Contribution of Molecular Structure to Self-Assembling and Biological Properties of Bifunctional Lipid-Like 4-(N-Alkylpyridinium)-1,4-Dihydropyridines

The design of nanoparticle delivery materials possessing biological activities is an attractive strategy for the development of various therapies. In this study, 11 cationic amphiphilic 4-(N-alkylpyridinium)-1,4-dihydropyridine (1,4-DHP) derivatives differing in alkyl chain length and propargyl moiety/ties number and position were selected for the study of their self-assembling properties, evaluation of their cytotoxicity in vitro and toxicity on microorganisms, and the characterisation of their interaction with phospholipids. These lipid-like 1,4-DHPs have been earlier proposed as promising nanocarriers for DNA delivery. We have revealed that the mean diameter of freshly prepared nanoparticles varied from 58 to 513 nm, depending upon the 4-(N-alkylpyridinium)-1,4-DHP structure. Additionally, we have confirmed that only nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6, and by 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 were stable after two weeks of storage. The nanoparticles of these compounds were found to be homogenous in size distribution, ranging from 124 to 221 nm. The polydispersity index (PDI) values of 1,4-DHPs samples 3, 6, 10, and 11 were in the range of 0.10 to 0.37. We also demonstrated that the nanoparticles formed by 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3, 6, and 9, and 4-(N-hexadecylpyridinium)-1,4-DHP derivatives 10 and 11 had zeta-potentials from +26.07 mV (compound 6) to +62.80 mV (compound 11), indicating a strongly positive surface charge and confirming the relative electrostatic stability of these nanoparticle solutions. Transmission electron microscopy (TEM) images of nanoaggregates formed by 1,4-DHPs 3 and 11 confirmed liposome-like structures with diameters around 70 to 170 nm. The critical aggregation concentration (CAC) value interval for 4-(N-alkylpyridinium)-1,4-DHP was from 7.6 µM (compound 11) to 43.3 µM (compound 6). The tested 4-(N-alkylpyridinium)-1,4-DHP derivatives were able to quench the fluorescence of the binary 1,6-diphenyl-1,3,5-hexatriene (DPH)—1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) system, demonstrating hydrophobic interactions of 1,4-DHPs with phospholipids. Thus, 4-(N-dodecylpyridinium)-1,4-DHP derivative 3 quenched the fluorescence of the DPH–DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHP derivatives. Likewise the compound 3, also 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 interacted with the phospholipids. Moreover, we have established that increasing the length of the alkyl chain at the quaternised nitrogen of the 4-(N-alkylpyridinium)-1,4-DHP molecule or the introduction of propargyl moieties in the 1,4-DHP molecule significantly influences the cytotoxicity on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines, as well as the estimated basal cytotoxicity. Additionally, it was demonstrated that the toxicity of the 4-(N-alkylpyridinium)-1,4-DHP derivatives on the Gram-positive and Gram-negative bacteria species and eukaryotic microorganism depended on the presence of the alkyl chain length at the N-alkyl pyridinium moiety, as well as the number of propargyl groups. These lipid-like compounds may be proposed for the further development of drug formulations to be used in cancer treatment.


