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Article

PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding

by
Olga Mostovaya
1,
Igor Shiabiev
1,
Daniil Ovchinnikov
1,
Dmitry Pysin
1,
Timur Mukhametzyanov
1,
Alesia Stanavaya
2,
Viktar Abashkin
2,
Dzmitry Shcharbin
2,
Arthur Khannanov
1,
Marianna Kutyreva
1,
Mingwu Shen
3,
Xiangyang Shi
3,4,
Pavel Padnya
1,* and
Ivan Stoikov
1,*
1
A.M. Butlerov Chemical Institute, Kazan Federal University, Kremlevskaya, 18, 420008 Kazan, Russia
2
Institute of Biophysics and Cell Engineering of NASB, 27 Akademicheskaya St., 220072 Minsk, Belarus
3
State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Shanghai Engineering Research Center of Nano-Biomaterials and Regenerative Medicine, College of Biological Science and Medical Engineering, Donghua University, Shanghai 201620, China
4
CQM—Centro de Química da Madeira, Universidade da Madeira, Campus Universitário da Penteada, 9020-105 Funchal, Portugal
*
Authors to whom correspondence should be addressed.
Pharmaceutics 2024, 16(11), 1379; https://doi.org/10.3390/pharmaceutics16111379
Submission received: 27 September 2024 / Revised: 24 October 2024 / Accepted: 24 October 2024 / Published: 27 October 2024
(This article belongs to the Special Issue Contribution of Dendrimers and Other Dendritic Polymers in Medical Nanotechnology)
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Abstract

Background/Objectives: Current promising treatments for many diseases are based on the use of therapeutic nucleic acids, including DNA. However, the list of nanocarriers is limited due to their low biocompatibility, high cost, and toxicity. The design of synthetic building blocks for creating universal delivery systems for genetic material is an unsolved problem. In this work, we propose PAMAM dendrimers with rigid thiacalixarene core in various conformations, i.e., PAMAM-calix-dendrimers, as a platform for a supramolecular universal constructor for nanomedicine. Results: Third generation PAMAM dendrimers with a macrocyclic core in three conformations (cone, partial cone, and 1,3-alternate) were synthesized for the first time. The obtained dendrimers were capable of binding and compacting calf thymus DNA, whereby the binding efficiency improved with increasing generation, while the influence of the macrocyclic core was reduced. A dramatic effect of the macrocyclic core conformation on the hemolytic activity of PAMAM-calix-dendrimers was observed. Specifically, a notable reduction in hemotoxicity was associated with a decrease in compound amphiphilicity. Conclusions: We hope the results will help reduce financial and labor costs in developing new drug delivery systems based on dendrimers.

