Triphenylphosphine Derivatives of Allylbenzenes Express Antitumor and Adjuvant Activity When Solubilized with Cyclodextrin-Based Formulations

Allylbenzenes (apiol, dillapiol, myristicin and allyltetramethoxybenzene) are individual components of plant essential oils that demonstrate antitumor activity and can enhance the antitumor activity of cytotoxic drugs, such as paclitaxel, doxorubicin, cisplatin, etc. Triphenylphosphine (PPh3) derivatives of allylbenzenes are two to three orders of magnitude more potent than original allylbenzenes in terms of IC50. The inhibition of efflux pumps has been reported for allylbenzenes, and the PPh3 moiety is deemed to be responsible for preferential mitochondrial accumulation and the depolarization of mitochondrial membranes. However, due to poor solubility, the practical use of these substances has never been an option. Here, we show that this problem can be solved by using a complex formation with cyclodextrin (CD-based molecular containers) and polyanionic heparin, stabilizing the positive charge of the PPh3 cation. Such containers can solubilize both allylbenzenes and their PPh3 derivatives up to 0.4 mM concentration. Furthermore, we have observed that solubilized PPh3 derivatives indeed work as adjuvants, increasing the antitumor activity of paclitaxel against adenocarcinomic human alveolar basal epithelial cells (A549) by an order of magnitude (in terms of IC50) in addition to being quite powerful cytostatics themselves (IC50 in the range 1–10 µM). Even more importantly, CD-solubilized PPh3 derivatives show pronounced selectivity, being highly toxic for the A549 tumor cell line and minimally toxic for HEK293T non-tumor cells, red blood cells and sea urchin embryos. Indeed, in many cancers, the mitochondrial membrane is more prone to depolarization compared to normal cells, which probably explains the observed selectivity of our compounds, since PPh3 derivatives are known to act as mitochondria-targeting agents. According to the MTT test, 100 µM solution of PPh3 derivatives of allylbenzenes causes the death of up to 85% of A549 cancer cells, while for HEK293T non-cancer cells, only 15–20% of the cells died. The hemolytic index of the studied substances did not exceed 1%, and the thrombogenicity index was < 1.5%. Thus, this study outlines the experimental foundation for developing combined cytostatic medications, where effectiveness and selectivity are achieved through decreased concentration of the primary ingredient and the inclusion of adjuvants, which are safe or practically harmless substances.


Introduction
Modern therapeutic strategies are in some cases ineffective against bacterial infections and cancers, with such cases being most often associated with multiple drug resistance (MDR) [1][2][3][4][5][6][7][8][9][10][11].Resistance mechanisms that reduce the likelihood of a patient's being cured can be divided into two groups [12]: (i) cellular metabolism (transferases, topoisomerases, growth factors), which alters the mechanism of action of the drugs or interferes with their action, and (ii) a decrease in the intracellular concentration of the drug.The drug enters the intracellular medium through the transport channels of the plasma membrane, in which Pharmaceuticals 2023, 16, 1651 2 of 20 pump proteins (ATP-binding cassette protein [4,13]) can be expressed, pumping the drug out of the cell and thus reducing its effect [4,5,[14][15][16][17].The main member of the efflux pump family, MDR1 (P-glycoprotein [3,13,14,18]), causes the resistance of various types of tumors to chemotherapy.Bacteria also have efflux pumps (for example, NorA [4,9,[19][20][21], P-glycoprotein) which cause the ineffectiveness of antibiotics.A number of substances that inhibit efflux (verapamil, reserpine, etc. [22]) are known to be rather toxic.Therefore, numerous studies are aimed at finding substances that inhibit efflux but at the same time are non-toxic.
Apiol analogues (myristicin, allyltetramethoxybenzene and dillapiol) have also demonstrated weak antitumor activity, but they have served as a booster for antitumor drugs (paclitaxel, doxorubicin, cisplatin) [49] due to the inhibition of mitochondrial enzymes [81,82], efflux pumps [49] and the increased permeability of the membrane of cancer cells [49,83].It was previously shown that dillapiol (25-50 µM) induced G0/G1 cell cycle arrest, the activation of a number of caspases and, accordingly, the apoptosis of cancer cells, while apiol and its analogues had virtually no effect on benign epithelial cells in vitro [46].Myristicin showed a similar but weaker effect.Recently, triphenylphosphine (PPh 3 ) derivatives of allylbenzenes were suggested for research into improving their antiproliferative potency toward cancer cells taking into account their tendency to preferential mitochondrial accumulation [46].The introduction of the PPh 3 moiety, possessing both hydrophobic and charged properties, enhances the conjugate's localization within the cell membrane and boosts inhibition against mitochondrial membrane enzymes.This was previously demonstrated by some of the authors of this article on a micellar model [82].Cancer cells have an altered metabolism, in particular the dynamics of mitochondria (the PPh 3 fragment can serve as an address label to cancer mitochondria), which provides many potential targets for cancer therapy [84][85][86].
Considering the action mechanism of PPh 3 derivatives of allylbenzenes, we can assume their potential synergistic effect with the main drug, paclitaxel.The action mechanism of paclitaxel is based on the suppression of the normal process of dynamic reorganization of the microtubule network, which is responsible for cell division.In addition, paclitaxel induces the formation of abnormal clusters and causes the formation of multiple microtubule stars during mitosis.Paclitaxel is used as a first-line drug in the treatment of ovarian, breast, lung, cervical cancer, etc.The combination of paclitaxel + adjuvant is expected to be more effective than a single drug due to the action of different mechanisms and an increase in the bioavailability of the cytostatic.In this paper, the key idea is to realize three main aspects to create enhanced antitumor activity: (i) a combination of the main cytostatic (paclitaxel) with an adjuvant (as efflux inhibitor) from the group of allylbenzenes, (ii) an increase in the mitochondrial bioavailability of the adjuvant by conjugating it with a PPh 3 fragment due to the depolarization of mitochondrial membranes in cancer cells, and (iii) the use of cyclodextrins derivatives and a heparin polysaccharide matrix as molecular containers to obtain soluble forms of drugs and increase their bioavailability.