Introduction
Over the past few decades, scientists worldwide have made efforts to expand the discovery and development of a broad range of nanoparticle delivery systems. These studies had been generally supported by the pharmaceutical and food industries as end-users. Liposomes have been widely studied as perspective delivery systems due to their efficiency, biocompatibility, and dual character-their ability to entrap either hydrophobic or hydrophilic drugs, improving their pharmacokinetic and pharmacodynamic properties [1]. Synthetic nanoparticle-forming cationic lipid-like compounds have been developed as delivery agents for the transfer of genetic materials, including plasmid DNA (pDNA) molecules, into cells [2][3][4] and recently also for therapy and diagnostic applications [5][6][7][8]. In general, among the synthetic cationic delivery systems, quaternary ammonium surfactants are more toxic than their analogues, with the cationic charge delocalised in a heterocyclic ring [9].
Diverse groups of nanoparticles have been reported to possess inherent antimicrobial properties [10,11]. Also, the intrinsic antitumor activity of a carrier material based on cationic lipids that have biguanide as a structural fragment have been reported [12]. Enhancement of the therapeutic efficiency of anticancer agents by their incorporation into vitamin E-based delivery systems is well-known approach with great potential [13][14][15]. The elaboration of nanoparticles that have inherent biological properties may have potential in the future development of antimicrobial and/or antitumor therapies [16,17].
Previously, our group elaborated and studied multiple liposomes forming cationic 1,4-dihydropyridine (1,4-DHP) amphiphiles, which were capable of transfecting pDNA into different cell lines in vitro. For an evaluation of the molecule architecture influence on gene delivery properties, various 1,4-DHP amphiphiles containing single or double cationic moieties were designed, synthesised, and studied ( Figure 1, groups 1 and 2). These studies demonstrated that the compounds with double pyridinium moieties (Group 2) showed high transfection efficiencies in vitro and revealed some important structure-activity relationships [18]. After these findings, more detailed studies of 1,4-DHP derivatives containing two pyridinium moieties were performed, and the influence of the cationic part of the molecule [19] and linker [20] on the self-assembling properties and structure-activity relationships were revealed. Additionally, it was established that several representatives of double pyridinium moieties containing compounds (Group 2) possessed antiradical activity [19], and one of them also worked as an antioxidant with the potent reversal of multidrug resistance blocking ability in murine lymphoma cells [21].
As a part of our current research program towards the development of novel non-viral delivery systems possessing biological activities, we continue studies on original single-charged cationic lipids containing a pharmacophore group as putative nanocarriers. Our previous studies have demonstrated that 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6 exhibited calcium antagonistic properties on neuroblastoma SH-SY5Y (IC50 about 5-14 µM) and vascular smooth muscle A7R5 (IC50 about 0.6-0.7 µM) cell lines [22]. Additionally, the memory-improving anxiolytic effects of 4-(N-dodecylpyridinium)-1,4-DHP 3 in transgenic Alzheimer's disease female mice were described [23], and it was shown that this compound crossed the blood-brain barrier and blocked neuronal and vascular calcium channels [24]. We also found a direct correlation between the length of the alkyl moiety at N-quaternised 4-pyridyl-1,4-DHP and improvement of membranotropic effects such as incorporation in the liposomal membranes and bilayer fluidity [25]. Furthermore, studies of the calcium channel antagonist and agonist activities of the 3,5-dipropargylcarbonyl moieties containing 4-(N-alkylpyridinium)-1,4-DHPs confirmed that these compounds targeted only calcium channels in vascular smooth muscle cells, and did not affect the calcium channels in cardiac cells [26].
In this work, we have studied in detail the ability to form nanoparticles by cationic 4-(Nalkylpyridinium)-1,4-DHP derivatives in aqueous media. Transmission electron microscopy (TEM) images of nanoparticles of selected compounds have been registered. We also established the size distribution and determined the stability of nanoparticles by dynamic light scattering (DLS) measurements. Additionally, critical aggregation concentration (CAC) has been estimated by the DLS technique. Moreover, the cytotoxicity of 4-(N-alkylpyridinium)-1,4-DHPs derivatives on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines has been evaluated, and an approximate LD50 value has been predicted. Additionally, toxicity on six prokaryotic (bacteria) species and one eukaryotic (yeast) microorganism species have been estimated. The hydrophobic interaction between 4-(N-alkylpyridinium)-1,4-DHP derivatives and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) model membranes has been studied. The obtained results will provide a basis for the further understanding of the structure-activity relationships of these compounds. These studies demonstrated that the compounds with double pyridinium moieties (Group 2) showed high transfection efficiencies in vitro and revealed some important structure-activity relationships [18]. After these findings, more detailed studies of 1,4-DHP derivatives containing two pyridinium moieties were performed, and the influence of the cationic part of the molecule [19] and linker [20] on the self-assembling properties and structure-activity relationships were revealed. Additionally, it was established that several representatives of double pyridinium moieties containing compounds (Group 2) possessed antiradical activity [19], and one of them also worked as an antioxidant with the potent reversal of multidrug resistance blocking ability in murine lymphoma cells [21].

4-(N-Alkylpyridinium)-1,4-Dihydropyridine (1,4-DHP) Derivatives
As a part of our current research program towards the development of novel non-viral delivery systems possessing biological activities, we continue studies on original single-charged cationic lipids containing a pharmacophore group as putative nanocarriers. Our previous studies have demonstrated that 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3 and 6 exhibited calcium antagonistic properties on neuroblastoma SH-SY5Y (IC 50 about 5-14 µM) and vascular smooth muscle A7R5 (IC 50 about 0.6-0.7 µM) cell lines [22]. Additionally, the memory-improving anxiolytic effects of 4-(N-dodecylpyridinium)-1,4-DHP 3 in transgenic Alzheimer's disease female mice were described [23], and it was shown that this compound crossed the blood-brain barrier and blocked neuronal and vascular calcium channels [24]. We also found a direct correlation between the length of the alkyl moiety at N-quaternised 4-pyridyl-1,4-DHP and improvement of membranotropic effects such as incorporation in the liposomal membranes and bilayer fluidity [25]. Furthermore, studies of the calcium channel antagonist and agonist activities of the 3,5-dipropargylcarbonyl moieties containing 4-(N-alkylpyridinium)-1,4-DHPs confirmed that these compounds targeted only calcium channels in vascular smooth muscle cells, and did not affect the calcium channels in cardiac cells [26].
In this work, we have studied in detail the ability to form nanoparticles by cationic 4-(N-alkylpyridinium)-1,4-DHP derivatives in aqueous media. Transmission electron microscopy (TEM) images of nanoparticles of selected compounds have been registered. We also established the size distribution and determined the stability of nanoparticles by dynamic light scattering (DLS) measurements. Additionally, critical aggregation concentration (CAC) has been estimated by the DLS technique. Moreover, the cytotoxicity of 4-(N-alkylpyridinium)-1,4-DHPs derivatives on HT-1080 (human fibrosarcoma) and MH-22A (mouse hepatocarcinoma) cell lines has been evaluated, and an approximate LD 50 value has been predicted. Additionally, toxicity on six prokaryotic (bacteria) species and one eukaryotic (yeast) microorganism species have been estimated. The hydrophobic interaction between 4-(N-alkylpyridinium)-1,4-DHP derivatives and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes has been studied. The obtained results will provide a basis for the further understanding of the structure-activity relationships of these compounds.

Cell Culture and Measurement of Cell Viability
Tumor cell lines HT-1080 (human connective tissue fibrosarcoma) and MH-22A (mouse hepatocarcinoma) were used. All of the cells were obtained from the American Type Culture Collection ((ATCC,) Rockville, MD, USA).

Basal Cytotoxicity Test
The neutral red uptake (NRU) assay was performed according to the standard protocol of Stokes [27] modified by NICEATM-ECVAM validation study [28]. The NRU cytotoxicity assay procedure was based on the ability of viable cells to incorporate and bind neutral red, which is a supravital dye.
NIH 3T3 (Mouse Swiss Albino embryo fibroblast) cells (9000 cells/well) were placed into 96-well plates for 24 h in DMEM medium containing 5% fetal bovine serum (FBS). Then, they were exposed to the test compound over a range of seven concentrations (1000 µg/mL, 316 µg/mL, 100 µg/mL, 31 µg/mL, 10 µg/mL, 3.0 µg/mL, and 1.0 µg/mL) for 24 h. Untreated cells were used as a control. After 24 h, the medium was removed from all plates. Then, 250 µL of neutral red (NR) solution was added (0.05 mg/mL NR in DMEM 24 h pre-incubated at 37 • C and then filtered before use through a 0.22-µm syringe filter). Plates were incubated for 3 h, and then, cells were washed three times with phosphate buffered saline (PBS). The dye within viable cells was released by extraction with a mixture of acetic acid, ethanol, and water (1:50:49). The absorbance of neutral red was measured using a spectrophotometer multiplate reader Infinite M1000 (Tecan Austria GmbH, Salzburg, Austria) at 540 nm. The optical density (OD) was calculated using the formula: OD (treated cells) × 100/OD (control cells). The IC 50 values were calculated using the program Graph Pad Prism ® 3.0.

Estimation of LD 50 from IC 50 Values
Data from the in vitro tests were used for estimating the starting dose for acute oral systemic toxicity tests in rodent. The in vivo starting dose is an estimated LD 50 value that is calculated by inserting the in vitro IC 50 value into a regression formula: log LD 50 (mM/kg) = 0.439 log IC 50 (mM) + 0.621 [29]. The value is recalculated to mg/kg, and compounds are evaluated in accordance with four toxicity categories [30]: category 1: LD 50 ≤ 5 mg/kg (highly toxic); category 2: 5 < LD 50 ≤ 50 mg/kg (moderately toxic); category 3: 50 < LD 50 ≤ 300 mg/kg (slightly toxic); category 4: 300 < LD 50 ≤ 2000 mg/kg (practically non-toxic). Using an alternative in vitro method enables comparing the possible toxicity of new compounds and selecting compounds for further study, which will vastly reduce the number of animal experiments.

Toxicity Screen
An estimation of the toxicity of 4-(N-alkylpyridinium)-1,4-DHP derivatives 3, 6, 7, and 9-11 was performed using six prokaryotic (bacteria) and one eukaryotic (yeast) microorganism species according to the procedure elaborated by Suppi et al. [31]. The microorganisms used in the study and their origin were: Bacillus subtilis subsp.

Microorganism Cultivation and Toxicity Test
For toxicity screening, a compound serial dilution-spot test method as suggested by Suppi et al. was used [31].
Bacteria and yeast cultures were cultivated until their exponential growth phase (optical density max 1); then, 100 µL of cell suspension was taken, washed in deionised (DI) water, and added to serially diluted samples of corresponding 4-(N-alkylpyridinium)-1,4-DHPs in DI water. All 1,4-DHPs were tested in five serially diluted concentrations (10 mM, 1 mM, 0.1 mM, 0.01 mM, and 0.001 mM). Test organisms were exposed to 1,4-DHP derivatives in DI water on 96-well microplates (Corning, Corning, NY, USA) at 28 • C for 24 h without shaking in the dark. Then, a 5 µL sample was taken from each well and spotted on yeast extract, bactopeptone, dextrose broth (YPD) (yeast extract 1%, bactopeptone 2%, glucose 2% and agar 2%) plates to test the growth of yeasts and lysogeny broth (LB) (yeast extract 0.5%, bactopeptone 1%, NaCl 0.5% and agar 2%) to test the growth for bacteria. Plates were left in 30 • C for 24 h, and assessed for microbial growth.
Boundary concentration was defined as the last dilution of 1,4-DHP, which did not fully inhibit the growth of microorganisms (colonies were present). If there were no colonies after incubation with 1,4-DHPs in the last concentration of (0.001 mM), then their boundary concentration was set to 0.0001 mM or 0.1 µM.
To characterise the hydrophobic interaction between the 1,4-DHP and phospholipids, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as a fluorescence probe. The lipid-bound DPH shows intense fluorescence at 440 nm (excitation, 368 nm), while the non-bound DPH does not exhibit fluorescence in aqueous solution. DPH was used as a probe to determine the hydrophobic interaction between the esters and the phospholipid. Stock solutions of DPH in acetone (15 × 10 −4 M) were freshly prepared and kept in the dark and diluted with TBS buffer (pH = 7.4) to concentration.

Self-Assembling Properties by Dynamic Light Scattering Measurements
Samples of compounds for dynamic light scattering (DLS) studies were prepared by thin-film hydration method in an aqueous solution at a concentration of 0.5 mg/mL for compounds 1-7, 0.25 mg/mL for compounds 8-10, and 0.1 mg/mL for compound 11. A corresponding amount of compounds was weighted in a round-bottom flask and dissolved in chloroform; then, the organic solvent was removed in vacuo, and the residue was dried in high vacuo for 1 h. An appropriate amount of deionised water was added to each flask for the preparation of stock solutions with the above-mentioned concentrations. Samples were prepared by sonication using a bath-type sonicator (Cole Parmer Ultrasonic Cleaner 8891CPX (Vernon Hills, IL, USA)). Samples were sonicated for 60 min at 50 • C.
The DLS measurements of the nanoparticles in an aqueous solution were carried out on a Zetasizer Nano ZSP (Malvern Panalytical Ltd., Malvern, UK) instrument with Malvern Instruments Ltd. Software 7.12, using the following specifications-medium: water; refractive index: 1.33; viscosity: 0.8872 cP; temperature: 25 • C; dielectric constant: 78.5; nanoparticles: liposomes; refractive index of materials: 1.60; detection angle: 173 • ; wavelength: 633 nm. Data were analysed using the multimodal number distribution software that was included with the instrument. The measurements were performed in triplicate in order to check their reproducibility.

Determination of CAC
The critical aggregation concentrations (CAC) of 4-(N-alkylpyridinium)-1,4-DHP derivatives were determined in aqueous media using a Zetasizer Nano S90 (Malvern Panalytical Ltd., Malvern, UK) instrument with Malvern Instruments Ltd. Software according to procedure described by Topel et al. and modified by our group [19,36]. Briefly, as stock solutions of tested compounds were used, samples were prepared for self-assembling experiments. All of the subsequent samples were prepared starting from the concentrated stock solution, which was subjected to a serial two-fold dilution each time with water. The intensity values of scattered light (kcps) as a function of concentration of amphiphiles were analysed. The scattering intensities detected for amphiphile concentrations below CAC have an approximately constant value corresponding to water. The intensity starts to show a linear increase with concentration at the CAC, since the amount of nanoparticles increases in the solution. The intersection of the best-fit lines drawn through the data points is the preliminary CAC value of the compounds.

Transmission Electron Microscopy (TEM)
The morphology of nanoparticles formed by selected 4-(N-alkylpyridinium)-1,4-DHP derivatives 3 and 11 (0.15 mM) was studied by TEM. Negative stained TEM samples were prepared using the side blotting method. The sample solution was placed on a carbon-coated grid (Agar AGS160-4); excess solution was drained with filter paper, and then it was negatively stained with 2% uranyl acetate, which was freshly prepared and filtered with a 0.22-µm filer. TEM measurements were performed using Tecnai G2 F20 (FEI, Hillsboro, OR, USA) microscope at 60 kV.

Statistical Analysis
Results are expressed as mean standard deviation (SD). All of the experiments were performed in triplicate.

4-(N-Alkylpyridinium)-1,4-Dihydropyridine Derivatives
The synthesis of selected 4-(N-alkylpyridinium)-1,4-dihydropyridine derivatives 1-11 differing in alkyl chain length and propargyl moiety/ties number and position was carried out by previously described methods [22,26]. Briefly, the 1,4-DHP derivatives were synthesised via typical synthetic routes, including the classical Hantzsch method [37], which involved the one-pot cyclocondensation of the corresponding esters of acetoacetic acid, and the corresponding aldehyde and ammonia or ammonium acetate as a nitrogen source in ethanol under reflux. The N-propargyl-substituted 1,4-DHP derivatives were obtained by analogy with the synthesis of other N-substituted 1,4-DHP ones via a modified Hantzsch-type cyclisation using propargyl amine hydrochloride as a nitrogen source instead of ammonia and pyridine as a solvent under reflux. The synthesis of all the 4-pyridinium moieties containing 1,4-DHP derivatives were performed by the alkylation of 4-pyridyl-1,4-DHP derivatives with the corresponding alkyl bromides in acetone under reflux. The typical procedure for the quaternisation of pyridyl-1,4-DHP derivatives was performed in analogy with the procedure described by Makarova et al. [38,39].
In order to evaluate the influence of the pharmacophore group on the structure-activity relationship, 11 1,4-DHP derivatives were divided into two groups considering structure elements (see Figure 2 The purities of the studied compounds were at least 97% according to high-performance liquid chromatography (HPLC) data. The detailed synthetic procedures, yields of products, nuclear magnetic resonance (NMR) spectrum data for 4-(N-alkylpyridinium)-1,4-dihydropyridine derivatives 1-11 were described by Rucins et al. [22,26].

Estimation of LD 50 from IC 50 Values
Antiproliferative activity and also a potential toxic effect were evaluated for 4-(Nalkylpyridinium)-1,4-DHP derivatives 1-9. Currently, it is recommendable to start toxicology studies with preliminary in vitro estimation before using animal models [40]. It has been proposed that the equation from the correlation of IC 50 (the concentration of a substance that causes 50% toxicity in vitro) could be applied to estimate unknown LD 50 values for a new compound from IC 50 values measured as basal cytotoxicity in vitro. This estimated LD 50 gives prior information regarding compound properties and would be used to select promising compounds and a starting dose for in vivo experiments.
The obtained data showed that 4-(N-ethylpyridinium)-1,4-DHP derivatives 1, 4, and 7 did not demonstrate any cytotoxic effect on tumor HT-1080 and MH-22A cell lines, and their estimated toxicity LD 50 was defined as practically non-toxic (basal cytotoxicity LD 50 ≥2000 mg/kg); LD 50 (Table 1)). Generally, it can be concluded that increasing the length of the alkyl chain from ethyl to dodecyl at the quaternised nitrogen atom at position 4 of the 1,4-DHP ring or the introduction of propargyl moiety/moieties into the 1,4-DHP molecule influences both the cytotoxicity against cancer cells and also the estimated basal cytotoxicity of tested 4-(N-alkylpyridinium)-1,4-DHP derivatives.

Toxicity Test
Several different species of microorganisms were chosen to evaluate toxicity of 4-(N-alkylpyridinium)-1,4-DHP derivatives 3, 6, 7, and 9-11 on microorganisms according to the procedure elaborated by Suppi et al. [31]. The panel of microorganisms was composed based on an ecotoxicological review by Egorova and Ananikov [38]. It included representatives of Gram-positive bacteria species (B. subtilis and M. luteus) and representatives of Gram-negative bacteria species (E. coli, P. miriabilis, P. aeroginosa, and K. pneumoniae) and one example of eukaryotic microorganism (S. cerevisiae). The obtained results (see Table S1 in the Supplementary Material) regarding the effect of 4-(N-alkylpyridinium)-1,4-DHP derivatives 3, 6, 7, and 9-11 on microbial survival were analysed by Principle Component Analyses (PCA) using the online ClustVis tool [33]. PCA is a mathematical procedure that reduces the dimensionality of large datasets, increasing their interpretability while at the same time minimizing information loss. Currently, PCA is one of the most important and powerful methods for standard statistical tests in biology, medicine, and other fields using statistical tests [41,42]. In our studies, PCA were used to evaluate how the influence of the molecular parameters (the presence of a propargyl group and/or length of the alkyl chain) of the 4-(N-alkylpyridinium)-1,4-DHP derivatives affected their toxicity in the tested microorganism panel. The boundary concentrations from the chosen 1,4-DHP derivatives that were tested in all the microorganisms were collected in a score matrix (Table S1 in the Supplementary Material), and the obtained data resulted in a four-dimension matrix (number of propargyl groups; side chain length; microorganism; boundary concentration).
The 4-(N-ethylpyridinium)-1,4-DHP 7 did not affect the survival of microorganisms in the whole range of the concentrations tested (10 to 0.001 mM). Similarly, this derivative turned out to be non-toxic to the tested cancer cell lines also. On the contrary, other tested 1,4-dihydropyridine derivatives containing N-dodecylpyridinium (compounds 3, 6, and 9) or N-hexadecylpyridinium (compounds 10 and 11) moieties were toxic at various degrees, affecting the viability of the microorganisms. A similar relationship was observed also for ionic liquids (ILs): the increase in the length of the alkyl chain on the ILs' heterocyclic ring significantly increased their toxicity in most test systems [43,44]. To evaluate the patterns of 4-(N-alkylpyridinium)-1,4-DHP alkyl chain effect on the survival of microorganisms PCA were carried out using each compound boundary concentrations for each microorganism. The obtained data are presented in Figure 3. concentrations from the chosen 1,4-DHP derivatives that were tested in all the microorganisms were collected in a score matrix (Table S1 in the Supplementary Material), and the obtained data resulted in a four-dimension matrix (number of propargyl groups; side chain length; microorganism; boundary concentration). The 4-(N-ethylpyridinium)-1,4-DHP 7 did not affect the survival of microorganisms in the whole range of the concentrations tested (10 to 0.001 mM). Similarly, this derivative turned out to be nontoxic to the tested cancer cell lines also. On the contrary, other tested 1,4-dihydropyridine derivatives containing N-dodecylpyridinium (compounds 3, 6, and 9) or N-hexadecylpyridinium (compounds 10 and 11) moieties were toxic at various degrees, affecting the viability of the microorganisms. A similar relationship was observed also for ionic liquids (ILs): the increase in the length of the alkyl chain on the ILs' heterocyclic ring significantly increased their toxicity in most test systems [43,44]. To evaluate the patterns of 4-(N-alkylpyridinium)-1,4-DHP alkyl chain effect on the survival of microorganisms PCA were carried out using each compound boundary concentrations for each microorganism. The obtained data are presented in Figure 3.
Data for the set of tested 4-(N-alkylpyridinium)-1,4-DHP derivatives and their toxicities ( Table  S1 in Supplementary Material), indicated that compounds were rather scattered across a twodimensional (2D) plot with no pattern clustering neither with respect to the number of propargyl groups and alkyl chain length at alkylpyridinium moiety/ies. The toxicity of 4-(N-ethylpyridinium)-1,4-DHP 7 substantially differs from the toxicities of N-dodecylpyridinium (compounds 3, 6, and 9) or N-hexadecylpyridinium (compounds 10 and 11) derivatives ( Figure 3A). However, if compound 7 was omitted from PCA analyses, it can be concluded that 4-(N-alkylpyridinium)-1,4-DHPs were grouped mainly based on their N-alkyl chain length rather than the number of propargyl groups ( Figure 3B (Table S1 in Supplementary Material); the results formed a four-dimension matrix (number of propargyl groups; side chain length; microorganism; boundary concentration). To find out which of the parameters (number of propargyl groups and/or side chain length) has the most significant impact on the compound's toxicity, the "artificial" set of variables that would help to visualise the main effects of data variance across a two-dimensional (2D) plot was sought. The data were analysed and visualised by online tool ClustVis [33]. Vector scaling is applied to rows; the Nonlinear Iterative Partial Least Squares (NIPALS) algorithm is used to calculate principal components [42]. The scores of individual compound's toxicities are plotted using the first two principal components, which explain most of the variance (PC1 63.3% and PC2 19.3% for A, and 39.8% and 24.7% for B). Green triangle: 4-(N-ethylpyridinium)-1,4-DHP 7.
Therefore we assume that the toxicity of the compound depends on the N-alkyl chain length at the pyridinium moiety and/or the number of propargyl groups. Boundary concentrations from all tested 1,4-DHP derivatives tested in all microorganisms were collected in a score matrix (Table S1 in Supplementary Material); the results formed a four-dimension matrix (number of propargyl groups; side chain length; microorganism; boundary concentration). To find out which of the parameters (number of propargyl groups and/or side chain length) has the most significant impact on the compound's toxicity, the "artificial" set of variables that would help to visualise the main effects of data variance across a two-dimensional (2D) plot was sought. The data were analysed and visualised by online tool ClustVis [33]. Vector scaling is applied to rows; the Nonlinear Iterative Partial Least Squares (NIPALS) algorithm is used to calculate principal components [42]. The scores of individual compound's toxicities are plotted using the first two principal components, which explain most of the variance (PC1 63.3% and PC2 19.3% for A, and 39.8% and 24.7% for B). Green triangle: 4-(N-ethylpyridinium)-1,4-DHP 7.
Data for the set of tested 4-(N-alkylpyridinium)-1,4-DHP derivatives and their toxicities (Table S1 in Supplementary Material), indicated that compounds were rather scattered across a two-dimensional (2D) plot with no pattern clustering neither with respect to the number of propargyl groups and alkyl chain length at alkylpyridinium moiety/ies. The toxicity of 4-(N-ethylpyridinium)-1,4-DHP 7 substantially differs from the toxicities of N-dodecylpyridinium (compounds 3, 6, and 9) or N-hexadecylpyridinium (compounds 10 and 11) derivatives ( Figure 3A). However, if compound 7 was omitted from PCA analyses, it can be concluded that 4-(N-alkylpyridinium)-1,4-DHPs were grouped mainly based on their N-alkyl chain length rather than the number of propargyl groups ( Figure 3B).
Therefore we assume that the toxicity of the compound depends on the N-alkyl chain length at the pyridinium moiety and/or the number of propargyl groups.

Phospholipid Binding Assay
Lipid bilayers are essential in the regulation of in vivo barriers, and surfactants are known to influence their organization and permeability [46]. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is a major membrane-forming phospholipid [47] in cell membranes, and is also widely used as a model membrane to provide valuable information regarding the interaction of cell membranes and lipids [48] and other various surfactants as antibacterial agents [49]. Therefore, in order to estimate the potential impact of 4-(N-alkylpyridinium)-1,4-DHP derivatives on microorganisms, the binding assay of DPPC with the cationic 1,4-DHP derivatives were performed.
DPPC bound to 1,6-diphenyl-1,3,5-hexatriene (DPH) shows enhanced fluorescence intensity at 440 nm (excited at 368 nm). According to the literature data, DPH interacts with DPPC through hydrophobic interaction, and compounds are also capable of binding with the phospholipids through hydrophobic interactions to quench the fluorescence of the lipid-DPH solution due to the competitive binding mechanism [34]. In this work, DPH was used as a fluorescent probe to characterise the interaction between the phospholipids and 4-(N-alkylpyridinium)-1,4-DHP derivatives.
According to obtained data (Figure 4), 4-(N-alkylpyridinium)-1,4-DHP derivatives quenched the fluorescence of the DPPH-DPH solution. The decrease of sample fluorescence in the presence of 1,4-DHPs indicated that 4-(N-alkylpyridinium)-1,4-DHPs were bound to the model membrane near the DPH binding sites. The 4-(N-alkylpyridinium)-1,4-DHP derivatives were able to quench the fluorescence of the binary DPH-DPPC, confirming that the tested 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-5 and 8-11 interact with the phospholipids via hydrophobic interactions in the order of 3 > 9 > 11 > 7 ≥ 1 ≥ 10 ≥ 2 > 5 ≥ 4. We failed to obtain comparable data for the influence of 4-(N-dodecylpyridinium)-1,4-DHP derivative 6 interaction on the binary DPH-DPPC system; therefore, the data for compound 6 was not included in Figure 4. In the case of 4-(N-dodecylpyridinium)-1,4-DHP derivative 3, the lowest relative fluorescence intensity was demonstrated, which confirmed the ability of this compound to quench the fluorescence of the DPH-DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHPs. Also, the ability of the 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 to quench the fluorescence of the DPH-DPPC system can be compared with the one obtained for compound 3. Our previous data showed that 4-(N-hexadecylpyridinium)-1,4-DHP derivative 10 and structurally related compounds caused the remarkable release of calcein from DPPC liposomes and induced the hemolysis of human erythrocytes, which confirmed correlation between the length of the alkyl moiety at N-quaternised 4-pyridyl-1,4-DHP and the improvement of membranotropic effects such as incorporation in the liposomal membranes and bilayer fluidity [25].

Self-Assembling Properties
The aim of this work was to characterise the self-assembling properties of 4-(Nalkylpyridinium)-1,4-DHP derivatives 1-11 as synthetic lipid-like compounds and study the formation of nanoparticles for the evaluation of their potential applications as nanocarriers. The detailed characterisation of nanosystems formed by lipid-like 1,4-DHPs is essential for the understanding of structure-activity relationships, which can be used for further rational design principles of delivery systems. According to our experience with dynamic light scattering (DLS) measurements, samples of dicationic 1,4-DHP amphiphiles (Figure 1, Group 2) and their structural analogues have to be prepared by dispersing a fixed amount of compound in an aqueous solution by sonication using a bath-type [20] or a probe-type sonicator [19].

Self-Assembling Properties
The aim of this work was to characterise the self-assembling properties of 4-(Nalkylpyridinium)-1,4-DHP derivatives 1-11 as synthetic lipid-like compounds and study the formation of nanoparticles for the evaluation of their potential applications as nanocarriers. The detailed characterisation of nanosystems formed by lipid-like 1,4-DHPs is essential for the understanding of structure-activity relationships, which can be used for further rational design principles of delivery systems. According to our experience with dynamic light scattering (DLS) measurements, samples of dicationic 1,4-DHP amphiphiles (Figure 1, Group 2) and their structural analogues have to be prepared by dispersing a fixed amount of compound in an aqueous solution by sonication using a bath-type [20] or a probe-type sonicator [19].
However, in the case of 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-11, both previously mentioned sample preparation methods did not work properly, and homogenous solutions of the tested compounds at required concentrations were not obtained. Therefore, for DLS measurements, the aqueous solutions of 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-11 were prepared by the thin-film hydration method. The final concentrations of the obtained samples of 1,4-DHP derivatives 1-7 were 0.5 mg/mL, and due to poor solubility of the other compounds, it was 0.25 mg/mL for compounds 8-10, and 0.1 mg/mL for compound 11. The hydrodynamic diameters, polydispersity index (PDI), zeta-potential, and stability of nanoparticles formed by 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-11 in aqueous medium were determined by the DLS method, and the data are presented in Table 2. The first set of DLS measurements represents the results obtained for freshly prepared samples. It was demonstrated that 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-4, 6-8, and 10 formed nanoparticles mainly as one population (over 95%), while 4-(N-alkylpyridinium)-1,4-DHP derivatives 9 and 11 formed two main populations at various ratios (Table 2), and 4-(N-hexylpyridinium)-1,4-DHP derivative 5 formed one main population (78%) and various smaller populations (up to 1-2%) ( Table 2). Table 2. Values of critical aggregation concentration (CAC), the mean diameter (D mean ), zeta-potential, and polydispersity index (PDI) of nanoparticles formed by 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-11 obtained by dynamic light scattering (DLS) measurements. Final compound concentrations were 0.5 mg/mL for compounds 1-7, 0.25 mg/mL for compounds 8-10, and 0.1 mg/mL for compound 11. The mean diameter (D mean ) depicts the hydrodynamic diameter of the main population of nanoparticles in the tested sample; the PDI value describes polydispersity of the sample; the zeta-potential gives information about the surface charge of nanoparticles. CAC is the concentration above which micelles and other nanoparticles are formed.
For the evaluation of the stability of nanoparticles, the second set of measurements was undertaken after two weeks of storage (samples were stored at r.t. between sets). It was found that the samples of 4-(N-ethylpyridinium)-1,4-DHP derivatives 1 and 4 and 4-(N-dodecylpyridinium)-1,4-DHP derivative 9 lost their homogeneity after two weeks of storage (Table 2). For these compounds, the PDI values were increased to 0.40, 0.41, and 0.57, respectively, compared to the initial values of 0.18, 0.16, and 0.36. The main point to be noted is that the measurements demonstrated the homogeneity of the particles, which were composed of 4-(N-dodecylpyridinium)-1,4-DHP derivatives 3, 6 and 4-(N-hexadecylpyridinium)-1,4-DHP derivative 10. Thus, after two weeks of storage, the PDI values were 0.17, 0.20, and 0.25, respectively. The sample for 4-(N-hexadecylpyridinium)-1,4-DHP derivative 11 maintained constant characteristic parameters: PDI values 0.37 and 0.33, and mean diameters of 145 nm (91%) and 143 (96%), respectively. The obtained data confirmed the stability of nanoparticles formed by 1,4-DHP derivatives 3, 6, 10, and 11 after two weeks of storage.
For self-assembling compounds, the concentration above which micelles and other nanoparticles are formed, which is called the critical aggregation concentration, is an important property. In this study, CAC was determined for 4-(N-alkylpyridinium)-1,4-DHP derivatives 1-7 and 9-11 by the DLS measurement. A representative example of the determination of CAC for 1,4-DHP 11 is presented at Figure S1 in the Supplementary Material. The determined CAC values for tested 4-(N-alkylpyridinium)-1,4-DHP derivatives 3, 6, and 9-11 were in intervals from 7.6 µM (compound 11) to 43.3 µM (compound 6) ( Table 2). CAC values for 4-(N-ethylpyridinium)-1,4-DHP derivatives 1, 4, 7, and 4-(N-hexylpyridinium)-1,4-DHP derivatives 2 and 5 cannot be determined clearly. An unclear CAC and negative and low zeta-potential value (interval from −0.78 mV until −9.96 mV) of particles formed by 4-(N-alkylpyridinium)-1,4-DHP derivatives 1, 2, 4, 5, and 7 also confirmed the weak stability of the nanoparticles formed by these compounds. Our previous data showed that the CAC values of double pyridinium moieties containing 1,4-DHP derivatives (Figure 2, Group 2) were in the range of 7 to 35 µM, depending on nature of substituents [19,51]. The obtained data of CAC has indicated that the increase of the N-alkyl chain length at the pyridinium moiety in the 1,4-DHP molecule decreased the CAC value. For 4-(N-dodecylpyridinium)-1,4-DHP derivative 3, the CAC value is comparable with the concentration of a substance that causes 50% toxicity in vitro (IC 50 ) on HT-1080 and MH-22A cell lines. It suggests that in this concentration, the cytotoxicity was estimated as the cytotoxicity of nanoparticles.
According to the obtained data, both samples contained spherical particles that were almost spherical in shape, with the diameters ranging in the interval from 70 to 170 nm. The obtained TEM data is also in agreement with the DLS measurement results, which enable making an assumption that the nanoparticles that are formed by selected 4-(N-dodecylpyridinium)-1,4-DHP 3 and 4-(N-hexadecylpyridinium)-1,4-DHP 11 are liposome-like structures. According to the obtained data, both samples contained spherical particles that were almost spherical in shape, with the diameters ranging in the interval from 70 to 170 nm. The obtained TEM data is also in agreement with the DLS measurement results, which enable making an assumption that the nanoparticles that are formed by selected 4-(N-dodecylpyridinium)-1,4-DHP 3 and 4-(Nhexadecylpyridinium)-1,4-DHP 11 are liposome-like structures.

Conclusions
The obtained results reveal that a variation of alkyl moiety lengths for the quaternised nitrogen atom at position 4 of the 1,4-DHP cycle and the propargyl moiety number and position in the 4-(Nalkylpyridinium)-1,4-DHP molecule strongly affects the self-assembling properties of compounds and characteristic parameters, and the stability of formed nanoparticles, as well as the toxicity of

Conclusions
The obtained results reveal that a variation of alkyl moiety lengths for the quaternised nitrogen atom at position 4 of the 1,4-DHP cycle and the propargyl moiety number and position in the 4-(N-alkylpyridinium)-1,4-DHP molecule strongly affects the self-assembling properties of compounds and characteristic parameters, and the stability of formed nanoparticles, as well as the toxicity of It can be concluded that the presence of N-dodecylpyridinium moiety and N-hexadecylpyridinium moiety at the 1,4-DHP cycle is essential for the formation of stable nanoparticles with mean diameter values in the range of 124 to 221 nm. However, the introduction of propargyl moieties at positions 3 and 5 of the 1,4-DHP ring decreases the stability and homogeneity of the formed nanoparticles. The obtained TEM data enables the suggestion that the nanoparticles formed by the selected 4-(N-dodecylpyridinium)-1,4-DHP 3 and 4-(N-hexadecylpyridinium)-1,4-DHP 11 are liposome-like structures.
The 4-(N-alkylpyridinium)-1,4-DHP derivatives quenched the fluorescence of the DPH of the binary DPH-DPPC system, confirming hydrophobic interaction with the phospholipids. 4-(N-Dodecylpyridinium)-1,4-DHP derivatives 3 and 9 quenched the fluorescence of the DPH-DPPC system more efficiently than the other 4-(N-alkylpyridinium)-1,4-DHPs. These findings are highly important, since 4-(N-dodecylpyridinium)-1,4-DHP 3 has already been reported to efficiently cross the blood-brain barrier, block brain calcium channels, and improve memory by enhancing the GABAergic and synaptic plasticity processes as well as influencing the levels of brain proteins, which contributes to synaptic plasticity [24]. After more detailed studies, some of these lipid-like 4-(N-dodecylpyridinium)-1,4-DHP derivatives can be proposed for use in cancer therapy as nanocarriers with intrinsic cytotoxic activity.