Graphical Abstract

1. Introduction

Interest in dendrimers has not waned since their discovery in the late 20th century [1,2]. These macromolecules differ favorably from traditional synthetic polymers due to their symmetrical and strictly defined structure, consisting of a core, branching units located around it, and terminal groups on a surface of macromolecule [3]. The size of dendrimer molecules in solution is typically less than a few nanometers, making them attractive objects for the design of drug and gene delivery systems. Dissolved dendrimers have a globular shape, while their internal cavities may be capable of binding hydrophobic compounds [4]. A large number of branches provides the presence of multiple terminal groups, which allows the formation of multiple binding centers and the realization of multivalent effect [5]. Thus, the combination of these features makes dendrimers excellent candidates for a wide range of applications, e.g., as catalysts, extraction agents, and nanocarriers [6,7]. A dendrimer functionalized with a sulfur-containing fragment is used in medical practice as the active substance in registered drugs made by Starpharma (Melbourne, Australia) [8,9]. However, along with a number of attractive properties, dendrimers have serious drawbacks, such as high cost and complexity of synthesis, which limit their application [10]. In this regard, an urgent task for increasing the use of dendrimers is to reduce their production cost, which can be achieved by decreasing the number of synthetic steps and using lower generations. In this case, a new challenge arises as the efficiency of interaction of classical dendrimers with different substrates is directly dependent on the generation, i.e., higher generations have more binding centers that can be involved in complexation by both electrostatic (terminal groups) and hydrophobic (internal cavities) interactions. At the moment, a significant barrier to the widespread use of dendrimer-based medical products is also the high hemo- and cytotoxicity of higher generations, which is especially characteristic of cationic poly(amidoamine) (PAMAM) and poly(propylene imine) dendrimers [11,12]. Reduced toxicity is usually achieved by functionalization of the dendrimer surface groups to reduce the positive charge, but this negatively affects their ability to bind to anionic substrates such as DNA and some proteins [13,14,15,16,17]. The importance of studying the interaction with nucleic acids of various compounds cannot be overemphasized. Such interactions are widely used in medical technologies. Binding to DNA, which disrupts its structure and reduces transcription, is used in the treatment of cancer; DNA-based drugs are used to treat a number of non-tumor diseases [18]. DNA binding and compaction has allowed the delivery of genetic material into the cell [19,20,21]. DNA- and RNA-based vaccines have been actively developed in recent years [22,23,24,25,26].
The replacement of a linear dendrimer core with a macrocyclic one proved to be a promising direction for the modification of dendrimers [27,28]. Macrocycles appear to be an excellent platform for the design of dendrimers, providing the desired derivatives with hydrophobic properties and an extensively branched surface by introducing an increased number of dendritic fragments into the molecule. Moreover, macrocycle-based dendrimers have several types of cavities, i.e., along with the cavity formed by dendrons, their structure includes an additional cavity provided by the macrocycle. For example, dendrimers based on β-cyclodextrin had seven dendrons and were capable of binding the hydrophobic antihelminthic drug Albendazole [29]. A dendrimer with a pillar[5]arene core demonstrated the potential for charge transfer in perovskite solar cells [30]. Previously, we found effective binding of drugs and biopolymers (proteins and DNA) by the first and second generation of PAMAM dendrimers with a thiacalix[4]arene core (PAMAM-calix-dendrimers) [31,32,33,34]. The efficiency of interaction of PAMAM-calix-dendrimers with lysozyme is higher than that of classical PAMAM dendrimers with ethylenediamine core [31]. It is worthy of note that the thiacalixarene is currently a popular platform for the design of compounds with a variety of applications [35,36,37,38]. The attractiveness of the thiacalixarene platform is due to easy functionalization and synthetic availability of various conformational isomers, which allow the binding centers of different natures to be fixed in a strictly defined way in space, as well as the possibility of varying the hydrophilic-hydrophobic balance of the resulting products within wide limits [39]. Successful use of the unique properties of thiacalixarene allows researchers almost unlimited access to new receptors, catalysts, sensors, etc. [40,41,42,43,44]. However, the examples of macrocycle-based dendrimers given in the literature were limited to low generation ones. Thus, most studies have been carried out for the first generation of macrocycle-based dendrimers, while only a few works are devoted to the second generation [32,45].
In summary, we can conclude that the development of universal methods for the synthesis of low and high generations of macrocycle-based dendrimers and discovery of the relationship between their generation and conformation and binding properties is an important research task (Figure 1). This article is devoted to the effect of generation (first, second, and third) and conformation of PAMAM-calix-dendrimers on calf thymus DNA binding. Special attention is paid to the relationship between the hemolytic activity of PAMAM-calix-dendrimers and their generation and conformation. The obtained data will help reduce the financial and labor costs associated with new drug delivery and receptor systems based on dendrimers.

2. Materials and Methods

General Experimental Information

Detailed information on the equipment, methods, synthesis, and physical–chemical characterization of the compounds studied is presented in the Supplementary Materials.

