Synthesis of N‐Sulfopropylated Hyperbranched Polyethyleneimine with Enhanced Biocompatibility and Antimicrobial Activity

Abstract

Hyperbranched polyethyleneimine having 25,000 Da molecular weight was functionalized by a simple sulfopropylation reaction, affording a novel N‐sulfopropylated PEI derivative (PEI‐SO₃ ⁻). The successful introduction of N‐sulfopropyl and sulfobetaine groups to the amino groups of PEI was spectroscopically confirmed. Furthermore, the antibacterial and anti‐cyanobacterial activity of PEI‐SO₃ ⁻ in comparison to the parent PEI were investigated on two type heterotrophic bacteria, i. e., Gram (−) Escherichia coli and Gram (+) Staphylococcus Aureus bacteria, and one type of autotrophic cyanobacterium, i. e. Synechococcus sp. PCC 7942. Both PEI‐SO₃ ⁻ and PEI showed an enhanced, concentration‐dependent antibacterial and anti‐cyanobacterial activity against the tested bacteria strains, with PEI‐SO₃ ⁻ exhibiting higher activity than the parent PEI, signifying that the introduction of the sulfopropyl and sulfobetaine groups to the PEI amino groups enhanced the antibacterial and the anti‐cyanobacterial properties of PEI. In the case of cyanobacteria, PEI‐SO₃ ⁻ was found to affect the integrity of the photosynthetic system by the inhibition of Photosystem‐II electron transport activity. Cytocompatibility and hemocompatibility studies revealed that PEI‐SO₃ ⁻ exhibits high biocompatibility, suggesting that PEI‐SO₃ ⁻ could be considered as an attractive antibacterial and anti‐cyanobacterial candidate for various applications in the disinfection industry and also against the harmful cyanobacterial blooms.

A novel N‐sulfopropylated hyperbranched polyethyleneimine derivative (PEI‐SO₃ ⁻) was prepared by a simple sulfopropylation reaction. This derivative exhibited high biocompatibility simultaneously with antibacterial and anti‐cyanobacterial activity. Thus, PEI‐SO₃ ⁻ could be considered as an attractive antibacterial and anti‐cyanobacterial candidate for various applications in the disinfection industry or against the harmful cyanobacterial blooms.

Introduction

Infections by pathogenic microorganisms are an ongoing and significant challenge for various sectors related to, for example, water purification systems, food packaging, synthetic textiles, constructions, healthcare and medical care, household sanitation, etc., as bacterial infections worldwide are known to kill more people than any other cause. Up to now, the use of antibiotics and is the main weapon in the fight against infectious diseases, but due to their misuse and overuse, antibiotic resistance becomes as one of the leading public health threats of the 21st century.[ ¹ , ² ] On the other hand, various disinfectants such as hypochlorite, triclosan, hydrogen peroxide, silver salts, etc., are used in daily life to fight against microbial contamination, but due to their short shelf life and various health‐related safety issues, their usage is restrained. ^[3] Thus, a need to discover new antimicrobial agents to fight microbial infections is now mandatory. ^[4]

In this concept, various polymers and polymer‐based materials have been proposed as excellent antibacterial agents due not only to their enhanced efficacy, but also to their low toxicity against mammalian cells, long shelf‐life, low probability of inducing drug resistance and low production cost.[ ⁵ , ⁶ , ⁷ , ⁸ ] Antibacterial polymers can either have inherent activity or after suitable functionalization with appropriate moieties or after incorporation of antibacterial compounds acquire the desired properties. ^[9] Their main mechanism of action involves either direct contact with the cell envelope, which affects the integrity of bacteria membrane or their metabolism, ultimately leading to cell death (active polymers) or inhibition of bacterial growth (passive polymers). ^[5] The antibacterial activity as well as the cytotoxicity of polycations are influenced by various factors like the type of repeating unit, positively charged group, hydrophilic‐lipophilic balance, molecular structure, and molecular weight.[ ¹⁰ , ¹¹ , ¹² , ¹³ ] The effect of molecular weight on antibacterial activity has been investigated for various types of polycation and found to be strongly dependent on the type of polymer, i. e. increasing its molecular weight either decreases or increases its antibacterial activity.[ ¹⁴ , ¹⁵ ] Furthermore, the type of repeating unit can also influence the antibacterial properties of the final polymer. Pachla's group found that linear polytrimethylenimine exhibited higher antibacterial activity than linear polyethyleneimine. ^[10] On the other hand, the hemolytic and cytotoxic activity of polymer can increase upon increasing its molecular weight,[ ¹⁶ , ¹⁷ ] while after suitable functionalization of the polymer's end groups, e. g. with polyethylene glycol chains, can reduce the hemolytic and cytotoxic activity of the parent polymer. ^[18]

Among these polymers, hyperbranched polyethyleneimine (PEI), which is an amine‐rich, low‐cost polymer having a highly branched tree‐like architecture and a large number of functional end groups, has been proposed as an antimicrobial agent. Specifically, PEI has exhibited antibacterial properties due to its polycationic amphiphilic character, which enables it to interact with the anionic components of cell envelop, i. e. teichoic acid and lipoteichoic acid in case of Gram (+) bacteria or liposaccharides and phospholipids in case of Gram (−) bacteria, inducing the disruption or permeabilization of bacterial membrane, leading finally to cell death.[ ¹⁹ , ²⁰ ] Kuroda's group ^[19] has also studied the antibacterial effects and the cytotoxicity of low molecular branched and linear polyethyleneimine polymers. They found that the effectiveness of PEI against E. coli and S. aureus bacteria was influenced by both the structure and molecular weight of the polymer. Specifically, the low molecular weight branched PEIs showed lower toxicity to human cells than linear analogous, while both branched and linear PEIs exhibited higher antibacterial activity against S. aureus than E. coli bacteria.

Moreover, its activity was found to improve upon suitable functionalization. Various quaternized PEI derivatives having also hydrophobic alkyl or aromatic groups have been synthesized and studied regarding their antibacterial properties.[ ²¹ , ²² , ²³ , ²⁴ , ²⁵ ] These derivatives that have a cationic amphiphilic character due to the presence of hydrophobic groups as well as hydrophilic quaternary moieties in the same molecule can effectively interact with the cell envelope, enhancing their antibacterial activity.[ ¹² , ²⁶ ] The hydrophobic/hydrophilic balance plays a key role in this interaction, and thus in the antibacterial properties of PEI derivatives. Specifically, the number of cationic and hydrophobic groups, the length of the alkyl chains and the molecular weight of the PEI derivatives tune the final antibacterial properties. ^[26]

Although PEI derivatives with cationic amphiphilic character have been extensively studied as antibacterial agents, to the best of our knowledge, there are no analogous studies on PEI functionalized with anionic groups, such sulfonates or with zwitterionic moieties such as sulfobetaines. Anionic polysaccharides, like heparin and chondroitin sulfate, exhibit not only excellent biological activity but also antibacterial properties.[ ²⁷ , ²⁸ ] In this context, sulfonated chitosan was prepared by grafting N‐sulfopropyl groups to the skeleton of chitosan. ^[29] The obtained derivatives showed enhanced antibacterial properties than the parent polymer. Specifically, sulfonated chitosan exhibited higher inhibitory activity against Gram (−) Escherichia coli and Gram (+) Staphylococcus aureus bacteria with the minimum inhibitory concentration of 0.13 mg/mL and 2.00 mg/mL, respectively, compare to those of chitosan with MIC of 0.50 mg/mL and 4.00 mg/mL. Moreover, polymers containing zwitterions, like sulfobetaines or carboxybetaines, showed enhanced antibacterial and antifouling property.[ ³⁰ , ³¹ , ³² ] Thus, Y. Chen et.al. introduced sulfobetaine groups to the backbone of chitosan obtaining a chitosan derivative, which exhibited better antibacterial activity along with lower cytotoxicity and hemolytic activity than chitosan. ^[33]

Recently, hyperbranched PEI was investigated as anti‐algal and anti‐cyanobacterial polymer in order to be used for prevention of the uncontrolled blue‐green bloom in fresh, salt, or brackish waters. ^[34] Algae and cyanobacteria are known to cause serious health, economic and technical problems in various applications such as water distribution systems or water reservoirs, as they can cause biofouling due to increased accumulation of microorganisms, leading to clogging of filters or pipes as well as other undesirable consequences, e. g., in the production of unpleasant odors and tastes, in reducing the systems performance, in inducing corrosion, etc.[ ³⁵ , ³⁶ ] Additionally, overgrowth of cyanobacteria in water reservoirs can cause production of harmful toxins that can seriously affect human health. ^[37] Therefore, nowadays the need to control and prevent the cyanobacterial bloom is imperative for human health, and various industrial sectors and thus the development of novel anti‐cyanobacterial agents is urgent. ^[38]

In this study, hyperbranched polyethyleneimine (PEI) of 25,000 Da molecular weight was functionalized by a simple sulfopropylation reaction, affording a novel N‐sulfopropylated PEI derivative (PEI‐SO₃ ⁻). After its structural characterization, the antibacterial activity of PEI‐SO₃ ⁻ was investigated against Gram (−) Escherichia coli and Gram (+) bacteria Staphylococcus Aureus bacteria, while its anti‐cyanobacterial activity was studied against cyanobacterium Synechococcus sp. PCC 7942. Furthermore, its biocompatibility was investigated through cytotoxicity on mammalian cell lines and haemolysis assays.

Results and Discussion

Synthesis and Characterization of N‐Sulfopropylated Hyperbranched Polyethyleneimine

Hyperbranched polyethyleneimine with molecular weight 25,000 Da was initially characterized by inverse‐gate decoupling ¹³C NMR. By the integration of the signals of carbons relative to the primary, secondary and tertiary amine groups, the ratio of primary to secondary to tertiary amines of PEI was calculated and it was found to be 1.00 : 1.18 : 1.00.[ ²⁵ , ³⁹ ] The degree of PEI branching was found to be 0.68 and its average number of primary, secondary and tertiary amine groups was determined as 183, 215 and 183, respectively. Based on the above, the introduction of sulfopropyl groups as well as sulfobetaine moieties to PEI was achieved following a simple sulfopropylation reaction (Scheme 1) between 1,3‐propane sultone and PEI, affording the N‐sulfopropylated polyethyleneimine derivative (PEI‐SO₃ ⁻). Specifically, the five‐membered ring of 1,3‐propane sultone reacted with nucleophilic primary, secondary and tertiary amines of PEI, producing N‐sulfopropylated substituted amines or sulfobetaines. ^[40] The introduction of sulfopropyl groups as well as sulfobetaine moieties to PEI was established by proton and carbon NMR spectroscopy (Figures S1 and S2) as well as by FTIR spectroscopy (Figure S3). Specifically, the substitution of PEI was confirmed by the appearance in the ¹H NMR spectrum of the new broad peaks centered at 2.80 and 1.85 ppm, attributed to the α‐ and β‐methylene protons adjacent to the sulfonic group. Additionally, the presence of the sulfobetaine groups to PEI scaffold was confirmed by the triplet peak at 3.50 ppm attributed to α‐methylene protons of quaternary ammonium groups. Furthermore, the introduction of sulfopropyl groups was confirmed by the new peaks in the ¹³C NMR spectrum at 48 ppm, attributed to the α‐ methylene carbons adjacent to the sulfonic group and at 24 and 21 ppm attributed to β‐methylene carbons of sulfopropyl group adjacent to the secondary and tertiary amines. On the other hand, the attachment of the sulfobetaine groups to PEI was established by the new peaks in the ¹³C NMR spectrum at 49 and 25 ppm, attributed to the α‐ and β‐methylene carbons relative to the sulfonate group. The total degree of substitution to the primary, secondary and tertiary amino groups was calculated by the integration of peaks in ¹H NMR spectrum at 1.85 ppm, attributed to the α‐ methylene protons adjacent to the sulfonic group and 2.20–2.75 ppm attributed to the methylene protons of PEI skeleton. It was found that, on the average, 80 sulfopropyl and sulfobetaine groups were attached to the dendritic scaffold. Precise determination of the degree of substitution to the primary, secondary and tertiary amino groups was achieved by inverse‐gate decoupling ¹³C NMR. Comparing the integrations of the signals of the β‐methylene carbons relative to the sulfonate group (21, 24 and 25 ppm) and the signals of the α‐methylene carbons relative to the primary amino groups of PEI (37 and 39 ppm), it was found that 37, 12 and 31 sulfopropyl groups were attached to the primary, secondary and tertiary amino groups of PEI, respectively.

Scheme 1.

Synthetic scheme of N‐sulfopropylated PEI (PEI‐SO₃ ⁻).

Moreover, FTIR spectrum of PEI‐SO₃ ⁻ was shown in Figure S3. The bands at 1354, 1165, 1036, 600 and 520 cm⁻¹ attributed to symmetric bending, asymmetric stretching, symmetric stretching and asymmetric deformation vibrations of the sulfonic group, respectively, as well as the band at 725 cm⁻¹ assigned to the C−S stretching vibrations, confirmed the presence of sulfopropyl groups to PEI scaffold. Additionally, the shoulder appeared at 1637 cm⁻¹ and the weak band at 938 cm⁻¹ can be attributed to the stretching and bending modes of C−N⁺ groups, respectively, corroborating the successfully attachment of sulfobetaine groups to polymeric backbone.

The size and polydispersity of the parent PEI and the N‐sulfopropylated PEI derivative were studied by dynamic light scattering (DLS). The hydrodynamic diameter of PEI was found to be, within experimental error, 3.7_±0.4 nm, with a polydispersity of 0.398, while the hydrodynamic diameter of PEI‐SO₃ ⁻ was found to be slightly higher, i. e. 4.1_±0.3 nm, with a polydispersity of 0.265 (Figure S4). As observed, the introduction of N‐sulfopropyl groups to the PEI scaffold did not significantly affect the PEI size. Furthermore, the ζ‐potential values of both polymers were measured at pH=7 and found to be, within experimental error, 33.8_±0.9 mV and 5.8_±0.8 mV for PEI and PEI‐SO₃ ⁻, respectively. These findings confirm the successful introduction of anionic N‐sulfopropyl groups to the PEI scaffold which, as expected, significantly reduce the surface charge of PEI.

Antibacterial Activity

The antibacterial activity of PEI‐SO₃ ⁻ was investigated against two types of bacteria, one gram positive (Staphylococcus aureus) and one gram negative (Escherichia coli). Additionally, the parent PEI was also screened for its antibacterial activity under the same conditions. Initially, the bacteria growth was studied in the presence of PEI‐SO₃ ⁻ and parent PEI at various concentrations ranging from 10 μg/mL to 150 μg/mL, by measuring the optical density (OD) of the treated cultures for 12 h. The growth kinetics curves of E. coli and S. aureus bacteria were plotted as a function of OD/OD₀ values versus incubation time (Figures 1 and 2). As observed, the logarithmic growth rate of both strains decreases in a concentration‐dependent manner, indicating that both polymers inhibit the bacteria growth. Specifically, the growth rate of E. coli bacteria treated with low concentrations of PEI‐SO₃ ⁻ (20 or 40 μg/mL) is lower than that of the untreated cells, while the growth of bacteria treated with higher concentrations (60 or 80 μg/mL) was almost completely inhibited (Figures 1A). On the other hand, as observed in Figure 1B, the growth rate of E. coli bacteria treated with PEI at concentrations ranged between 20 to 100 μg/mL, is lower than that of the treated bacteria with PEI‐SO₃ ⁻, while completely inhibition of bacteria growth is observed at higher concentration than PEI‐SO₃ ⁻, i. e. 150 μg/mL, indicating that PEI‐SO₃ ⁻ exhibits better antibacterial activity against E. coli than the parent polymer. Analogous results are observed in case of S. aureus bacteria (Figure 2). Thus, bacterial growth inhibited after treatment with PEI‐SO₃ ⁻ and PEI in a concentration range between 10 to 60 μg/mL and 10–100 μg/mL, respectively, while when S. aureus bacteria were treated with 80 μg/mL PEI‐SO₃ ⁻ or 150 μg/mL PEI, there is no apparent bacterial growth within 12 h, which means that almost all bacterial cells can be inactivated at a lower concentration of PEI‐SO₃ ⁻ concentration than that of the parent polymer.

Figure 1.

Growth curves of Escherichia coli bacteria in absence (control) or presence of PEI‐SO₃ ⁻ (A) and PEI (B).

Figure 2.

Growth curves of Staphylococcus Aureus bacteria in absence (control) or presence of PEI‐SO₃ ⁻ (A) and PEI (B).

The MIC and MBC values of PEI‐SO₃ ⁻ and PEI against E. coli and S. aureus bacteria as determined by the broth dilution and colony‐counting methods in accordance to M07‐A9 and M26‐A protocols published by the Clinical Laboratory Standards Institute (CLSI),[ ⁴¹ , ⁴² ] respectively, are summarized in Table 1. Specifically, the MIC values of PEI‐SO₃ ⁻ are 200 and 120 μg/mL for E. coli and S. aureus, respectively, while for PEI, MIC values of 300 and 200 μg/mL are recorded for E. coli and S. aureus. On the other hand, MBC values of PEI‐SO₃ ⁻ are 400 and 200 μg/mL for E. coli and S. aureus, respectively, while for PEI, these MIC values are higher (i. e. 500 and 300 μg/mL were recorded for E. coli and S. aureus). It is obvious that PEI‐SO₃ ⁻ shows better antibacterial activity than the parent PEI, indicating that the introduction of the sulfopropyl and sulfobetaine groups to the PEI amino groups enhanced the antibacterial properties of PEI. Analogous results have been observed when sulfobetaine groups introduced to chitosan scaffold. ^[34] It was found that the presence of sulfobetaine groups not only improved the antibacterial properties of chitosan, but also enhanced its aqueous solubility and biocompatibility. Similarly, in another work, N‐sulfopropylated chitosan derivatives were proposed as safe alternatives to antibiotics and chemical compounds able to inhibit microorganism growth. ^[43]

Table 1.

MIC and MBC values of PEI‐SO₃ ⁻ and PEI against E. coli and S. aureus bacteria.

Samples	E. coli		S. aureus
	MIC (μg/mL)	MBC (μg/mL)	MIC (μg/mL)	MBC (μg/mL)
PEI‐SO3 −	200	400	120	200
PEI	300	500	200	300

To investigate the antibacterial mechanism of PEI‐SO₃ ⁻, SEM was employed to study the surface morphology as well as the membrane structure changes of E. coli bacteria after 12 h treatment at 37 °C with PEI‐SO₃ ⁻ at 1/2 MIC. SEM images of the untreated (control) and treated bacteria with PEI‐SO₃ ⁻ are presented in Figure 3. All untreated bacteria appear intact with a smooth surface, while, on the other hand, the surface of treated bacteria is rougher and some of their membranes have been ruptured, signifying that the bacteria have lost their cellular integrity, while it is observed leakage of intracellular components suggesting that cell lysis has occurred, possibly resulting in bacterial death.

Figure 3.

SEM images of E. coli bacteria: untreated cells (A) and cells after 12 h incubation time at 37 °C with PEI (B) or PEI‐SO₃ ⁻ (C) at 1/2 MIC.

It is known that the mechanism of antibacterial activities of various zwitterionic compounds has been attributed either to the strong electrostatic interaction of their positively charged groups with the negatively charged groups located on the bacteria cell envelop or to the strong ionic interaction or chelation between the positively or negatively charged moieties with the metal ions of the outer bacterial membrane such as magnesium, iron, zinc, copper and calcium, which are essential for the bacterial survival. As a result, the integrity and selectivity of the membrane is altered, leading to bacterial dysfunction, precipitation of cytoplasmic components and eventually cell damage or death.[ ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷ ] Since PEI‐SO₃ ⁻ has a zwitterionic character as it contains both positively and negatively charged moieties, its antibacterial mechanism may be similar to that of the zwitterionic polymers, viz. it could be attributed to the strong interaction of the amino groups of PEI‐SO₃ ⁻ with the negatively charged groups of bacteria cell membranes and walls or between amino or sulfonate groups with the metal ions of the outer bacteria membrane, causing an antibacterial effect.

Anti‐Cyanobacterial Activity

The anti‐cyanobacterial activity of PEI‐SO₃ ⁻ was studied against the cyanobacterium Synechococcus sp. PCC 7942. Cyanobacteria are a phylum of prokaryotes that are able to perform oxygen‐evolving photosynthesis in an analogous manner to that of higher plants. Cyanobacteria with the exception of prochlorophytes, contain only one type of chlorophyll, chlorophyll a (Chl α), which is a green pigment that allows photosynthesis to transform solar energy into chemical energy. Although, cyanobacteria are one of the oldest organisms and also one of the most important bacterial groups in the world as they are responsible for the oxygenation of the atmosphere and oceans, it is known that several cyanobacteria strains can produce a variety of toxic secondary metabolites such as dermatotoxins, cytotoxins, neurotoxins, etc., which may be harmful to animals even to humans. ^[48]

Herein, cell proliferation of cyanobacteria Synechococcus sp. PCC 7942 was monitored by measurement of the Chl α concentration every 24 h, for seven days. Figure 4 shows the growth curves of cyanobacteria as a function of concentrations of PEI or PEI‐SO₃ ⁻. As observed, both polymeric derivatives inhibit the cyanobacteria cell proliferation in a dose‐dependent manner. Comparing the effect of both derivatives on the cyanobacteria growth, it is clearly observed that PEI‐SO₃ ⁻ causes a higher inhibition of the cyanobacterial cell proliferation than that of parent PEI, as the growth of Synechococcus sp. PCC 7942 in the presence of 20 μg/mL PEI‐SO₃ ⁻ fully inhibited. Moreover, to quantify these observations, the half maximal effective inhibitory concentration (IC‐50) as well as the minimum inhibitory concentration (MIC) were calculated by the area under the growth curves for each concentration of each derivative, using non‐linear regression of a 4‐parameters logistic function (Figure S5 and Table 2). It was found that the IC‐50 value of PEI‐SO₃ ⁻ is much lower (4.14 μg/mL) than that of the parent PEI (16.50 μg/mL) as well as the MIC value of PEI‐SO₃ ⁻ was found to be 18 μg/mL, considerably lower than that of PEI (37 μg/mL). These findings imply that as in cases of E. coli and S. aureus bacteria, the introduction of the sulfopropyl and sulfobetaine groups to the PEI amino groups enhanced the anti‐cyanobacterial properties of PEI.

Figure 4.

Effect of PEI and PEI‐SO₃ ⁻ on cell proliferation of cyanobacteria Synechococcus sp. PCC 7942. Growth curves of untreated cyanobacteria (control) or treated cyanobacteria with different concentrations of: (A) PEI and (B) PEI‐SO₃ ⁻). Error bars represent mean ± SD for at least three independent experiments.

Table 2.

IC‐50 and MIC values of PEI and PEI‐SO₃ ⁻ on cyanobacterium Synechococcus sp. PCC 7942.

Samples	IC‐50 (μg/mL)	MIC (μg/mL)
PEI	16.50	37
PEI‐SO3 −	4.14	18

Comparing the activity of PEI‐SO₃ ⁻ on the heterotrophic bacteria (E. coli and S. aureus) with that on the autotrophic bacteria (cyanobacteria Synechococcus sp. PCC 7942), it is obvious that PEI‐SO₃ ⁻ exhibits higher inhibitory activity on the cyanobacteria, which is probably attributed to the effect of PEI‐SO₃ ⁻ on the photosynthetic apparatus of cyanobacteria. Thus, the selective evaluation of the Photosystem (PS) I ^[49] and II ^[50] activity in the presence of PEI‐SO₃ ⁻ was assessed in order to study the effect of PEI‐SO₃ ⁻ on the integrity of the photosynthetic system regarding the photoinduced electron transport. In detail, initially cyanobacteria Synechococcus sp. PCC7942 were treated with lysozyme at room temperature resulting in permeaplasts formation, i. e., ion‐permeable modified membranes. ^[51] Thus, rapid internalization of PEI‐SO₃ ⁻ into cyanobacteria was achieved. Then, photoinduced electron transport activities in both PSI and PSII were measured using cyanobacterial permeaplasts after treatment with various PEI‐SO₃ ⁻ concentrations by determination of oxygen evolution. As observed in Table S1, only in case of the PSII, the oxygen evolution rate decreases upon increase in PEI‐SO₃ ⁻ concentration, revealing that only PSI electron transport activity depends on the polymer concentration, while PSII electron transport activity remains almost unaffected. In Figure 5, the inhibition of the PSI and PSII by PEI‐SO₃ ⁻ is shown. It is obvious that PEI‐SO₃ ⁻ causes a minor inhibition of the PSI even at the highest tested concentration (50 μg/mL). On contrary, a 50 % inhibition of PSII is observed at concentration of 10 μg/mL, while at the highest tested concentration (50 μg/mL), the inhibition of PSII reaches the value of approximately 80 %. These results reveal that the photosynthetic electron transport of PSII is functionally affected by PEI‐SO₃ ⁻ in cyanobacteria, justifying its higher toxicity against cyanobacteria than that against E. coli and S. aureus.

Figure 5.

Effect of PEI‐SO₃ ⁻ on the photosynthetic electron transport activities of PSII and PSI in Synechococcus sp. PCC 7942 permeaplasts.

Biocompatibility Studies

Since the biocompatibility of antibacterial agents is a critical parameter so that they can find application in various fields, the in vitro cytotoxicity of PEI‐SO₃ ⁻ and the parent PEI was initially studied on human breast MCF‐7 and SKBR‐3 cell lines. Specifically, these cells were treated with various concentrations of PEI or PEI‐SO₃ ⁻ and then cell viability was estimated using the standard MTT assay after 24 h of incubation time. As shown in Figure 6, PEI was found to be toxic in concentration‐dependant manner against both tested cell lines. Specifically, PEI was found to be non‐toxic (survival: 80–90 %) only at low concentrations (up to 20 μg/mL), while PEI exhibited significant toxicity (survival ~20 %) at the highest tested concentration (400 μg/mL). On contrary, PEI‐SO₃ ⁻ was found to be non‐toxic up to ~200 μg/mL, while at higher concentrations (300–400 μg/mL) is only subtoxic (survival>60 %).

Figure 6.

Comparative toxicities of PEI‐SO₃ ⁻ and PEI on SKBR‐3 (A) and MCF‐7 (B) cells following incubation at various concentrations for 24 h as determined by MTT assays. Data are expressed as mean ± SD of six independent values obtained from at least three independent experiments. The statistical significance, obtained from Student's paired two‐tailed t‐tests, follows the assignment: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns, not significant.

Moreover, the biocompatibility of PEI‐SO₃ ⁻ and the parent PEI was assessed on human red blood cells by measuring their hemolytic activity. Specifically, red blood cells were treated with various concentrations of PEI‐SO₃ ⁻ or PEI and subsequently the released hemoglobin was spectroscopically measured. Triton X‐100 and PBS were used as positive (100 % hemolysis) and negative control (~0 % hemolysis), respectively. The hemolytic activities of both polymeric derivatives are shown in Figure 7. As observed, the hemolytic percentage of both derivatives increases in concentration‐dependant manner. Nevertheless, PEI‐SO₃ ⁻ is less hemolytic than the parent PEI as it causes 40 % hemolysis at the highest tested concentration (200 μg/mL), which is much lower than that of PEI (~80 % hemolysis at 200 μg/mL). It is obvious that the introduction of sulfopropyl groups and sulfobetaine groups to PEI scaffold significantly improves the biocompatibility of the parent polymer.

Figure 7.

Concentration‐response curves of the hemolytic activities of PEI‐SO₃ ⁻ and PEI. The statistical significance, obtained from Student's paired two‐tailed t‐tests, follows the assignment: *p<0.05, **p<0.01, and ****p<0.0001.

Conclusions

In this study, hyperbranched polyethyleneimine having 25,000 Da molecular weight was functionalized, following a simple and known from the literature sulfopropylation reaction, affording an N‐sulfopropylated PEI derivative (PEI‐SO₃ ⁻). Its chemical structure was characterized by FTIR and NMR spectroscopies, establishing the successful introduction of N‐sulfopropyl and sulfobetaine groups to the amino groups of PEI. Additionally, DLS and ζ‐potential measurements revealed that PEI size was slightly affected upon functionalization, in contrast, its surface charge decreased due to the presence of anionic N‐sulfopropyl groups in PEI‐SO₃ ⁻. To investigate the antibacterial and anti‐cyanobacterial activity of PEI‐SO₃ ⁻ in comparison to the parent PEI, two type heterotrophic bacteria, i. e. Gram (−) Escherichia coli and Gram (+) Staphylococcus Aureus bacteria, and one type of autotrophic cyanobacterium, i. e. Synechococcus sp. PCC 7942, were used. It was found that both PEI‐SO₃ ⁻ and PEI showed an enhanced, concentration‐dependent antibacterial and anti‐cyanobacterial activity against the tested bacteria strains, with PEI‐SO₃ ⁻ exhibiting higher activity than the parent PEI. Specifically, PEI‐SO₃ ⁻at a concentration of 80 μg/mL completely inhibited the bacteria growth within 12 h treatment, while in the case of PEI, a concentration of 150 μg/mL was required to achieve complete growth inhibition under the same treatment conditions. Additionally, MIC and MBC values of PEI‐SO₃ ⁻ were found to be lower than those of the parent PEI, revealing that the introduction of the sulfopropyl and sulfobetaine groups to the PEI amino groups enhanced the antibacterial properties of PEI. These enhanced properties may be attributed to the zwitterionic character of PEI‐SO₃ ⁻, which allows the strong interaction of the amino groups of PEI‐SO₃ ⁻ with the negatively charged groups of bacteria cell membranes and walls or between amino or sulfonate groups with the metal ions of the outer bacteria membrane. On the other hand, in the case of the cyanobacteria, it was found that PEI‐SO₃ ⁻ affected the integrity of the photosynthetic system regarding the photoinduced electron transport and specifically the photosynthetic electron transport of PSII, justifying also its higher toxicity against Synechococcus sp. PCC 7942 cyanobacteria than that against E. coli and S. aureus. Furthermore, cytocompatibility and hemocompatibility studies on human breast MCF‐7 and SKBR‐3 cell lines and human red blood cells, respectively, revealed that PEI‐SO₃ ⁻ exhibit higher biocompatibility than the parent PEI, especially in the concentration range in which it also exhibited both high antibacterial and anti‐cyanobacterial activity. Therefore, PEI‐SO₃ ⁻ could be considered as an attractive antibacterial and anti‐cyanobacterial candidate for various applications in the disinfection industry and also against the harmful cyanobacterial blooms.

Experimental Section

Materials

Hyperbranched polyethyleneimine with a molecular weight of 25,000 Da (Lupasol® WF, water‐free, 99 %) was kindly donated by BASF GmbH (Ludwigshafen, Germany). 1,3‐propanesultone, dialysis tube (molecular weight cut‐off (MWCO): 1200), glutaraldehyde, sodium cacodylate, and resazurin were purchased from Sigma‐Aldrich (St. Louis, MA, USA). Dulbecco's modified Eagle's medium (DMEM) low glucose with phenol red, Dulbecco's phosphate buffered saline (PBS), penicillin/streptomycin, L‐glutamine, trypsin/EDTA and fetal bovine serum (FBS) were purchased from Biochrom GmbH (Berlin, Germany). Thiazolyl blue tetrazolium bromide (MTT), Triton X‐100 and isopropanol were obtained from Merck KGaA (Calbiochem®, Darmstadt, Germany). Red Hydrogen Peroxide Assay Kit was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). All used solvents (Riedel‐de Haën, Seelze, Germany) were of analytical grade and distilled prior to use.

Synthesis of N‐Sulfopropylated Polyethyleneimine Derivate

Hyperbranched polyethyleneimine (PEI) was functionalized by a simple sulfopropylation reaction by a method analogous to one previously reported. ^[52] Thus, 4 mmol of 1,3‐propanesultone dissolved in 3 mL of dry acetonitrile, were slowly added to 0.04 mmol PEI dissolved in 10 mL of dry methanol. The mixture was allowed to react for three days under argon atmosphere at 40 °C. Subsequently, the solution was concentrated by solvent distillation under reduced pressure and the product was received after precipitation with diethyl ether. The crude product was dissolved in water and was subjected to dialysis (mol weight cut‐off: 1200) to remove by‐products. Lyophilization afforded the final N‐sulfopropylated PEI derivative, PEI‐SO₃ ⁻. The introduction of sulfopropyl groups as well as sulfobetaine moieties to the PEI scaffold was confirmed by proton and carbon NMR spectroscopy using a Bruker Avance DRX spectrometer operating at 500 and 125.1 MHz, respectively. The degree of N‐sulfopropylated substitution was determined by inverse gated ¹³C NMR. Additionally, its structure was established by FTIR spectroscopy using an Attenuated Total Reflectance accessory (ATR) with diamond crystal (Smart Orbit™, Thermo Electron Corporation). A minimum of 32 scans were collected and the recorded signal averaged.

¹Η ΝΜR (500 MHz, D₂O): δ(ppm)=3.50 (t, γ‐CH₂ of sulfobetaine group relative to SO₃ ⁻), 2.75–3.00 (m, α‐CH₂ relative to SO₃ ⁻), 2.20–2.75 (m, γ‐CH₂ of sulfopropyl group and CH₂ of PEI scaffold), 1.85 (m, β‐CH₂ relative to SO₃ ⁻).

¹³C ΝΜR (125.1 MHz, D₂O): 52.5 (N⁺‐CH₂, N‐CH₂CH₂‐NH and N‐CH₂CH₂‐NH₂), 52 (N‐CH₂ CH₂‐N), 51(NH‐CH₂CH₂‐NH₂), 49 (Ν⁺‐CH₂CH₂ CH₂SO₃ ⁻), 48 (Ν‐CH₂CH₂ CH₂SO₃ ⁻ and ΝH‐CH₂CH₂ CH₂SO₃ ⁻), 47 (NH‐CH₂ CH₂‐NH), 45 (NH‐CH₂CH₂‐N), 39 (NH‐CH₂ CH₂‐NH₂), 37(N‐CH₂ CH₂‐NH₂), 25 (ΝH‐CH₂ CH₂CH₂SO₃ ⁻), 24 (Ν‐CH₂ CH₂CH₂SO₃ ⁻), 21 (Ν⁺‐CH₂ CH₂CH₂SO₃ ⁻).

FTIR (cm⁻¹): 3340 (m) ν _as(NH), 3275 (m) ν _s(NH₂), 2930 (m) ν _s(CH₂), 2880 (m), 2810 (s) ν _as(CH₂), 1637(shoulder) ν(C−N⁺), 1630 (w) ν _s(NH), 1545 (w) δ(NH), 1450 (s) δ(CH₂), 1354 (m) δ(SO₃), 1165(s) ν _as(SO₃), 1110(m) ν _as(C−N), 1036 (s) ν _s(SO₃), 938 (w) δ(C‐N⁺), 790 (m) ρ(CH₂), 725 (m) ν(C−S), 600 (m) δ _s(SO₃), 520(m) δ _as(SO₃).

Physicochemical Characterization

The size, the polydispersity and the charge of the parent PEI and PEI‐SO₃ ⁻ was determined at 25 °C by dynamic light scattering (DLS) and ζ‐potential, respectively, applying a Zetasizer Nano apparatus (Malvern Instruments Series, Nano‐ZS with multipurpose titrator). For these experiments, aqueous solutions of PEI or PEI‐SO₃ ⁻ (20 mg/mL) were used. For each solution, twenty measurements, for each measurement, were collected, and the results were averaged.

Assessment of Antibacterial Properties

The antibacterial activity of PEI‐SO₃ ⁻ in comparison with PEI was initially assessed by a bacteria growth inhibition assay and then by determination of MIC and MBC values employing the microdilution method in combination of the colorimetric resazurin assay and the colony‐counting method, respectively. Escherichia coli (strain DH5a) and Staphylococcus Aureus (strain ATCC 25923) bacteria were grown in lambda broth (LB) and tryptic soy broth (TSB) medium, respectively, at 37 °C for 16 h, in a Stuart SI500 orbital shaker (~200 rpm shaking speed) in aerobic conditions. The obtained bacteria suspensions diluted with media to a concentration equal to 0.5 McFarland Standard (~10⁸ CFU/mL) and used for the followed experiments.

Bacterial growth study: The culture of E. coli and S. aureus bacteria were diluted with LB and TSB, respectively, to a concentration of 10⁵ CFU/mL as determined by the suspensions’ optical density (OD) measured at 600 nm using a Cary 100 Conc UV‐visible spectrophotometer (Varian Inc., Mulgrave, Australia). Then, bacteria were inoculated into PEI and PEI‐SO₃ ⁻ solutions at concentrations ranging from 5 to 100 μg/mL and incubated at 37 °C for 12 h. Untreated bacteria were used as control. During the incubation period, bacterial growth was monitored by measuring the OD of the bacterial suspensions at 600 nm every 1 h for St. Aureus or 2 h for E. Coli for 12 h. The bacteria growth curves were presented as a function of the obtained OD/OD₀ values versus incubation time.

MIC determination: Overnight cultures of E. coli and S. Aureus were diluted to approximately 10⁵ CFU/mL and then PEI or PEI‐SO₃ ⁻ were added at final concentrations ranging from 5 to 500 μg/mL. The assay was carried out in a 96‐well plate format in a 100 μL final volume. Two controls, one negative (100 μL broth medium without cells), and one positive (100 μL of bacteria suspension at 10⁵ CFU/mL) were also included in the plate. After 24 h incubation at 37 °C, 5 μL of resazurin solution (6.72 mg/mL) was added to each well and mixed thoroughly. After 4 h incubation at 37 °C, the plate was subjected to fluorescence measurement at an excitation wavelength of 530 nm and emission wavelength of 590 nm using an Infinite M200 plate reader (Tecan group Ltd., Männedorf, Switzerland).

MBC determination: Fifty microliters of treated bacteria from the MIC experiment were obtained from the well at MIC value and two wells above the MIC value and inoculated onto LB agar plates. Agar cultures were incubated at 37 °C for 18 h and then the colonies on the plates were counted. MBC endpoint is considered the concentration as a lowest concentration killing 99.9 % of the initial bacterial inoculum.

Bacteria morphological analysis by scanning electron microscope: The bacteria morphology after treatment with PEI or PEI‐SO₃ ⁻ was studied by scanning electron microscopy (Jeol JSM 7401 F Field Emission SEM). In brief, cells were incubated with PEI or PEI‐SO₃ ⁻ at 1/2 MIC for 24 h and then fixed with 3 % glutaraldehyde in sodium cacodylate buffer (100 mM, pH=7.1) for 12 h. Subsequently, the cells were pelleted, washed to remove the excess of glutaraldehyde, and resuspended in the same buffer. 50 μL of the obtained cell dispersion was transferred to a poly(L‐lysine) coated glass cover slip, dehydrated using ethanol gradient (twice of 50 %, 70 %, 95 %, and 100 % ethanol for 10 min each), dried, and coated with gold in a sputter coater. ^[53]

Assessment of Anti‐Cyanobacterial Properties

Anti‐cyanobacterial properties of PEI‐SO₃ ⁻ was assessed on Synechococcus sp. PCC 7942 bacteria (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France). Cyanobacteria were grown in BG‐11 medium containing 20 mM HEPES buffer (pH=7.5). The cultures were incubated under white light (100 μmol photons m⁻² s⁻¹), in an orbital incubator (Galenkamp INR‐400) in a humidified atmosphere containing 5 % CO₂ at 32 °C. ^[54]

Cyanobacteria Growth Inhibition Assay: Cyanobacteria were treated with various PEI‐SO₃ ⁻ concentrations ranging from 2 to 20 μg/mL. The cyanobacteria culture was inoculated in each test solution in the exponential growth phase at concentrations of approximately 1 μg Chl α/mL. Cyanobacterial growth was monitored in terms of concentration of chlorophyll a (Chl α) determined in N,N‐dimethylformamide (DMF) extracts. To extract Chl α from the treated cyanobacteria, cells were centrifuged and the obtained pellet was dispersed in DMF. The resulted dispersion was centrifuged again and the clear supernatant DMF extract was used to measure the Chl α concentration. ^[55] The PEI‐SO₃ ⁻ toxicity was determined as the effective concentrations (μg/mL) that inhibits the cell proliferation by 50 % (IC‐50) relative to the control. The IC‐50 values were calculated by the area under the growth curves for each PEI‐SO₃ ⁻ concentration, using non‐linear regression of a 4‐parameters logistic function. ^[25] The experiments were performed in three replicates.

Assessment of Photosystem I and II electron transport activities: Photo‐induced electron transport rates were assessed in Synechococcus permeaplasts at room temperature oxymetrically (for each Photosystem) with a Clark‐type oxygen electrode (DW1; Oxygraph, Hansatech, King's Lynn, UK). Cyanobacteria permeaplasts were obtained after cell treatment with lysozyme before the assessment of the photosynthetic electron transport activities. ^[52] The instrument was equipped with a slide projector to provide actinic illumination of samples (4.0 mE m⁻² s⁻¹). Photosystem I (PSI) activity was assessed by the determination rate of the oxygen evolution, in the presence of the post‐ Photosystem II (PSII) electron transfer inhibitor 3‐(3,4‐dichlorophenyl)‐1,10‐dimethylurea (DCMU), using Na ascorbate/diaminodurene as an electron donor to PSI and methyl viologen as a post‐PSI electron acceptor and mediator of oxygen uptake. ^[56] The reaction mixture in BG11 medium consisted of permeaplasts (5 μg Chl α/mL), Na‐ascorbate (2 mM), DCMU (0.01 mM), diaminodurene (1 mM) and methyl viologen (0.15 mM). On the other hand, PSII activity was assessed by measuring the oxygen evolution rate, with water as electron donor and p‐benzoquinone as post‐PSII electron acceptor. The reaction mixture in BG11 medium consisted of permeaplasts (5 μg Chl α/mL) and p‐benzoquinone (1 mM). The experiments were performed in three replicates.

Biocompatibility Studies

Cytotoxicity studies: The cytotoxicity of PEI‐SO₃ ⁻ and parent PEI was assessed on human breast MCF‐7 and SKBR‐3 cell lines, using the standard MTT assay. Cells were grown in D‐MEM high glucose supplemented with 10 % FBS, penicillin (100 U/mL)/ streptomycin (100 μg/mL) solution and L‐Glutamine (2 mM). Cells were incubated in a humidified atmosphere containing 5 % CO₂ at 37 °C and sub‐cultured twice a week after detaching with a trypsin (0.05 % w/v)/EDTA (0.02 % w/v) solution. Cells were inoculated (10⁴ cells/well) in 96‐well plates and left to incubate in complete media containing 10 % FBS for 24 h. Cells were then treated with various concentrations of PEI‐SO₃ ⁻ and parent PEI for 24 h. The mitochondrial redox function (translated as cell viability) of all cell groups was assessed by the MTT assay. In brief, cell media was replaced with MTT solution (10 μg/mL in complete media) and incubating at 37 °C in a 5 % CO₂ humidified atmosphere for 4 h. Then, MTT media were removed and the produced formazan was solubilized in 2‐isopropanol (100 μL/well). The absorbance at 540 nm was measured using an Infinite M200 microplate reader (Tecan group Ltd., