading of dendrimers in water. Dendrimer

n indo per Dendrimer

Loading [wt%]*

G1-Pyr

1.5

17.9

G2-Pyr

4

21.3

G3-Pyr

5

14.1

*wt%=wt(indo)/(wt(Den)+wt(indo))


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The Pyrrolidone dendrimers presented a generation dependent loading capacity for indomethacin. The small Pyr-dendrimer (G1), was capable of solubilizing 1.5 indomethacin per dendrimer in average, which suggests that two dendrimers share three indomethacin molecules. The generation two Pyr-dendrimer could dissolve four and the G3 dendrimer five indomethacin molecules. Due to the doubling of molecular weight for each dendrimer generation the loading in wt% gives more useful information than the loading number. Here, the G2 Pyr-dendrimer had by far the best loading capacity with 21.3 wt%, followed by G1 Pyr-dendrimer with 17.9 wt% and G3 Pyr-dendrimer with the lowest loading (14.1 wt%). This data seems comparable with a previous study where indomethacin was dissolved in water using G4 PAMAM dendrimers, which are comparable in molecular weight to the G3 Pyrdendrimer, with –NH2 (25 wt%), -OH (23 wt%) and –COOH (6.5 wt%).9 It appears, that indomethacin preferable undergoes interaction with positively charged dendrimers, due to electrostatic interaction of the indomethacin carboxylic acid with the primary amines of the dendrimer surface. However, these dendrimers come with the disadvantage of an unfavorable toxicity profile.12, 22 The neutral charged dendrimers (-OH in the previous study and –Pyr in the current study), seem to have loading capacities only marginally below the –NH2 dendrimers. The negatively surface charged carboxylic acid dendrimer seems to have a repulsive effect on the deprotonated indomethacin under physiological conditions and is thus unfavorable for an efficient drug delivery system. While the former study focused only on generation 4 dendrimers, we found a generation dependent binding number. This allows picking the right dendrimer generation to optimize loading capacity and to use dendrimer generations with a suitable in vivo circulation time, which is strongly associated with nanoparticle size.29-30


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NMR Evaluation of indomethacin binding behavior to dendrimers The aim of this study, was not only to find binding stoichiometries of indomethacin to Pyrrolidone dendrimers, but also to investigate the binding mode and strength of the complex. This was accomplished by applying a previous developed NMR binding model,24 which correlated changes in chemical shift, within a NMR titration series, to a binding constant. In order to calculate reliable binding constants, equilibrium between complex and free molecule (free dendrimer and free indomethacin) is necessary in the concentration range of NMR (0.05 mM to 200 mM). Indomethacin, as a very lipophilic drug, is virtually insoluble in polar media like pure water (Figure S1, Supporting Information), which restricts the information that can be extracted from this system. In order to overcome this obstacle, we investigated the dendrimerindomethacin relationship in the solvent that is closest to water, which is methanol, where one of the water –OH groups is changed to an –OCH3 unit. This allows solubility to indomethacin and an equilibrium between unbound and bound form that can be investigated. Furthermore, the results were compared to a study in chloroform, a solvent which is less polar and more lipophilic. A 1H-NMR assignment for a G2 pyrrolidone dendrimer and Indomethacin in deuterated methanol is presented in Figure 2 and Figure 3 respectively. The NMR assignments for all dendrimer generations (G1-G3) are depicted in the supporting information.


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Figure 2. 1H-NMR peak assignment of G2-Pyr in deuterated methanol.

Figure 3. 1H-NMR peak assignment of Indomethacin in deuterated methanol.


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An association of indomethacin with the pyrrolidone surface functionalized dendrimers was elucidated by a titration series of indomethacin to a Pyr-dendrimer sample. As shown in the Figure 4, small addition amounts of indomethacin led already to a line broadening of the interior dendrimer signals and a change in chemical shift, which indicates a complex formation. Interestingly, the dendrimer protons next to the tertiary amines (3, 8, and 13) of the dendrimer structure were affected first by addition of indomethacin, which indicates an electrostatic interaction between tertiary amine and the carboxylic acid of indomethacin. Higher concentration of indomethacin lead to further line broadening and a change in chemical shift of lipophilic backbone (4, 9, 14) protons and surface protons (19, 23), which could indicate lipophilic interactions. The close binding interaction was further confirmed with NOESY-spectroscopy (Figures S2, Supporting Information), which correlates interactions of protons within very close proximity. Correlation signals were found between the aromatic indomethacin protons and the dendrimer backbone, which further indicates a lipophilic binding mode, besides the obvious carboxylate amine interaction between dendrimer and indomethacin.