3. Results and Discussion

3.1. Synthesis

Previously, we developed an efficient approach to obtain first (G1) and second (G2) generation PAMAM-calix-dendrimers containing the thiacalix[4]arene core in various conformations (cone, partial cone, and 1,3-alternate) [32,33]. In this work, we used the described G1 and G2 PAMAM-calix-dendrimers as initial compounds. The previously developed methods were successfully adapted for the synthesis of G3 PAMAM-calix-dendrimers (Scheme 1). The first step of the synthesis was the reaction of G2 PAMAM-calix-dendrimers with methyl acrylate for 48 h at 25 °C (Scheme 1). The ninhydrin test (1% ninhydrin solution in ethanol) to determine the presence of primary amino groups of the mixture was negative after 48 h and indicated the absence of terminal amino groups. As a result, G2.5 half-generations were obtained at a 90–94% yield and were further reacted with excess ethylenediamine. The reaction was carried out for 110 h at 25 °C to ensure complete reaction and complete substitution of all 32 terminal ester groups. Such mild conditions and the use of a large excess of ethylenediamine minimized the probability of side reactions, e.g., incomplete aminolysis, retro-Michael reactions, and intra- and intermolecular cross-linking [46,47,48]. As a result, third generation (G3) PAMAM-calix-dendrimers containing the macrocyclic core in various conformations (cone (G3-cone), partial cone (G3-paco), and 1,3-alternate (G3-alt)) were obtained with an 80–87% yield.
The synthesized G2.5 and G3 PAMAM-calix-dendrimers were fully characterized by physical methods, i.e., 1H and 13C{1H} NMR, FTIR spectroscopy, and high-resolution electrospray ionization mass spectrometry (ESI HRMS) (Figures S1–S21). For comparative evaluation of the spectral characteristics of the first, second, and third generation dendrimers, the 1H NMR spectra of G1-alt, G2-alt, and G3-alt are presented in Figure 2. The 1H NMR spectra show an increase in the signal intensity of dendritic fragments relative to the signal intensity of the thiacalixarene platform in the series G1-alt < G2-alt < G3-alt, which confirms the increase in the degree of branching of the macromolecule. The broadening of the proton signals of the macrocyclic core and close (tert-butyl, aromatic, and oxymethylene) fragments with increasing dendrimer generation is also noteworthy. This is associated with an increase in the overall conformational mobility of the molecule during the growth of the dendritic structure.
IR spectroscopy was also applied to analyze the structures of G2.5 and G3 PAMAM-calix-dendrimers. The IR spectra of the obtained compounds show bands at 1087–1095 cm–1, characteristic of arylalkyl esters and corresponding to the dendron linkage to the macrocyclic platform of thiacalix[4]arene (Ar-O-CH2). The IR spectrum of G3 completely lacks the intense band at 1732 cm–1, which is present in the IR spectrum of G2.5 and corresponds to the vibrations of the ester (COOMe) group. This further confirms the complete substitution of all the ester moieties during the synthesis of G3 PAMAM-calix-dendrimers. The synthesized dendrimers were additionally characterized by ESI HRMS. An intense peak of polyprotonated molecular ion [M + 16H + Na]17+ is observed in the ESI mass spectrum of G3 PAMAM-calix-dendrimers. Thus, third generation PAMAM-calix-dendrimers containing a macrocyclic core in various conformations (cone, partial cone, and 1,3-alternate) were successfully obtained.

3.2. Hemolytic Activity and Platelets Aggregation

Under physiological conditions, the outer surface of the erythrocyte membrane, like that of other cells, is negatively charged due to the presence of glycolipids and glycoproteins in its structure. As a result, cationic particles can interact with the membrane, leading to significant changes or damage to the structure and the formation of pores [49,50]. Previously, we found an unusual behavior in PAMAM-calix-dendrimers compared to classical PAMAM dendrimers; namely, a decrease in hemolytic activity with increasing generation [32]. However, that study only focused on two generations of dendrimers with a macrocyclic core in 1,3-alternate conformations. The hemolytic activity of G1-alt (20 µM) was 36% (after 3 h) and 61% (after 24 h), while the hemolytic activity of G2-alt (10 µM) was less (4% after 3 h and 20% after 24 h) [32]. The study of hemolytic activity of the third generation PAMAM-calix-dendrimers confirmed the maintenance of the trend of decreasing toxicity of PAMAM-calix-dendrimers with increasing generation, i.e., the hemolytic activity of G3-alt (5–50 µM) was less than 5% after 24 h (Figure 3). We also investigated all three stereoisomers of the third (least toxic) generation of PAMAM-calix-dendrimers to reveal the influence of the macrocyclic core conformation on the hemolytic activity of the dendrimers (Figure 3). G3-cone was found to have the highest hemolytic activity. Typically, cone conformation is the most toxic due to its amphiphilic structure (the substituents are located on one side of the macrocyclic platform), and therefore better ability to integrate into RBC bilayer membrane [51]. In the case of G3-alt, the hemotoxic effect was insignificant and did not exceed 5% after 24 h. This effect can be explained by the lower density of the cationic charge, which was distributed evenly on the surface leading to a decrease in negative effects during interaction with RBCs.
The mechanisms of platelet aggregation under the action of associates and nanoparticles, which are not agents for the treatment of diseases associated with problems of hematopoiesis and thromboembolism, are generally poorly understood and can differ greatly for different classes of nanomaterials [52,53]. In our work, we also studied the ability of G3 PAMAM-calix-dendrimers to induce platelet aggregation. As a result of measuring aggregation in platelet-rich plasma, no differences were found between samples treated with compounds and control samples. Since the studies were carried out in blood plasma, where all coagulation factors are present, it can be concluded that G3 PAMAM-calix-dendrimers did not interact with platelets and did not interfere with the work of factors.