Article Design
The present work is aimed at developing and studying complex antitumor formulations based on three components: the main drug (paclitaxel), the adjuvant (apiol-PPh 3 and its analogues) and the molecular container (cyclodextrins (CD) for the formation of inclusion complexes with paclitaxel and its adjuvant in the hydrophobic cavity of CD or heparin polyanion to stabilize the cationic triphenylphosphine fragment).In previous studies, the authors have shown that cytostatics (paclitaxel, doxorubicin, etc.) are enhanced by allylbenzenes, which can also act as promising anticancer drugs [49,87].In this paper, PPh 3 derivatives of allylbenzenes are used to enhance the effect of cytostatics due to the depolarization of mitochondrial membranes and considering their tendency to preferential mitochondrial accumulation.However, because PPh 3 derivatives are poorly soluble, to realize their potential, it is necessary to develop the optimal container, providing an increase in allylbenzene-PPh 3 solubility and obtaining double-drug inclusion complexes, which can provide synergism of the action of the main antibiotic and the adjuvant (cyclodextrin derivatives or anionic polysaccharides are proposed in this work).To achieve this, the following tasks were realized: (1) the spectral characterization of PPh 3 derivatives of allylbenzenes and the study of their solubility, (2) the characterization of double-drug inclusion complexes of these compounds and paclitaxel with various cyclodextrins derivatives or heparin and the determination of dissociation constants of complexes, (3) analysis of the cytotoxic activity of the cytostatic agents alone and in the complex drug formulations against A549 using the MTT test and (4) analysis of the selectivity of the cytostatic activity and the safety of drugs for HEK293T non-cancerous cells in vitro using FTIR spectroscopy, red blood cells and sea urchin embryos in vivo safety study.

The Spectral Characteristics of PPh 3 Derivatives of Allylbenzenes
Allylbenzenes (apiol, myristicin, etc.) have a number of important biological activities, including experimental prerequisites to be synergists (enhancers) of the action of cytotoxic drugs.To increase the bioavailability of allylbenzenes, the modified form of allylbenzenes with a PPh 3 fragment (Figure 1) was obtained according to the methodology described recently [46].Confirmation of the success of synthesis follows from NMR and FTIR spectroscopy data (Figures 2 and S1, Table 1).The original substances (apiol and analogues) are characterized by the main signals: aromatic protons (6 and 6.5 ppm), protons at the double bond of the allyl group (5 ppm), protons of methoxy groups and/or methylene bridges (3.3-4 ppm).After the introduction of the PPh 3 residue into these molecules, the proton signals of the allyl group double bond disappear, but the proton signals of phenyl substituents (7.6-8.1 ppm) as well as the alkyl spacer (1.7-3 ppm) appear.In the FTIR spectra (Figure 2c) of the initial allylbenzenes, the most significant are the oscillation bands C=C 1660 cm −1 (allyl group) and 1450-1550 cm −1 (aromatic system).After the modification of allylbenzenes with PPh 3 , the peak of the oscillations of the C=C allyl group disappears, but peaks corresponding to the deformation fluctuations of the C-H triphenylphosphine fragment (1400-1480 cm −1 ) and fluctuations of C=C bonds (1500-1600 cm −1 ) appear.