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Figure 4. 1H-NMR Titration series of different concentrations of Indomethacin in 1 mM G3-Pyr dendrimer in deuterated methanol. Line broadening and a concentration dependent shift is visible for internal dendrimer peaks (3, 8, 13, 14) and dendrimer surface peaks (19).

The binding stoichiometry in the water experiments could be extrapolated from the number of indomethacin which was dissolved due to the dendrimer. However, since indomethacin is itself soluble in methanol, the stoichiometry has to be extracted in a different manner. A modified version of the original Job plot was used to determine the binding stoichiometries of G1-3 pyrrolidone dendrimers with indomethacin in deuterated methanol.24 The binding stoichiometry was calculated by plotting the change in chemical shift, observed for indomethacin proton signals, while varying the mole fraction between indomethacin and dendrimer. A stacked NMR


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spectrum, visualizing the change in indomethacin peaks, for a 1H-NMR Job Plot series is presented in Figure 5 and the corresponding Job plots are shown in Figure 6. The smallest generation Pyr-dendrimer G1 was capable of binding only one indomethacin molecule, which is similar to the result obtained in water. The G2 and G3 Pyr-dendrimers had a binding stoichiometry of two indomethacin molecules per dendrimer. This suggests that the binding is in general less favorable in methanol than in water, since indomethacin can be dissolved by the solvent and is easier released from the dendrimer. However, a similar observation was made, smaller generation dendrimers seems to have a higher uptake capacity if expressed in weight percent, G1: 12.6 wt%, G2: 11.9 wt%, G3: 6.2 wt%.

Figure 5. Stacked spectra of a 1H-NMR Job plot of G2-Pyr with Indomethacin in different mole fractions in deuterated methanol.


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G3-Pyr G2-Pyr G1-Pyr

0.68

0,015

∆ppm x Mole fraction

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


0.66 0,010

0.52

0,005

0,000

0,0

0,2

0,4

0,6

0,8

1,0

Mole fraction [Indo]

Figure 6. NMR Job plot of G1-3 Pyrrolidone dendrimers with indomethacin in deuterated methanol. A ratio between dendrimer and indomethacin was found as follows: A) G1-Pyr:Indo 1:1 B) G2-Pyr:Indo 1:2 C) G3-Pyr:Indo 1:2. The dissociation constants for the Pyr-dendrimer-indomethacin complexes were determined with 1

H-NMR titration experiments with constant dendrimer concentrations and varied indomethacin

concentrations. By plotting the change in chemical shift (∆) versus the concentration of indomethacin a binding curve was obtained. This binding curve was subsequently fitted with the model that was previously derived by us.24 The equations behind the binding model are depicted in the Supporting Information. This model makes two assumptions; each host has a number of 'n' independent binding sites and all binding sites have the same dissociation constant (Kd). The number of binding sites (n) was determined before by means of Job plots (Figure 6), ∆ is the measured change in chemical shift, ∆

max

is the maximum change in chemical shift. The ∆

max

and Kd values were determined by least square fitting using Origin 9.0. The fitted binding curve for pyrrolidone dendrimer generations G1-3 in deuterated methanol is shown in Figure 7.


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Furthermore, this figure presents a comparison of a G3 pyrrolidone dendrimer in deuterated chloroform to visualize the impact of a more lipophilic solvent system. The Job plot for the chloroform study is presented in the Supporting information.

Figure 7. Binding curves for the binding of indomethacin to G1 (top left), G2 (top right), and G3 (bottom left) in CD3OD and G3 (bottom right) in CDCl3.