3.3. Interaction of PAMAM-Calix-Dendrimers with Calf Thymus DNA

We have previously shown the ability of first generation PAMAM-calix-dendrimers to interact with salmon sperm DNA [33]. However, the high hemolytic activity of first generation PAMAM-calix-dendrimers requires the investigation and comparison of the complexation of different generations (G2 and G3) with DNA. Electron absorption (UV–Vis) spectroscopy was initially used to prove the interaction of the obtained PAMAM-calix-dendrimers with calf thymus DNA. To increase the affinity for the polyanionic DNA molecule, PAMAM-calix-dendrimers were converted to the hydrochloric ammonium salt form. The UV–Vis spectra of the ammonium salt and neutral forms of first and second generation PAMAM-calix-dendrimers have been described in detail previously [31,32,33]. The absorbance of PAMAM-calix-dendrimers at 200–315 nm was due to π-π* transitions of the aromatic rings of the macrocyclic fragment and n-π* transitions of the carbonyl groups. The UV–Vis spectra of all three generations were similar, differing only in the value of optical density. The intensity increased significantly in the order G1 < G2 < G3 due to an increase in the number of chromophoric amide fragments (from 12 to 68, respectively), while the number of aromatic rings remained the same. Calf thymus DNA absorbed in the same spectral region, which was due to π-π* electron transitions in the purine and pyrimidine bases of the DNA molecule [54,55]. The UV–Vis spectra of mixtures of DNA with PAMAM-calix-dendrimers for all conformations and generations showed a small hyperchromic effect at 260 nm, indicating the interaction of the components (Figure 4 and Figures S22–S30). The rise of the baseline in the long-wave region of the spectrum additionally confirmed the association of components with the formation of associates [56,57].
The complexity of the UV–Vis spectra made it impossible to quantify binding by this method. Another convenient method for studying compound interactions is fluorescence spectroscopy [58]. However, the lack of fluorescent properties in DNA made it necessary to use a fluorescent marker, i.e., ethidium bromide (EB), in this study. EB intercalates into the DNA molecule with a significant increase in emission intensity. We prepared the DNA/EB systems beforehand, and after half an hour of incubation, the dendrimer was added. When the DNA/EB system was irradiated at 525 nm, intense emission with a maximum at 600 nm was observed. The fluorescence intensity decreased significantly in the presence of increasing amounts of PAMAM-calix-dendrimers (0.5–60 μM) (Figure 5a, Figures S31, S33, S35, S37, S39, S41, S43 and S45). Next, we recorded fluorescence spectra of free EB in the presence of dendrimers (Figure 5b) to verify that the fluorescence quenching was caused by the formation of the ternary DNA/EB/PAMAM-calix-dendrimers system and not by the interaction of EB with the dendrimers. The absence of changes in the intensity of the emission spectra in the EB/PAMAM-calix-dendrimers system confirmed the nature of the interaction.
The Stern–Volmer plots had a complex and nonlinear shape with several bends, which significantly complicated the calculation of binding constants (Figure 6, Figures S32, S34, S36, S38, S40, S42, S44 and S46). The presence of the hyperchromic effect in the electronic absorption spectra indicates the static nature of the quenching due to the formation of complexes (Figure 4 and Figures S22–S30). However, the Stern–Volmer plots clearly indicated several simultaneous processes, both static (with the formation of different types of complexes) and dynamic. Therefore, we decided to qualitatively evaluate the efficiency of the interaction between PAMAM-calix-dendrimers and the DNA. For this purpose, we determined PAMAM-calix-dendrimer concentrations (C50, µM) at which a 50% decrease in the emission intensity, F50, of the DNA/EB system from the initial one was observed (F50 = (F0 − Fmin)/2) (Figures S47–S55). There was a consistent decrease in these concentrations (from 8.44 to 1.10 μM) with increasing generation (Table 1). The differences between the C50 of various conformations were maximal for G1 (3.56–8.44 μM), and was negated for G2 (3.00–3.22 μM) and G3 (1.10–1.31 μM). Thus, the influence of the macrocyclic core was highest for the first generation, and G1-alt best bound the DNA. This appeared to be due to the lower steric arrangement of the binding ammonium groups in space [59]. With increasing generation, the influence of the macrocyclic platform decreased significantly due to its shielding by substituents, and the C50 concentrations became close.
The secondary structure of the biopolymers determines their biological properties. Circular dichroism (CD) spectroscopy is the most sensitive and relatively inexpensive method for establishing this structure. The CD spectrum of the double-stranded calf thymus DNA molecule had a maximum at 275 nm and a minimum at 247 nm, which was characteristic of a mixture of canonical A- and B-forms of DNA [60,61]. The least toxic G3 PAMAM-calix-dendrimers were selected to study the effect of dendrimers on the CD spectrum of DNA. The CD spectra of the DNA/G3 mixtures showed only weak changes with a slight bathochromic shift of the maxima of the CD signals relative to those of the pure DNA solution (Figure 7). Such character of the CD spectra changes indicated non-intercalative binding of PAMAM-calix-dendrimers in the grooves of the DNA, since more significant changes could be observed in the CD spectra in the case of intercalation [62,63]. The results obtained correlated well with previously published data for G1 PAMAM-calix-dendrimers [33], in the presence of which the preservation of the DNA’s secondary structure was shown.
Significant changes in the CD spectra of the DNA/EB system were observed in the presence of G3 PAMAM-calix-dendrimers. In the CD spectrum of the DNA/EB system, the appearance of an additional positive signal with a maximum at 308 nm was observed, in addition to a significant increase in the amplitude intensities of the CD signals of DNA (Figure 8). This band corresponds to the absorption of molecules of the initially nonchiral intercalator, which acquired chirality when incorporated into an optically active biopolymer [64,65]. When PAMAM-calix-dendrimers were added to the DNA/EB system, the new band of intercalated EB disappeared. This indicated the loss of chirality of EB due to its removal from the biopolymer molecule. At the same time, the structure of the biomolecule was preserved, as evidenced by the preservation of signals at 245 and 275 nm, characteristic of free DNA. Thus, we can exclude the possibility of loss of chirality of the intercalator due to “unfolding” of the biopolymer molecule. A similar trend was observed for all three conformations of G3 PAMAM-calix-dendrimers. This effect can be explained if we take into account the fact that EB is not only intercalated in the DNA molecule, but at the same time its binding is realized mainly along the small groove due to electrostatic interactions with DNA phosphate backbone [66,67]. Previously, thiacalixarenes containing terminal amino groups were found to bind to double-stranded DNA along the major and minor grooves [68]. Thus, we suggest PAMAM-calix-dendrimers displaced EB located in the DNA grooves upon binding to DNA, thereby shifting the equilibrium between them and the intercalated dye molecules. This process led to the release of the intercalator from the biopolymer.
Next, the effect of PAMAM-calix-dendrimer generation was determined using the least toxic compounds with a macrocylic core in 1,3-alternate conformation. The CD spectra of the DNA/EB systems in the presence of G1-alt, G2-alt, and G3-alt showed that the maximum decrease of the intercalator signal at 308 nm was observed for G3-alt, which may indicate its almost complete removal from the biopolymer molecule (Figure 9). This fact could be easily explained by the increase in the number of ammonium groups binding to the DNA with increasing dendrimer generation.
An important characteristic of compounds is the size of the particles they form in solutions [69,70]. The size of the particles determines both the rate of their deposition and their retention time in the target organs, e.g., at the site of tumor [71]. Dynamic light scattering (DLS) was used to determine the size of the particles formed in solution upon the interaction of DNA with PAMAM-calix-dendrimers. All studied PAMAM-calix-dendrimers did not form stable self-associates (10 mM Tris-HCl, pH = 7.4) (Tables S1–S3), which correlates well with previously published data for G1 PAMAM-calix-dendrimers [33]. However, supramolecular systems DNA/PAMAM-calix-dendrimers with monomodal particle distributions were formed when calf thymus DNA (20 µM in nucleotide base pairs) was added to the dendrimers (Figures S56, S58–S63). Only the DNA/G1-alt systems were not monodisperse and had visually distinguishable colloidal aggregates in solution (Table S1). The DNA/G1-cone and DNA/G1-paco (concentrations of dendrimers equal to 50, 100, and 500 μM) systems were monodisperse with the lowest polydispersity indices (PDI). The smallest particles (160 nm, PDI 0.21) of the DNA/G1-cone system were formed at 50 μM of G1-cone (Table S1, Figure S59). The smallest particles (121 nm, PDI 0.19) of the DNA/G1-paco system were formed at 500 μM of G1-paco (Table S1, Figure S61).
