key: cord-0639273-xosojr92
authors: Mel, Judith U. De; Gupta, Sudipta; Harmon, Sydney; Stingaciu, Laura; Roth, Eric W.; Siebenbuerger, Miriam; Bleuel, Markus; Schneider, Gerald J.
title: Acetaminophen Interactions with Phospholipid Vesicles Induced Changes in Morphology and Lipid Dynamics
date: 2021-06-03
journal: nan
DOI: nan
sha: a3b2d2fe050ed1e0a12746ff10ba7b7c5e2091de
doc_id: 639273
cord_uid: xosojr92

Acetaminophen (APAP) or Paracetamol, despite its wide and common use for pain and fever symptoms, shows a variety of side effects, toxic effects, and overdose effects. The most common form of toxic effects of APAP is in the liver where phosphatidylcholine is the major component of the cell membrane with additional associated functionalities. Although this is the case, the effects of APAP on pure phospholipid membranes have been largely ignored. Here, we used DOPC, a commonly found phospholipid in mammalian cell membranes to synthesize large unilamellar vesicles to investigate how the incorporation of APAP changes pure lipid vesicle structure, morphology, and fluidity at different concentrations. We used a combination of dynamic light scattering (DLS), small-angle neutron and X-ray scattering (SANS, SAXS), cryo TEM for structural characterization, and neutron spin-echo (NSE) spectroscopy to investigate dynamics. We showed that the incorporation of Acetaminophen in the lipid bilayer significantly impacts the spherical phospholipid self-assembly in terms of its morphology as well as influences the lipid content in the bilayer, causing a decrease in bending rigidity. We discussed how the overall impact of APAP molecules on the pure lipid membrane may play a significant role in the drug's mechanisms of action. Our results showed the incorporation of APAP reduces membrane rigidity as well as changes the spherical unilamellar vesicles into much more irregularly shaped vesicles. Although bilayer structure did not show much change when observed by SAXS, NSE and cryo-TEM results showed the lipid dynamics change with the addition of APAP in the bilayer which causes the overall decreased membrane rigidity. A strong effect on the lipid tail motion was also observed. rigidity. A strong effect on the lipid tail motion was also observed.

Acetaminophen (APAP) and popular NSAIDs (Nonsteroidal anti-inflammatory drugs) such as Aspirin (ASA), and Ibuprofen (IBU) have been used for decreasing inflammation and pain relief for centuries. Commercially available from the 1950s, many Over-the-counter (OTC) antiinflammatory and pain medications are extensively used worldwide without prescription control.

Among these, Acetaminophen is the most commonly used which has a profound presence in antipyretic and analgesic usage that it is almost to a point of over-use. 1 The danger arises due to the toxic effects and side effects on mammals these drugs can have. [2] [3] [4] [5] [6] It has also been shown that despite the low dosages consumed, the drugs tend to accumulate and concentrate in different tissues where the therapeutic effects, side effects, and toxic effects are detected. Therefore, researchers from various disciplines have attempted to unravel their mechanisms of actions in humans and other animal and tissue models using a variety of approaches.

APAP or Paracetamol is considered the first-line choice for pain relief while drugs such as ASA and IBU are considered anti-inflammatory counterparts. 7 Despite the differences in application, one thing these drugs have in common is that their main mechanism of action is connected to a membrane-bound protein family called cyclooxygenase (COX) which regulates prostaglandin formation which then in return regulates inflammatory responses and pain. 8 Over the years, the focus of understanding underlying mechanisms of actions of APAP has been in connection to the COX (Cyclooxygenase) enzyme centers and other related proteins. Despite the long history of use in modern medicine, mechanisms of action of APAP are complicated and not completely understood. [9] [10] [11] [12] Although this is the case, APAP is currently known as a COX inhibitor by competitive inhibition to the active site which binds arachidonic acid, 9 as opposed to the action of other drugs such as IBU which is related to non-specific inhibition of COX 13 , or ASA which has shown activity related to chemopreventive effects and platelet aggregation in addition to being a COX inhibitor by covalently modifying COX active site. 14 Therefore, despite the common relationship with the COX enzymes, these drugs have diverse interaction pathways which call for an independent investigation of the impacts of the drugs to understand their unique effects. One aspect that requires continuous attention is the drug-lipid membrane interactions. Studies have shown different impacts of small drug molecules on lipid membranes such as induced fusion 15 , membrane permeability 16, 17 , and changes in membrane rigidity. [18] [19] [20] Particularly, drug-induced membrane rigidity changes are important to explore due to the relationship of membrane rigidity with multiple functions such as the metastatic potential of cancer cells [21] [22] [23] , apoptosis 24 , erythrocyte morphology 25 , etc.