Solubility of PPh 3 -Modified Allylbenzenes Adjuvants and Complex Formation with Cyclodextrins and Heparin
As already mentioned, PPh 3 derivatives are poorly soluble, so to realize its potential, the formation of the inclusion complexes with cyclodextrins (CDs) (with an external hydrophilic shell and an internal hydrophobic cavity) or with heparin is suggested.This can provide the possibility of using aromatic adjuvants (which are otherwise not applicable) as an antitumor agent and to obtain an efficient combined antitumor formulation.CDs and heparin can protect the substance from destruction and inactivation, increase the half-life, and in addition, due to adsorption on cell membranes, increase the membrane permeability [49,77].
Loading both the main cytostatic agent and its adjuvants (apiol-PPh 3 and its analogues) into molecular containers is suggested as a perspective approach to increase the solubility of substances in aqueous solutions, to increase the bioavailability, and consequently, improve the effectiveness of the antitumor formulation.Previously, we studied allylbenzenes as independent antitumor preparations and adjuvants to paclitaxel, where we obtained soluble forms due to complex formation with M-β-CD [77] (otherwise, these substances cannot be used at all due to insolubility and oil-water phase separation).Here, we suggest using cyclodextrins or non-cyclic polysaccharide for the preparation of soluble formulations of triphenylphosphine derivatives.We consider M-β-CD, which was efficient for allylbenzenes solubilization, as well as γ-CD, which has a larger size of the inner cavity.We have chosen heparin as a polyanion to form electrostatic complexes with positively charged PPh 3 .In addition, heparin as an antithrombotic agent in the tumor microenvironment could have an additional therapeutic effect, since the tumor's development is a thrombosis-associated process.FTIR spectroscopy provides valuable data on the interaction of molecules, including those applicable to characterize the non-covalent complexes of apiol-PPh 3 (and analogues) with cyclodextrins and heparin.In the FTIR spectra of apiol-PPh 3 and analogues (Figure 3a), one characteristic is the bands of valence oscillations of the bonds C=C of the aromatic system (1450-1650 cm −1 ) overlapping with the bands of deformation oscillations C-H (1400-1500 cm −1 ).The intensity of these peaks increases with the formation of non-covalent complexes of apiol-PPh 3 and its analogues with cyclodextrins and heparin due to the transition of the solid phase into solution.Linear fitting of the intensity of peaks in the FTIR spectra on the cyclodextrin or heparin concentration (Section 2.3) in Hill coordinates allows determining the dissociation constants of the complexes (Table 2).The interactions of triphenylphosphine derivatives of allylbenzenes with γ-CD is rather weak (K d 10 mM values).In the case of M-β-CD, the dissociation constants reach millimolar values, which is sufficient to obtain soluble forms of adjuvants (Table 2).Thus, the β-cyclodextrin derivatives are more suitable in terms of size for inclusion of the adjuvants studied.Heparin forms rather strong complexes due to multipoint electrostatic interactions: K d 10 −3 -10 −4 M per heparin monomeric unit or 10 −5 M per heparin molecule.Comparing the values of the dissociation constants of alkylbenzenes and PPh 3 -derivatives complexes with cyclodextrins, we observed that these K d values are close, which means that it is the allylbenzene-fragment (of apiol-PPh 3 ) that plunges into the cyclodextrin cavity, and the triphenylphosphine radical looks outward (Figure 3b), which would provide the implementation of mitochondrial targeting of the developed formulations.