Further study of the association of G2 PAMAM-calix-dendrimers with calf thymus DNA in the same concentration range showed that the DNA/G2-cone systems showed the highest PDI in contrast to G1-cone (Table S2, Figures S64–S69). In the case of G2-cone, the formation of stable systems was observed at 1, 50, and 500 μM, while the PDI was in the range of 0.30–0.40, with a minimum particle diameter of 567 nm. In the case of G2-paco, the formation of monodisperse systems (PDI 0.25–0.29) was observed at 1, 50, and 500 μM of G2-paco. The smallest particles (148 and 166 nm) for this stereoisomer were recorded at 500 and 50 μM, respectively (Table S2, Figure S67). The DNA/G2-alt systems were least polydisperse (PDI 0.20–0.38). At the same time, the smallest particles (206 nm, PDI 0.20) for this stereoisomer were recorded at 500 μM of G2-alt (Table S2, Figure S69).
For G3 PAMAM-calix-dendrimers, the trend was retained (Table S3, Figures S70–S78). Thus, the least polydisperse systems were DNA/G3-cone with dendrimer concentrations of 2 μM (182 nm, PDI 0.16) and 0.16 μM (281 nm, PDI 0.26) (Figure S71). Similar characteristics were observed for the DNA/G3-paco systems (2 μM–185 nm, PDI 0.20; 0.16 μM–244 nm, PDI 0.26) (Figure S74). The polydispersity of the DNA/G3-alt systems was higher, and the particle size also exceeds that of analogs (from 202 to 532 nm, PDI 0.30–0.42) (Figure S77). Reducing the DNA concentration to 10 μM (in nucleotide base pairs) (Figure S57) resulted in stable monodisperse systems in a wide range of G3 concentrations. At the same time, the particle sizes decreased significantly. The diameter of the formed particles of the DNA/G3-cone system (2 μM of G3-cone) was 102 nm (PDI 0.17) (Figure S72). The particles of the DNA/G3-paco system were slightly larger (245 nm, PDI 0.26–0.27), especially at low (up to 0.33 μM) dendrimer concentrations. At higher concentrations of G3-paco, the particle sizes were 123–185 nm (PDI 0.17–0.23) (Figure S75). The polydispersity of the DNA/G3-alt system also decreased (PDI 0.15–0.17) with particle sizes of 106 and 119 nm at 5 and 3.3 μM of G3-alt, respectively (Figure S78).
Thus, all three generations of PAMAM-calix-dendrimers with calf thymus DNA formed monodisperse systems with submicron (<200 nm) particles. In the case of G1 PAMAM-calix-dendrimers, the most significant DNA compaction is achieved for G1-cone, which was consistent with the data obtained earlier in the study of the thiacalixarene complexes with calf thymus DNA [72]. Shielding of the macrocyclic core of G3 PAMAM-calix-dendrimers by dendrons led to a change in the size characteristics of the formed particles of DNA/G3. The particle sizes were found to be close, with the greatest compactification achieved for G3-alt, which is the most similar in structure to classical PAMAM dendrimers.
The electrokinetic potentials of DNA complexes (10 μM in nucleotide base pairs) with the third generation PAMAM-calix-dendrimers indicated the stability of the as-formed associates (Table S4). The resulting complexes are generally positively charged (electrokinetic potential ranges from +30.5 to +40.0 mV), with the exception of complexes with very low concentrations of PAMAM-calix-dendrimers (1 μM, range from –19.8 to –28.3 mV) (Figures S79–S90). This high positive charge on G3 PAMAM-calix-dendrimers without DNA (Table S4) put them on par with cationic polymers with excellent abilities in transfection [73].
Next, we used the electron microscopy method to evaluate the morphology of the formed particles. We chose DNA-based systems and the least toxic G3 PAMAM-calix-dendrimers, which formed particles of minimal size (<200 nm). Figure 10a shows the formation of filamentous structures of a calf thymus DNA solution (0.6 μM in nucleotide base pairs). G3 PAMAM-calix-dendrimers formed amorphous particles regardless of core conformation (Figure 10c, Figures S91a and S92a). For the DNA/G3-cone system, monodisperse particles with a diameter of approximately 50 nm are presented in TEM images (Figure 10b), while the particle size of the DNA/G3-paco system reached ~100 nm (Figure S91b). TEM images of the DNA/G3-alt system (Figure S92b) show conglomerates of particles composed of smaller particles, which is characteristic of this conformation [74]. Some discrepancy in the sizes of nanoparticles obtained by DLS and TEM was not a surprising fact and could be explained by different methods of sample preparation and study. The DLS method measures the hydrodynamic particle diameter of the colloidal system, while the TEM method measures the size of nanoparticles formed by concentration and subsequent drying on the surface.

4. Conclusions

Thus, a simple and efficient method for the synthesis of the third generation PAMAM-calix-dendrimers with a macrocyclic core based on p-tert-butylthiacalix[4]arene has been developed. The replacement of the linear core by a macrocyclic core led to a dramatic decrease in hemolytic activity with increasing generation, which is in contrast to the results for classical PAMAM dendrimers. The conformation of the macrocyclic core also had an effect on the hemolytic activity, which decreased in the series cone > partial cone > 1,3-alternate. The macrocyclic core served as a template for the fixation of positively charged ammonium groups. Therefore, the lowest hemotoxicity was observed for 1,3-alternate stereoisomers possessing the lowest charge density due to the even distribution of substituents on different sides of the macrocycle, while the highest hemotoxicity was found for the most amphiphilic cone stereoisomers with maximally separated hydrophilic (positively charged) and hydrophobic parts. Apparently, the influence of the macrocyclic core was due to its hydrophobicity, which allowed hydrophobic interactions to be additionally realized in addition to electrostatic and hydrogen interactions characteristic of classical PAMAM dendrimers. As a result, even the first generation of PAMAM-calix-dendrimers was capable of DNA binding. Increasing the generation of PAMAM-calix-dendrimers led to an increase in DNA binding efficiency, while the influence of the macrocyclic platform was negated. Thus, the third generation PAMAM-calix-dendrimers are analogues of classical PAMAM dendrimers with significantly reduced hemolytic activity. We found that PAMAM-calix-dendrimers formed small (d < 200 nm) associates with calf thymus DNA regardless of the generation and conformation of the macrocyclic core. Thus, G3-alt with both low hemolytic activity and high DNA binding efficiency was the most promising for the creation of nucleic acid compacting agents among all investigated PAMAM-calix-dendrimers. We hope our study will open a wide range of possibilities for the design and synthesis of new macrocyclic dendrimers capable of binding nucleic acids, as well as serve to further expand the range of applications of dendrimer compounds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics16111379/s1. References [32,33] are cited in the Supplementary Materials.

Author Contributions

Investigation, Methodology, Data curation, Writing—original draft preparation, O.M.; Investigation, Methodology, Data curation, Writing—original draft preparation, Visualization, I.S. (Igor Shiabiev); Investigation, Methodology, Writing—original draft preparation, D.O. and D.P.; Investigation, Validation, Data curation, T.M.; Investigation, Methodology, Data curation, A.S.; Investigation, Visualization, Validation, Writing—original draft preparation, V.A.; Formal analysis, Validation, Writing—review & editing, D.S.; Investigation, Resources, Data Curation, A.K. and M.K.; Formal analysis, Supervision, Writing—review & editing, M.S. and X.S.; Data curation, Writing—original draft preparation, Supervision, P.P.; Conceptualization, Writing—review & editing, Supervision, Funding acquisition, Project administration, I.S. (Ivan Stoikov). All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by Russian Science Foundation, Russian Federation (grant 21-73-20067, https://rscf.ru/en/project/21-73-20067/ (accessed on 27 September 2024)).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Pharmaceutics 16 01379 g001
Figure 1. The sketch image of design and application of macrocycle-based dendrimers.
Figure 1. The sketch image of design and application of macrocycle-based dendrimers.
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Scheme 1. Synthesis of G3 PAMAM-calix-dendrimers.
Scheme 1. Synthesis of G3 PAMAM-calix-dendrimers.
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Figure 2. Stacked 1H NMR spectra of G1-alt, G2-alt, and G3-alt (CD3OD, 298 K, 400 MHz).
Figure 2. Stacked 1H NMR spectra of G1-alt, G2-alt, and G3-alt (CD3OD, 298 K, 400 MHz).
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Figure 3. Hemolysis induced by G3 PAMAM-calix-dendrimers after (a) 1 h, (b) 3 h, and (c) 24 h of incubation. Data presented as percentage of hemolysis, mean ± SD, n = 4. Statistical significance between negative control (untreated RBC) and samples (at * p < 0.05) was estimated by the post-hoc Newman–Keuls test.
Figure 3. Hemolysis induced by G3 PAMAM-calix-dendrimers after (a) 1 h, (b) 3 h, and (c) 24 h of incubation. Data presented as percentage of hemolysis, mean ± SD, n = 4. Statistical significance between negative control (untreated RBC) and samples (at * p < 0.05) was estimated by the post-hoc Newman–Keuls test.
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Figure 4. UV–Vis spectra of DNA (20 µM in nucleotide base pairs), G3-alt (10 µM), and their mixture (10 mM Tris-HCl, pH = 7.4).
Figure 4. UV–Vis spectra of DNA (20 µM in nucleotide base pairs), G3-alt (10 µM), and their mixture (10 mM Tris-HCl, pH = 7.4).
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Figure 5. Fluorescence spectra of (a) the DNA/EB system (18 µM in nucleotide base pairs/5 µM) in the presence of G3-alt (0–60 μM) and (b) EB (5 µM) with/without G3-alt (60 μM), (10 mM Tris-HCl, pH = 7.4).
Figure 5. Fluorescence spectra of (a) the DNA/EB system (18 µM in nucleotide base pairs/5 µM) in the presence of G3-alt (0–60 μM) and (b) EB (5 µM) with/without G3-alt (60 μM), (10 mM Tris-HCl, pH = 7.4).
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Figure 6. Stern–Volmer plot of the DNA/EB/G3-alt system (10 mM Tris-HCl, pH = 7.4).
Figure 6. Stern–Volmer plot of the DNA/EB/G3-alt system (10 mM Tris-HCl, pH = 7.4).
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Figure 7. CD spectra of calf thymus DNA (20 µM in nucleotide base pairs) with/without G3 PAMAM-calix-dendrimers (10 µM) (10 mM Tris-HCl, pH = 7.4).
Figure 7. CD spectra of calf thymus DNA (20 µM in nucleotide base pairs) with/without G3 PAMAM-calix-dendrimers (10 µM) (10 mM Tris-HCl, pH = 7.4).
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Figure 8. CD spectra of the DNA/EB system (20 µM in nucleotide base pairs/10 µM) with/without G3 PAMAM-calix-dendrimers (10 µM) (10 mM Tris-HCl, pH = 7.4).
Figure 8. CD spectra of the DNA/EB system (20 µM in nucleotide base pairs/10 µM) with/without G3 PAMAM-calix-dendrimers (10 µM) (10 mM Tris-HCl, pH = 7.4).
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Figure 9. CD spectra of the DNA/EB system (20 µM in nucleotide base pairs/10 µM) with/without G1-alt, G2-alt, and G3-alt (10 µM) (10 mM Tris-HCl, pH = 7.4).
Figure 9. CD spectra of the DNA/EB system (20 µM in nucleotide base pairs/10 µM) with/without G1-alt, G2-alt, and G3-alt (10 µM) (10 mM Tris-HCl, pH = 7.4).
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Figure 10. TEM images of (a) DNA (0.6 µM in nucleotide base pairs); (b) DNA (0.6 µM in nucleotide base pairs) with G3-cone (1 µM); (c) G3-cone (1 µM).
Figure 10. TEM images of (a) DNA (0.6 µM in nucleotide base pairs); (b) DNA (0.6 µM in nucleotide base pairs) with G3-cone (1 µM); (c) G3-cone (1 µM).
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Table 1. Concentrations of PAMAM-calix-dendrimers (C50, µM) at which 50% fluorescence quenching (F50) of the DNA/EB system is observed.
Table 1. Concentrations of PAMAM-calix-dendrimers (C50, µM) at which 50% fluorescence quenching (F50) of the DNA/EB system is observed.
DendrimerC50, µM
conepartial cone1,3-alternate
G15.778.443.56
G23.003.223.15
G31.311.281.10
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MDPI and ACS Style

Mostovaya, O.; Shiabiev, I.; Ovchinnikov, D.; Pysin, D.; Mukhametzyanov, T.; Stanavaya, A.; Abashkin, V.; Shcharbin, D.; Khannanov, A.; Kutyreva, M.; et al. PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding. Pharmaceutics 2024, 16, 1379. https://doi.org/10.3390/pharmaceutics16111379

AMA Style

Mostovaya O, Shiabiev I, Ovchinnikov D, Pysin D, Mukhametzyanov T, Stanavaya A, Abashkin V, Shcharbin D, Khannanov A, Kutyreva M, et al. PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding. Pharmaceutics. 2024; 16(11):1379. https://doi.org/10.3390/pharmaceutics16111379

Chicago/Turabian Style

Mostovaya, Olga, Igor Shiabiev, Daniil Ovchinnikov, Dmitry Pysin, Timur Mukhametzyanov, Alesia Stanavaya, Viktar Abashkin, Dzmitry Shcharbin, Arthur Khannanov, Marianna Kutyreva, and et al. 2024. "PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding" Pharmaceutics 16, no. 11: 1379. https://doi.org/10.3390/pharmaceutics16111379

APA Style

Mostovaya, O., Shiabiev, I., Ovchinnikov, D., Pysin, D., Mukhametzyanov, T., Stanavaya, A., Abashkin, V., Shcharbin, D., Khannanov, A., Kutyreva, M., Shen, M., Shi, X., Padnya, P., & Stoikov, I. (2024). PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding. Pharmaceutics, 16(11), 1379. https://doi.org/10.3390/pharmaceutics16111379

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