Acetaminophen is a small drug molecule (C8H9NO2, 151.1626 g mol -1 ) that consists of an aromatic core with an acetanilide functional group and a hydroxyl functional group in para positioning to each other. The pKa value of 9.38 renders the molecule to be charge-neutral in physiological pH as shown in Figure 1 . 26 Molecular dynamics simulations predict APAP molecules are located close to the carbonyl group region of the phospholipids, in an intermediate location between the hydrophobic and hydrophilic parts of the phospholipid. 27 Understanding the unique effects of APAP on mammalian cells particularly, molecular-level details of the drug's influences on physicochemical properties of the cell membrane integrity and fluidity is critical for future therapeutic advancements (e.g. decreasing side-effects due to toxicity). APAP overdose effects have also been researched extensively. 28 The most common form of APAP toxicity occurs in the liver where phosphatidylcholine (PC) is the primary component of the cell membrane in addition to its other functions such as being a precursor of signaling molecules as well as being a key element in lipoprotein and bile. 29 Many studies have shown a connection between phospholipids with the APAP activity. Bhattacharyya et al. showed the presence of PCs and lysoPCs with very long fatty acids significantly decreased in overdose conditions indicative of a structure-activity relation with enzymes responsible for phospholipid metabolism. 30 Recently, Yamada et al. showed mechanisms where acute liver failure induced by APAP toxicity is ferroptosis-mediated which is driven by polyunsaturated fatty acids. 31 Ming et al. have also reported that the APAP overdose conditions induce dramatic changes to PC and PE profiles of plasma and liver through possible hepatocyte damage and interferences to phospholipid metabolism. 29 Therefore, the importance of investigating APAP effects on pure phospholipid bilayer structures becomes an important research question from a fundamental, material, and chemical standpoint.

Here we have investigated the structural and dynamic details of drug-lipid vesicle interactions in hope of broadening the knowledge of biophysical processes that may carry links to many long-standing questions in the realm of the drug's side-effects and toxicity effects at the molecular basis. We hypothesized the contribution from incorporated Acetaminophen to the phospholipid bilayer is enough to contribute to changes in physicochemical properties of the bilayer, the morphology of the vesicle self-assembly as well as lipid dynamics at nanometer length-scale and nanosecond timescale which will result in functional changes to the membranebound enzymes, etc. regulating Acetaminophen mechanisms of action and its side/overdose effects. To investigate these structural, morphological, and dynamical changes we used DOPC large unilamellar vesicles with APAP ( Figure 1 ) and conducted dynamic light scattering (DLS), small-angle X-ray and neutron scattering (SAXS/SANS), cryo-transmission electron microscopy (cryo-TEM), and neutron spin-echo spectroscopy studies (NSE). 

Vesicle structure: The vesicle form factor is modeled using an extension of the core-shell model used in our previous studies. [32] [33] [34] [35] [36] For unilamellar vesicles the core-shell model consists of a water core of radius , encapsulated by three shells with (i) lipid inner-head, (ii) tail region (hydrocarbon core), and (iii) outer-head. The 1D scattering pattern is given by:

with is the lipid volume fraction. The scattering contribution from three different shells are given by 2 ( ) = 1 2 + 2 2 + 3 2 + 12 + 23 + 13 (2) with 12 = 1 2 , 12 = 2 3 , and 13 = 1 3 are the cross-terms for inner-head-tail, tailouter-head and the two outer lipid head layers, respectively. 

Here, 1 = + ℎ , 2 = + ℎ + , 3 = + 2 ℎ + . The membrane thickness of the bilayer from SANS is given by, ( ) = 2 ℎ + . For DOPC we used the neutron scattering length density (NSLD) of the hydrocarbon tail, tail = −2.08 × 10 9 cm -2 , and for phosphatidylcholine (PC) head group, head = 1.73 × 10 10 cm -2 has been used. 37 For D2O the NSLD of the solvent of, solv = 6.36 × 10 10 cm -2 has been used. Each shell thickness and scattering length density is assumed to be constant for the respective shell. The macroscopic scattering cross-section is obtained by

For the size polydispersity, ( ), we used a log-normal distribution. 38 A Gaussian distribution was used to include polydispersity of the bilayer and the water layer in the case of multilamellar vesicles.

Bilayer structure: To get direct access to the macroscopic scattering cross-section given by the SAXS scattering intensity from random lamellar sheet consisting of lipid heads and tails of thicknesses are and , respectively is given by

with particle volume fraction, , and the lamellar repeat distance, d. The form factor is given by:

The scattering contrasts for the head and tail are ∆ H and ∆ T , respectively. The corresponding thicknesses are and , respectively. The head to head bilayer thickness is given by, ( ) = 2( + ). For unilamellar structure, = 1, the repeat distance in equation 6 is given by the membrane thickness, . The Caille structure factor is given by

with the number of lamellar plates, ( > 1), and the correlation function for the lamellae, ( ), defined by 

The Q-dependent decay rate, Γ , can be used to determine the intrinsic bending modulus, , by 44-46

Here is the viscosity, the Boltzmann constant, the temperature. For lipid bilayers, [45] [46] [47] To obtain model-independent data we analyze the mean squared displacement (〈Δ ( ) 2 〉, MSD), and the non-Gaussianity parameter, 2 ( ) = +2 〈Δ ( ) 4 〉 〈Δ ( ) 2 〉 2 − 1, from the measured dynamic structure factor, ( , ), using a cumulant expansion given by, 33, 39, 48, 49 

The non-Gaussianity parameter 2 is essentially defined as a function of the fourth 〈Δ ( ) 4 〉 and the second moment squared 〈Δ ( ) 2 〉 2 , Here we use the space dimension, Table 1 .

The cryo-TEM data were obtained at the BioCryo facility of Northwestern University Nuance Center (remote access due to COVID19 restrictions). The samples were applied to 200 mesh Cu grids with a lacey carbon membrane.

Before plunge-freezing, grids (EMS Cat# LC200-CU-100) were glow discharged in a Pelco easiGlow glow discharger (Ted Pella Inc., Redding, CA, USA) using an atmosphere plasma generated at 15 mA for 15 seconds with a pressure of 0.24 mbar. This treatment created a negative charge on the carbon membrane, allowing liquid samples to spread evenly over the grid. 

SAXS data were obtained on two occasions. SAXS results displayed in Figure 3 were obtained at the Stanford Synchrotron Radiation Light source (SSRL), beamline 4-2. 51 An automated sampler system was used to load 40 μL aliquots of samples to the capillary cell where the sample was exposed to the X-ray radiation 24 times in 1 s exposures as the flow cell gently moves back and forth to minimize any potential radiation damage by continuous spot exposure. The scans were then statistically averaged, and radial averaging over the same data yield the intensity as a function of the momentum transfer. The data were obtained at a beam energy of 11 keV, at the detector distance of 1 m to explore a range of 0.01 -1 Å -1 using a Pilatus 3 × 1 M detector.

Data reduction was done using standard SSRL protocols implemented in the software Blu-Ice. 52 

First, the structure was investigated using a combination of DLS, cryo-TEM, SANS, and SAXS.

DLS results showed a slight decrease in the hydrodynamic radius of the vesicles with increasing Acetaminophen (APAP) concentration in the lipid bilayer, Table 1 . Size distribution increased with the addition of APAP, (Figure 2 .a), as reflected by the polydispersity of the vesicles calculated by log-normal distribution fits, Table 1 . SANS data were modeled (Figure 2b) Table 1 . While SANS provides information on the liposome diameter, due to its resolution small-angle Xray scattering (SAXS) can be used to obtain an accurate estimate of the lipid head and tail thickness as well as the number of bilayers. This sensitivity is achieved due to the reduced influence from vesicle form-factor over the relevant -range, higher X-ray contrast for phosphorous atoms in the lipid heads, and excellent instrumental resolution. Figure 3 illustrates SAXS diffraction patterns for 1 wt% DOPC with 0 to 0.12 wt% Acetaminophen concentrations. Vesicles appeared more fluid-like and had irregular sphere shapes as well as occasional tubular shapes indicated by the yellow arrows. Such deviations appear to be more pronounced when the Acetaminophen concentration was increased from 0.06 wt% to 0.12 wt%. Furthermore, overall vesicle sizes decreased in the presence of Acetaminophen. Figure 4d) shows the size distribution of vesicles in each sample measured using multiple cryo-TEM images. More than 1500 size measurements were used for the size analysis using the log-normal distribution model to describe data. The trend of size variation was similar to DLS and SANS results previously presented in Table 1 . The polydispersity of the particles increased agreeing with results obtained by DLS and SANS. More images and details of the analysis are provided in the SI.