Anticancer Activity of PPh3 Derivatives and Formulations
Previously, we demonstrated the antitumor activity of apiol, eugenol and their analogues from the allylbenzene class, and we showed the ability of these substances to act as efflux pump inhibitors and as a membrane-penetrating enhancer agent [49].Apparently PPh3 derivatives effectively penetrate into cancer cells along a potential gradient, inhibit efflux proteins and mitochondrial enzymes, causing the apoptosis of cancer cells, with IC50 values being lower by two orders of magnitude than the corresponding allylbenzene (Table 3).Surprisingly, PPh3 derivatives (especially apiol-PPh3) in the complex with M-β-CD are close in terms of the cytostatic efficiency to the well-known cytotoxic drug paclitaxel (Figure 4a, Table 3); in addition, they demonstrate synergy with paclitaxel, with a synergy coefficient over 2 (indicating strong synergy).For allylbenzenes, a synergy close to additivity (the cytostatic effect of adjuvant + paclitaxel is almost equal to the sum of their individual contributions) was observed; for PPh3 derivatives, a pronounced increase in the action of paclitaxel is characteristic (the cytostatic effect of adjuvant + paclitaxel is much higher (>) than the sum of their individual contributions).For apiol-PPh3, the most pronounced effect of increasing the activity of paclitaxel was observed (Figure 4b).
The mechanism of synergistic action is that the main cytostatic and its adjuvant act on different targets.The PPh3 fragment serves as an intermediary for the delivery of allylbenzenes to the mitochondria of cancer cells, which is where the inhibition of enzymes (dehydrogenases) occurs.Paclitaxel disrupts the cycle of cell division.Meanwhile, CD Table 2. Dissociation constants of complexes of adjuvants and cyclodextrins or heparin.Solubility of X and X-PPh 3 in PBS and solubility of their complexes with M-β-CD in PBS.Comparison of unmodified "apiols" and the PPh 3 derivatives.Figure 3c shows the UV spectra of myristicin-PPh 3 , apiol-PPh 3 and their complexes with M-β-CD: PPh 3 derivatives due to their low solubility in water do not have a clearly defined spectrum.On the contrary, their complexes with M-β-CD are soluble, and a clear peak in the UV spectrum is pronounced (225 nm).Based on UV spectra data in the presence of M-β-CD, the increase in solubility of apiol-PPh3 and analogues by a second order of magnitude is observed (Table 2).

Substance X-PPh
Visually, the dissolution of PPh 3 derivatives of allylbenzenes is observed in a light microscope (Figure 3d-g): with an increase in the molar excess of cyclodextrin, an increasing number of inclusion complexes are formed and, consequently, solubility increases (1:1 molar ratio) and crystals decrease to complete dissolution (10-fold molar excess of M-β-CD).

Anticancer Activity of PPh 3 Derivatives and Formulations
Previously, we demonstrated the antitumor activity of apiol, eugenol and their analogues from the allylbenzene class, and we showed the ability of these substances to act as efflux pump inhibitors and as a membrane-penetrating enhancer agent [49].Apparently PPh 3 derivatives effectively penetrate into cancer cells along a potential gradient, inhibit efflux proteins and mitochondrial enzymes, causing the apoptosis of cancer cells, with IC 50 values being lower by two orders of magnitude than the corresponding allylbenzene (Table 3).Surprisingly, PPh 3 derivatives (especially apiol-PPh 3 ) in the complex with M-β-CD are close in terms of the cytostatic efficiency to the well-known cytotoxic drug paclitaxel (Figure 4a, Table 3); in addition, they demonstrate synergy with paclitaxel, with a synergy coefficient over 2 (indicating strong synergy).For allylbenzenes, a synergy close to additivity (the cytostatic effect of adjuvant + paclitaxel is almost equal to the sum of their individual contributions) was observed; for PPh 3 derivatives, a pronounced increase in the action of paclitaxel is characteristic (the cytostatic effect of adjuvant + paclitaxel is much higher (>) than the sum of their individual contributions).For apiol-PPh 3 , the most pronounced effect of increasing the activity of paclitaxel was observed (Figure 4b).The mechanism of synergistic action is that the main cytostatic and its adjuvant act on different targets.The PPh 3 fragment serves as an intermediary for the delivery of allylbenzenes to the mitochondria of cancer cells, which is where the inhibition of enzymes (dehydrogenases) occurs.Paclitaxel disrupts the cycle of cell division.Meanwhile, CD improves the solubility of drugs and increases adsorption and absorption by cancer cells, which in total affects the increase in the effectiveness of the cytostatic drug.The potential of inclusion of antitumor drugs in CD in terms of enhancing penetration into cancer cells was demonstrated also in our previous work [49].
Thus, allylbenzene-PPh 3 inclusion complexes with cyclodextrin are potentially applicable in medicine as antitumor drugs.At the same time, it is important to find out the selectivity of the cytostatic action of the formulation developed against cancer cells and the safety of these formulations for normal cells.