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Table 2. Summary of the ∆δmax and Ka values obtained by fitting the binding curve of G1-3 pyrrolidone dendrimers with indomethacin at 300 K. Dendrimer / Solvent

n

∆ max

Ka [M-1]

G1-Pyr / MeOD

1

0.094 ± 0,005

67.02 ± 9,40

G2-Pyr / MeOD

2

0.158 ± 0,009

57.60 ± 2.62

G3-Pyr / MeOD

2

0.067 ± 0,002

33.55 ± 1.92

G3-Pyr / CDCl3

2

0.089 ± 0,010

14.68 ± 1.10

The obtained dissociation constants are listed in Table 2. Remarkable is the generation dependent decrease of Ka with increasing dendrimer generation in methanol. The higher generation Pyrdendrimers bind more guest molecules, however, the individual binding interaction per guest molecules is weaker. Furthermore, the difference of the association constant of the third generation dendrimer in CD3OD and CDCl3 is notable; the Ka in MeOD is more than double the value of the Ka in CDCl3 which suggest a weaker complex in CDCl3. This phenomenon is possibly due to the hydrophobicity of the solvent, where methanol is hydrophilic (polarity index: 5.1)31 which allows more favorable lipophilic interaction with the dendrimer than in case of chloroform (polarity index = 4.1).31


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Figure 8. Van't Hoff plot of G3 pyrrolidone terminated dendrimer with indomethacin as guest molecule at different temperatures in deuterated methanol .

A closer look on thermodynamic parameters like entropy, binding enthalpy and Gibbs free energy was gained by performing a 1H-NMR titration at different temperatures and evaluating the results with Van’t Hoff plots. This was achieved using the generation one to three dendrimers in deuterated methanol as model system. The NMR experiments for the Van’t Hoff plot were obtained by varying the temperatures ranging from 300 K to 330K, which is a reasonable temperature range for in vivo applications, since 310 K would represent the body temperature of a human being. The association constants (1/Kd) were obtained as described before, by NMR binding curves for each temperature. Subsequently, the logarithmic Ka values were plotted against reciprocal temperature values (Figure 8). The slope of the Van’t Hoff plot represents ∆H/2.303RT, and the interception corresponds to ∆S/2.303R (Equation 1), the Gibbs free energy can subsequently be calculated from these values using Equation 2.32 log

∆ .

∆ ∆ ∆

∆ .

Equation 1 Equation 2


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Table 3. Thermodynamic values ∆ , ∆ and ∆ for the complexation of G1 to G3 pyrrolidone dendrimers with indomethacin in deuterated methanol. Dendrimer

∆ [kJ mol1 ]

∆ [kJ mol1 ]

Ka [M-1]

T [K]

67.02 ± 9,40

300

-10.71 ± 0.42

59.71 ± 7.24

310

-9.91 ± 0.13

G1-Pyr

-34.78 ± 9.12

∆ [J mol-1K-1]

-80.22 ± 29.01

24.50 ± 5.52

320

-9.11 ± 0.16

22.05 ± 4.73

330

-8.30 ± 0.45

57.60 ± 2.62

300

-10.31 ± 0.20

55.60 ± 5.38

310

-10.00 ± 0.06

G2-Pyr

-19.67 ± 4.34

-31.21 ± 13.82

36.67 ± 1.72

320

-9.69 ± 0.08

29.71 ± 5.24

330

-9.37 ± 0.22

33.55 ± 1.92

300

-8.77 ± 0.01

34.90 ± 2.51

310

-9.14 ± 0.01

G3-Pyr

2.17 ± 0.23

36.49 ± 0.73

35.50 ± 2.64

320

-9.50 ± 0.01

36.42 ± 2.38

330

-9.87 ± 0.01


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The calculated thermodynamic values are presented in table 3. The ∆ present e similar values for all three systems, and demonstrates that the complex formation is exergonic and can form spontaneously without applying work to the system. Again a generation dependency was found, were the Gibbs free energy decreases with increasing dendrimer generation.

A deviation in ∆ and ∆ values was found. The G1 and G2 Pyr-dendrimers have negative values for both ∆ and ∆ while, the G3 Pyr-dendrimer shows positive values for both parameters. This results in a decrease of Ka with an increased temperature for the first and second generation Pyr-dendrimer, while the third generation Pyr-dendrimer presents slightly increasing Ka values with increasing temperature. At physiological temperature (310 K) the G1 and G2 Pyr-dendrimers present a very similar thermodynamic dataset. The G1 and G2 Pyrdendrimers have a more favorable complex formation (G1: ∆ = 9.91 kJ/mol, G2: ∆ = 10.00 kJ/mol) and higher associating constant (G2: Ka=59.71 mol-1, G2: Ka=55.60 mol-1) than the G3 Pyr-dendrimer with ∆ = 9.14 kJ/mol and Ka=34.90 mol-1 respectively. From a drugdelivery point of view the G1-Pyr and G2-Pyr have a very interesting feature, at higher temperatures, such as common in inflamed tissue the association constant drops, which makes a drug release more likely. The individual ∆ and ∆ values give information about the various kinds of interactions possible during complex formation. Where ∆ > 0 and ∆ > 0, as it is the case for G3-Pyr, indicates that hydrophobic interactions are the dominate factor in the binding of the host and guest. A situation with ∆ < 0 and ∆ < 0, which was observed for G1 and G2 Pyrdendrimers, indicates that Van der Waals forces and hydrogen bonding are the main driving forces.33-36


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From Table 3, it can be concluded that the complex of the G3 Pyr-dendrimer with indomethacin is most likely formed because of hydrophobic association, which means a withdrawal of nonpolar groups from the polar solvent environment. This is in agreement with the results in table 2 where the complexation in methanol was found to be more stable compared to chloroform. The complexation reaction can be assumed as entropy driven endothermic complex formation. The G1- and G2 Pyr-dendrimers show an almost inversed set of thermodynamic parameters; the ∆ and ∆ suggest an exothermic enthalpy driven complex formation with Van der Waals interactions and hydrogen bonding as dominating interactions. 33-36

In-Vitro Testing In order to evaluate cytotoxicity and anti-inflammatory properties of the dendrimer-indomethacin complexes, in-vitro studies on human monocytic cell lines were performed. For this purpose, we chose THP-1 and U937 cell lines, which are commonly used myeloid models for studying inflammatory changes in vitro. These cell lines are well characterized and we previously applied them in a study showing that Pyrrolidone modified dendrimer (Pyr-dendrimer) have no proinflammatory characteristics, which makes them suitable carriers for anti-inflammatory drugs.23 The cytotoxicity of the Pyr-dendrimer-indomethacin complexes were investigated using the two cell lines in a resazurin assay (Figure 9). Both free indomethacin and Pyr-dendrimerindomethacin complexes had a comparable toxicity profile. Nevertheless, a small generation dependent trend on the toxicity can be seen in both cell lines. The G1 Pyr-dendrimer complex has no significant effect on the cell viability. However, the G3 and especially the G2 Pyr-


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dendrimer seem to increase the indomethacin toxicity slightly, which is a valuable feature for possible in vivo applications.

Figure 9. Influence of PAMAM Pyr-dendrimers loading with indomethacin on the viability of THP-1 and U937 cells. Viability was determined by the resazurin assay after 24 h of treatment.

As one of the potential immunomodulatory effects of indomethacin involves downregulation of cytokine levels,37-38 we decided to evaluate the impact of the drug and drugloaded Pyr-dendrimers on the expression of interleukin-1β -6 and -10 at mRNA level.


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First, unstimulated cells were tested. In this control setup, indomethacin and drug-loaded Pyr-dendrimers had no impact on the cytokine expression level (Table S1 - Supplementary Materials). However, the treatment with LPS, highly pro-inflammatory endotoxins, significantly induced the expression of selected genes in both cell lines (Figure 10, first column).

As

expected, indomethacin was able to decrease the LPS-induced expression of IL-6 and IL-10 (Figure 10, third column). Similar results were obtained for indomethacin-carrying Pyrdendrimers (Figure 10, column 4-6), indicating that the encapsulated drug retains its activity. Surprisingly, drug-loaded Pyr-dendrimers were able to inhibit the expression IL-1β in LPStreated THP-1 cell line, which was not observed in case of free indomethacin. In this case, complexes exhibited enhanced inhibition of gene expression in comparison to the free drug, which is a significant improvement of the inflammatory response due to the dendrimer complexation of the drug.


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Figure 10. Effect of indomethacin and dendrimers on cytokine-related gene expression at the mRNA level. The concentrations of Indomethacin and Indomethacin-dendrimer complex were 200 µM. The relative level of gene expression was evaluated using quantitative real-time RTPCR. Data presented as percentage of cognate mRNA expression in control (untreated) cells (number of cognate mRNA copies per 1 copy of geometric-averaged mRNA for reference genes), average ± S.E.M of 3 experiments. * Statistically signiﬁcant difference towards untreated control at p < 0.05. † Statistically signiﬁcant difference towards control stimulated with LPS at p < 0.05.


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4. Conclusion

The complexation of indomethacin with generation one to three pyrrolidone terminated dendrimers was characterized by 1H-NMR. It was found that indomethacin is capable of forming stable complexes with pyrrolidone dendrimers in deuterated water, methanol and chloroform. The binding stoichiometry depends on the solvent system and dendrimer generation. In general, higher generation dendrimers and more hydrophilic (polar) solvents led to an increase in binding ratio and binding constant. Van’t Hoff plots elucidated that the G3-Pyr dendrimer had an entropy driven binding with indomethacin, which was explained with hydrophobic interactions between indomethacin and dendrimer. In contrary, the G1-Pyr and G2-Pyr dendrimers had an enthalpy driven binding mode, which was assumed as driven by polar interactions like hydrogenbonding between dendrimer and indomethacin guest molecule. The binding constant of the G1/2Pyr dendrimers drops with increasing temperatures, such as found in inflamed tissues sites, which makes a release of the encapsulated drug more likely and could lead to a temperature dependent drug delivery. In-vitro evaluation of the different generation dendrimers showed that the G1-Pyr complex had a similar cell toxicity profile as free indomethacin. However, the G2-Pyr and G3-Pyr complexes showed a small decline in cytotoxicity. Both free Indomethacin and the dendrimer-complexes were found to inhibit pro-inflammatory cytokines on LPS treated cell lines, which demonstrates that the indomethacin is capable of keeping its activity when associated with the dendrimer. In the monocytic THP-1 cell line the dendrimer-indomethacin complexes achieved a significant inhibition of IL-1β, which was not presented with free indomethacin, which shows the beneficial properties as dendrimers for the delivery of indomethacin. In conclusion, it was found that


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pyrrolidone terminated PAMAM dendrimers are potential drug delivery agents for indomethacin, that can form stable complexes with a generation dependent number of indomethacin molecules. ASSOCIATED CONTENT Supporting Information. Detailed experimental procedures (Dendrimer Synthesis, Gene Expression Assay) and additional analytical data (Dendrimer characterization and data from the NMR binding studies) are depicted in the supporting information PDF file. AUTHOR INFORMATION Corresponding Author *Corresponding author email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

REFERENCES 1.

Medzhitov, R.; Janeway, C. A., Decoding the patterns of self and nonself by the innate immune

system. Science 2002, 296 (5566), 298-300. 2.

Iwasaki, A.; Medzhitov, R., Regulation of adaptive immunity by the innate immune system.

science 2010, 327 (5963), 291-295. 3.

Willoughby, D. A.; Moore, A. R.; Colville-Nash, P. R., COX-1, COX-2, and COX-3 and the future

treatment of chronic inflammatory disease. The Lancet 2000, 355 (9204), 646-648.


28

Page 29 of 44 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


4.

Weber, C.; Noels, H., Atherosclerosis: current pathogenesis and therapeutic options. Nat. Med.

(N. Y., NY, U. S.) 2011, 17 (11), 1410-1422. 5.

Berliner, J. A.; Navab, M.; Fogelman, A. M.; Frank, J. S.; Demer, L. L.; Edwards, P. A.; Watson, A.

D.; Lusis, A. J., Atherosclerosis: Basic Mechanisms: Oxidation, Inflammation, and Genetics. Circulation 1995, 91 (9), 2488-2496. 6.

Singh, G., Gastrointestinal complications of prescription and over-the-counter nonsteroidal anti-

inflammatory drugs: a view from the ARAMIS database. American journal of therapeutics 2000, 7 (2), 115-122. 7.

Wallace, J. L., Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach

digest itself? Physiol. Rev. 2008, 88 (4), 1547-1565. 8.

Zarghi, A.; Arfaei, S., Selective COX-2 inhibitors: a review of their structure-activity relationships.

Iranian Journal of Pharmaceutical Research 2011, (4), 655-683. 9.

Chauhan, A. S.; Sridevi, S.; Chalasani, K. B.; Jain, A. K.; Jain, S. K.; Jain, N.; Diwan, P. V.,

Dendrimer-mediated transdermal delivery: enhanced bioavailability of indomethacin. J. Controlled Release 2003, 90 (3), 335-343. 10.

Chauhan, A. S.; Jain, N. K.; Diwan, P. V.; Khopade, A. J., Solubility enhancement of indomethacin

with poly (amidoamine) dendrimers and targeting to inflammatory regions of arthritic rats. J. Drug Targeting 2004, 12 (9-10), 575-583. 11.

Vasconcelos, T.; Sarmento, B.; Costa, P., Solid dispersions as strategy to improve oral

bioavailability of poor water soluble drugs. Drug Discov. Today 2007, 12 (23), 1068-1075. 12.

Wu, L.-p.; Ficker, M.; Christensen, J. B.; Trohopoulos, P. N.; Moghimi, S. M., Dendrimers in

Medicine: Therapeutic Concepts and Pharmaceutical Challenges. Bioconjugate Chem. 2015, 26 (7), 11981211.


29


13.

Page 30 of 44

Chauhan, A. S.; Diwan, P. V.; Jain, N. K.; Tomalia, D. A., Unexpected In Vivo Anti-Inflammatory

Activity Observed for Simple, Surface Functionalized Poly(amidoamine) Dendrimers. Biomacromolecules 2009, 10 (5), 1195-1202. 14.

Hayder, M.; Fruchon, S.; #xE9; verine; Fourni; #xE9; , J.-J.; Poupot, M.; Poupot, R.; #xE9; my,

Anti-Inflammatory Properties of Dendrimers per se. TheScientificWorldJOURNAL 2011, 11, 1367-1382. 15.

Tomalia, D. A.; Christensen, J. B.; Boas, U., Dendrimers, dendrons, and dendritic polymers:

discovery, applications, and the future. Cambridge University Press: 2012. 16.

Goldberg, D. S.; Ghandehari, H.; Swaan, P. W., Cellular entry of G3. 5 poly (amido amine)

dendrimers by clathrin-and dynamin-dependent endocytosis promotes tight junctional opening in intestinal epithelia. Pharm. Res. 2010, 27 (8), 1547-1557. 17.

Perumal, O. P.; Inapagolla, R.; Kannan, S.; Kannan, R. M., The effect of surface functionality on

cellular trafficking of dendrimers. Biomaterials 2008, 29 (24), 3469-3476. 18.

Maingi, V.; Kumar, M. V.; Maiti, P. K., PAMAM dendrimer-drug interactions: effect of pH on the

binding and release pattern. J. Phys. Chem. B 2012, 116 (14), 4370-6. 19.

Fleming, C. J.; Yin, N.-N.; Riechers, S. L.; Chu, G.; Liu, G.-y., High-Resolution Imaging of the

Intramolecular Structure of Indomethacin− Carrying Dendrimers by Scanning Tunneling Microscopy. ACS Nano 2011, 5 (3), 1685-1692. 20.

Ciolkowski, M.; Petersen, J. F.; Ficker, M.; Janaszewska, A.; Christensen, J. B.; Klajnert, B.;

Bryszewska, M., Surface modification of PAMAM dendrimer improves its biocompatibility. Nanomedicine 2012, 8 (6), 815-7. 21.

Janaszewska, A.; Studzian, M.; Petersen, J. F.; Ficker, M.; Christensen, J. B.; Klajnert-Maculewicz,

B., PAMAM dendrimer with 4-carbomethoxypyrrolidone--in vitro assessment of neurotoxicity. Nanomedicine 2015, 11 (2), 409-11.


30

Page 31 of 44 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


22.

Jain, K.; Kesharwani, P.; Gupta, U.; Jain, N., Dendrimer toxicity: Let's meet the challenge. Int. J.

Pharm. 2010, 394 (1), 122-142. 23.

Janaszewska, A.; Gorzkiewicz, M.; Ficker, M.; Petersen, J. F.; Paolucci, V.; Christensen, J. B.;

Klajnert-Maculewicz, B., Pyrrolidone Modification Prevents PAMAM Dendrimers from Activation of ProInflammatory Signaling Pathways in Human Monocytes. Mol. Pharm. 2018, 15, 12-20 (2018) 24.

Hansen, J. S.; Ficker, M.; Petersen, J. F.; Nielsen, B. E.; Gohar, S.; Christensen, J. B., Study of the

Complexation of Oxacillin in 1-(4-Carbomethoxypyrrolidone)-Terminated PAMAM Dendrimers. The Journal of Physical Chemistry B 2013, 117 (47), 14865-14874. 25.

Ficker, M.; Petersen, J. F.; Hansen, J. S.; Christensen, J. B., Guest-Host Chemistry with

Dendrimers—Binding of Carboxylates in Aqueous Solution. PLoS One 2015, 10 (10), e0138706. 26.

Fréchet, J.; Tomalia, D., Dendrimers and other Dendritic Compounds. John Wiley & Sons:

Chichester, UK: 2001. 27.

Ficker, M.; Paolucci, V.; Christensen, J. B., Improved large-scale synthesis and characterization of

small and medium generation PAMAM dendrimers. Can. J. Chem. 2017, 95 (9), 954-964. 28.

Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I., Color test for detection of free terminal

amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 1970, 34 (2), 595-598. 29.

He, C.; Hu, Y.; Yin, L.; Tang, C.; Yin, C., Effects of particle size and surface charge on cellular

uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010, 31 (13), 3657-3666. 30.

Albanese, A.; Tang, P. S.; Chan, W. C., The effect of nanoparticle size, shape, and surface

chemistry on biological systems. Annu. Rev. Biomed. Eng. 2012, 14, 1-16. 31.

Lalman, J. A.; Bagley, D. M., Extracting long-chain fatty acids from a fermentation medium. J.

Am. Oil Chem. Soc. 2004, 81 (2), 105-110. 32.

Atkins, P. W.; De Paula, J., The elements of physical chemistry. Oxford University Press New York,

NY, USA:: 2005; Vol. 3.


31


33.

Page 32 of 44

Bekale, L.; Chanphai, P.; Sanyakamdhorn, S.; Agudelo, D.; Tajmir-Riahi, H., Microscopic and

thermodynamic analysis of PEG–β-lactoglobulin interaction. RSC Advances 2014, 4 (59), 31084-31093. 34.

Chanphai, P.; Bekale, L.; Tajmir-Riahi, H., Conjugation of steroids with PAMAM nanoparticles.

Colloids and Surfaces B: Biointerfaces 2015, 136, 1035-1041. 35.

Ross, P. D.; Subramanian, S., Thermodynamics of protein association reactions: forces

contributing to stability. Biochemistry 1981, 20 (11), 3096-3102. 36.

Folch-Cano, C.; Guerrero, J.; Speisky, H.; Jullian, C.; Olea-Azar, C., NMR and molecular

fluorescence spectroscopic study of the structure and thermodynamic parameters of EGCG/βcyclodextrin inclusion complexes with potential antioxidant activity. J. Inclusion Phenom. Macrocyclic Chem. 2014, 78 (1-4), 287-298. 37.

Franklin, I.; Walton, L.; Greenhalgh, R.; Powell, J., The influence of indomethacin on the

metabolism and cytokine secretion of human aneurysmal aorta. European journal of vascular and endovascular surgery 1999, 18 (1), 35-42. 38.

Bour, A.; Westendorp, R.; Laterveer, J.; Bollen, E.; Remarque, E., Interaction of indomethacin

with cytokine production in whole blood. Potential mechanism for a brain-protective effect. Exp. Gerontol. 2000, 35 (8), 1017-1024.


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TABLE OF CONTENS / GRAPHICAL ABSTRACT


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Molecular Pharmaceutics 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


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Complexes of Indomethacin with 4-Carbomethoxy-pyrrolidone

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