Membrane dynamics were measured by neutron spin-echo (NSE) spectroscopy. Data were modeled by the multiplicative model (equation 9) which includes vesicle translational diffusion, membrane fluctuations, and confined motion of lipid tails for dynamic structure factor ( , ) analysis. 39 All experiments were conducted at room temperature. Therefore, DOPC lipids are in the fluid phase. The model agreed well with the data in the and Fourier time range used for the experiment ( Figure 5 ). It should be noted that within the NSE time window the translational diffusion, , is almost by a factor 5 slower than the ZG decay as observed from the dynamic structure factor illustrated in Figure S7 in SI. The details of the NSE data analysis are presented in the SI.

Bending rigidity values, κ η , calculated from the analysis are displayed in Table 2 Figure S9b ). This change points to more spatial freedom for the tail motion once APAP has been added to the system. The transition to the ZG region seems to depend on the concentration of the APAP as well.

Considering the fast dynamics at < 8 ns, we have calculated ⁄ using equation 11

as a function of the Fourier time over the entire NSE time window. The results for 0, 0.06% and 0.12% acetaminophen concentrations are presented in Figure 7 . The results are calculated using = and for = 0. 33, 39, 40 The average bending rigidity values found from the analysis within the ZG region are displayed in Figure 7 and are similar to that observed from the multiplicative model (equation 9) as displayed in Table 2 . comparable. To obtain model-independent insights, we have compared the normalized membrane rigidities. In order to see a trend with molar fraction of drugs in the lipid membrane, and decrease in membrane rigidity, the κ η the ratio is obtained by normalizing compared to the pure phospholipid vesicle systems used in the respective studies (Table 3) . 

In summary, we have investigated the concentration-dependent impact of Acetaminophen (APAP) or commonly known as paracetamol on DOPC LUVs in the fluid phase. We observed a slight decrease in vesicle size by DLS and SANS. Our cryo-TEM experiments illustrated further morphological changes. Vesicle shapes became more and more irregular with the increasing concentration of Acetaminophen which might be an explanation of the increasing polydispersity of the vesicle populations observed by DLS. In SAXS, surprisingly, we did not observe much change in the bilayer structure. Previous studies have shown substantial differences in SAXS profiles when small drug molecules alter the melting temperature of the lipid bilayers. 61 We assume that the relatively small changes in the SAXS profile may relate to the fluid phase (experiments are conducted well above the of DOPC).

We observe a change in aggregation number and find a decrease by almost 28% (0.06 wt% APAP) and 19% (0.12 wt% APAP) compared to the pure DOPC (0 wt% APAP). At the same time, we find a decrease in the diameter, which leads to the surprising effect that the radius ascribed to a lipid stays almost constant within the experimental accuracy. Eventually, a slight change of 1.06 can be calculated. Surprisingly, simulations on the system APAP and DPPC, and DMPC revealed a virtually constant value. 27 Though, simulations and our experiments are different both results point to a little to no effect on the area per lipid.

Surprisingly, we observe a stronger change of the mean square displacement of the lipid tail motion. It seems that the space explored by the lipid tails increases by a factor of 1.45 (0.06%), simulations find a substantial decrease, which the authors ascribe to drug molecules entering the space between the lipids. Our results point also to drug molecules increasing the space between lipids, which would explain the larger MSD of the tails. As the distance between the molecules determines the molecular interactions [Nademi] the reduced bending modulus seems to be a direct consequence of the increased spatial freedom. Again, we point to the surprising area per lipid from the SANS experiments which is virtually unchanged (at most changed by a factor of 1.06). So, despite little changes in the static structure strong changes of the tail motion and bending elasticity seem to be a consequence of drug molecules penetrating the free space between the lipids.

We compared our bending rigidity values with other existing work and established a general trend in membrane rigidity changes induced by structurally similar drugs on phospholipid vesicles in the fluid phase. Since Acetaminophen toxicity has been related to multiple cellular functions such as oxidative stress, lipid peroxidation 62 , etc., the changes in membrane dynamics and fluidity may be directly or indirectly connected to these cellular functions hence should not be ignored. This also opens up therapeutic avenues to explore the usage of clinically well-established "old" drugs such as Acetaminophen in cancer therapeutic drug delivery systems as an agent to manipulate membrane rigidity. 63

Physicochemical details of Acetaminophen, Cryo-TEM additional images and analysis details, SAXS data from CAMDlab X-ray source, NSE data modeling using ZG model, Impact of translational diffusion on NSE data, ZG decay rate Γ Q variation with and without the translational diffusion of the vesicles.

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