Selectivity of Action and Safety of Cytotoxic Formulations Developed 2.5.1. HEK293T as Normal Cell Model
HEK293T are model normal (non-cancer) cells that are widely used to compare the selectivity of cytostatic formulations on cancer cells [49].Quantitative data on the safety and selectivity of action for the formulations based on PPh 3 derivatives are presented in Table 4.According to the MTT test, the concentration of cytostatics of 100 µM causes the death of up to 85% of cancer cells A549 (Figure 4a), while for non-cancer cells (HEK293T), the dying rate is only 15-20%.Earlier, we showed that the data of FTIR spectroscopy correlate with the data of the MTT test on cell survival [49,83].The main cell structural units that contribute to the absorption of IR diapason can be assigned as follows (Figure 5): lipids of the cell membrane (2800-3000 cm −1 ), proteins, especially transmembrane (1500-1700 cm −1 ), phosphate groups of DNA (1240 cm −1 ) and carbohydrates, including lipopolysaccharides (900-1100 cm −1 ).Previously, we developed a technique for tracking the penetration and adsorption of the drug into cells using FTIR spectroscopy: dramatic changes in the intensity of the peaks of amide 1 and amide 2 indicate effective penetration of the drug into cells and vice versa [49,87,88].
Here, we present the real-time data of FTIR spectroscopy during the incubation of a suspension of HEK293T cells with apiol-PPh 3 in M-β-CD (Figure 5).Comparing the red spectrum (at 0 min incubation) and the black spectrum (after 60 min), it is obvious that there are practically no changes in the intensity of the peaks of amide I and II, characterizing the interaction of the drug with trans-membrane proteins, and indicating drug penetration.There is only a shift of the peak of amide 1 to the low-frequency region (inserts in Figure 5, the normalized intensity is shown) and amide 2 to the high-frequency region with the simultaneous appearance of the shoulder.This indicates the only adsorption of drug molecules on the cell surface, which is also confirmed by a weak increase in the intensity of the peaks at 2850-3000 cm −1 corresponding to the valence vibrations of the CH 2 groups (lipid bilayer).Thus, PPh 3 derivatives show only marginal activity against normal cells.
For comparison, we present a positive control of the active and inactive reagent on the HEK293T cells (Figure 5b).Dramatic changes in the intensity of the amide 1 and amide 2 peaks (Figure 5b, left) indicate the penetration of the model cells' membrane-penetrating drug (doxorubicin) into the cells and effective cytostatic effect (according to MTT test).On the contrary, small changes in the intensity of the peaks of amide 1 and amide 2 indicate the weak penetration of the drug into the cells and the non-cytostatic effect of doxorubicin in the composition with "intelligent" micelles (Figure 5b, right).2.5.2.Hemolytic Activity, Thrombogenicity and Phenotypic Sea Urchin Embryo Assay Hemolytic activity and thrombogenicity are the primary parameters for evaluating the safety of medical formulations [89][90][91].The phenotypic sea urchin embryo assay developed by colleagues is a visual way to study the toxicity of formulations in vivo.The sea urchin and human genomes contain more than 7000 common genes, including orthologs associated with a number of human diseases.Therefore, sea urchins can be considered as a reliable and versatile model organism for studying the safety of new and existing cytotoxic formulations in vivo.Table 5 presents data on the % of erythrocyte hemolysis, the degree of whole blood thrombosis of apiol-PPh 3 and analogues, and data of phenotypic sea urchin embryo assay.Thus, the non-toxicity of PPh 3 derivatives of allylbenzenes for normal non-cancer cells, as well as the selectivity of action against cancer cells, is shown.The selectivity of cytotoxic action against cancer cells in comparison with normal cells can be explained by the fact that the PPh3-cation provides selective accumulation and reduction in the mitochondrial membrane potential of the transformed cancer cells [92].Cyclodextrin (M-β-CD) and heparin are non-toxic (and approved by the FDA for intravenous application).

Hemolytic Activity, Thrombogenicity and Phenotypic Sea Urchin Embryo Assay
Hemolytic activity and thrombogenicity are the primary parameters for evaluating the safety of medical formulations [89][90][91].The phenotypic sea urchin embryo assay developed by colleagues is a visual way to study the toxicity of formulations in vivo.The sea urchin and human genomes contain more than 7000 common genes, including orthologs associated with a number of human diseases.Therefore, sea urchins can be considered as a reliable and versatile model organism for studying the safety of new and existing cytotoxic formulations in vivo.Table 5 presents data on the % of erythrocyte hemolysis, the degree of whole blood thrombosis of apiol-PPh3 and analogues, and data of phenotypic sea urchin embryo assay.Thus, the non-toxicity of PPh3 derivatives of allylbenzenes for normal non-cancer cells, as well as the selectivity of action against cancer cells, is shown.The selectivity of cytotoxic action against cancer cells in comparison with normal cells can be explained by the fact that the PPh3-cation provides selective accumulation and reduction in the mitochondrial membrane potential of the transformed cancer cells [92].Cyclodextrin (M-β-CD) and heparin are non-toxic (and approved by the FDA for intravenous application).
The synthesis of triphenylphosphine derivatives of allylbenzenes was performed as described earlier in the work [46].

Characterization of PPh 3 Adjuvants Using NMR Spectroscopy
The 1 H and 13 C NMR spectra of the apiol, apiol-PPh 3 and analogues in d 6 -DMSO were recorded on a Bruker Avance 400 spectrometer (Bruker Biospin, Rheinstetten, Germany) at an operating frequency of 400 MHz.The chemical shifts are shown in ppm on the δ scale relative to hexamethyldisiloxane as an internal standard.The analysis and processing of the NMR spectra were performed with the program MestReNova v.12.0.0-20080).

Non-Covalent Complexes of Apiol, Apiol-PPh 3 and Analogues with Cyclodextrins and Heparin, Preparation and Characteristics
Non-covalent complexes of apiol, apiol-PPh 3 and analogues with cyclodextrins (with different molar ratios) and heparin (15 kDa, 1:1 w/w) were obtained by adding solutions of cyclodextrins (100 mg/mL) or heparin (20 mg/mL) in PBS to apiol or apiol-PPh 3 (or analogues) samples (2-3 mg).The excess of CDs or heparin was varied from 0.04 to 10 mol/mol.Then, mixtures were incubated for 1 h at 40 • C. Cyclodextrin or heparin are necessary for the solubilization of extremely poorly soluble compounds.
Concentrations of the active substance varied from 10 −2 to 10 −4 M. For MTT assay and biological experiments, substances in the concentration range from 10 −3 to 10 −9 M were studied by the dilution of initial ones in a cell growth medium or buffer.
The solubility of apiol and apiol-PPh 3 (or analogues) in aqueous solution was determined by UV spectroscopy.UV spectra were recorded on the UltraSpec 2100 pro device (AmerSham Biosciences, Cambridge, UK) three times.The substances were dissolved in acetonitrile followed by recording the UV spectra at various cytostatics' concentrations, and then we plotted calibration dependencies.Next, saturated solutions of substances in water were prepared, and the spectra of the aqueous solutions were recorded.Considering the extinction coefficients in water and acetonitrile to be approximately equal, the solubility was determined.

MCD Inclusion Complexes Synthesis
The inclusion complexes of Paclitaxel with MCD were prepared as described in our previous paper [49].

Determination of the Dissociation Constants of Complexes of Apiol-PPh 3 and Analogues with Cyclodextrins and Heparin Using FTIR Spectroscopy
The ATR-FTIR spectra of samples (Section 3.3) were acquired using a Bruker Tensor 27 spectrometer equipped with a liquid N 2 cooled MCT (mercury cadmium telluride) detector.Samples were placed in a thermostatic cell BioATR-II with ZnSe ATR element (Bruker, Germany).FTIR spectra were recorded from 850 to 4000 cm −1 with 1 cm −1 spectral resolution; 50 scans were accumulated and averaged.Spectral data were processed using the Bruker software system Opus 8.2.28 (Bruker, Germany).The spectrum of cyclodextrin or heparin in the corresponding concentration was subtracted from the spectra of the complexes.Then, the dependencies of the peak intensities of the corresponding C=C oscillation (aromatic system of apiol-PPh 3 and analogues (1475-1510 cm −1 )) was constructed, which least overlaps with the spectrum of cyclodextrin and heparin.

Confirmation of Particle Formation and Their Characterization
Confirmation of particle formation was carried out using Atomic Force Microscopy (NTEGRA II Moscow, Russia), and Fourier Infrared Microscopy (Micran-3 IR microscope, Simex, Novosibirsk, Russia).
Topography, phase and magnitude signal images of the micelles deposited onto freshly cleaved surface of mica were obtained by AFM microscopy using a scanning probe microscope NTEGRA operated in a semi-contact mode with 15-20 nm peak-topeak amplitude of the "free air" probe oscillations.Using AFM, the formation of ordered particles of heparin complexes with drugs (100-150 nm) from disordered heparin aggregates (50-480 nm) was observed.
Using IR microscopy, dry powders of drugs and their complexes with CD and heparin were studied: they showed a uniform distribution of the drug over the area of the studied sample (i.e., evenly inclusion in molecular containers).
Circular dichroism spectra of heparin were recorded on the Jasco J-815 CD Spectrometer (Tokyo, Japan) for the determination of heparin in the tested formulations.

Cell Cultivation and Determination of Cytotoxic Activity
Adenocarcinomic human alveolar basal epithelial cells A549 cell lines (Manassas, VA, USA) were cultured in RPMI-1640 medium, linear cells of the embryonic kidney human epithelium (HEK293T) were grown in DMEM medium as described earlier [49].Cell lines were obtained from Lomonosov Moscow State University Depository of Live Systems Collection and Laboratory of Medical Biotechnology, Institute of Biomedical Chemistry (Moscow, Russia).

Phenotypic Sea Urchin Embryo Assay
Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast and kept in an aerated seawater tank and were used to study the cleavage alteration of Apiol-PPh 3 and analogues [46,93].Experiments with sea urchin embryos comply with the requirements of biological ethics.Artificial spawning does not lead to the death of animals, embryos develop outside the female body, and both adult sea urchins after spawning and an excess of intact embryos return to the sea, which is their natural habitat.

Study of the Safety of Formulations (Hemolytic Activity and Thrombogenicity)
The hemolytic activity and thrombogenicity of apiol-PPh 3 and analogues were studied using an earlier published technique [83].

Statistical Analysis
Statistical analysis of cytotoxicity and spectral data was performed using Student's ttest Origin 2022 software (OriginPro 2022 v.9.9.0.225,OriginLab Corporation, Northampton, MA, USA).Values are given as the mean ± SD of three or five experiments.

Figure 4 .
Figure 4. Viability of A549 cells in the presence of paclitaxel and allylbenzenes' PPh 3 derivatives in the form of complexes with M-β-CD (1:5 mol/mol).MTT assay.(a) Dose-effect graphs.(b) MTT assay results for 100 nM paclitaxel alone and combined with allylbenzenes' PPh 3 derivatives in the form of complexes with M-β-CD (1:5 mol/mol).

Figure 5 .
Figure 5. (a) FTIR spectra of HEK293T cells during online incubation (with step 5 min) with apiol-PPh 3 in the form of inclusion complexes with M-β-CD.The inserts show enlarged fragments of peaks of amide I and II with a normalized intensity for the better visualization of shifts of maxima.T = 37 • C. The inserts show enlarged fragments of peaks of amide I and II with a normalized intensity for better visualization of shifts of maxima.(b) FTIR spectra of HEK293T cells pre-incubated with doxorubicin (left), doxorubicin in "intelligent" micelles [87] (right) as a control of the correlation of changes in the intensity of peaks with the penetration and cytostatic effect of the drug.

Position of the Characteristic Peak in the FTIR Spectra, cm −1 Octane-Ethanol (50:50 v:v) Water-Ethanol (50:50 v:v)
* the atoms whose bond oscillations are observed are underlined.

Table 4 .
Anti-HEK293T activity of PPh 3 derivatives of allylbenzenes in M-β-CD as a criterion for the safety of medicinal formulations.MTT assay.RPMI-1640 medium supplemented with 5% fetal bovine serum and 1% sodium pyruvate at 5% CO 2 /95% air in a humidified atmosphere at 37 • C.

Table 5 .
Safety data on triphenylphosphine derivatives and paclitaxel in the complex with M-β-CD.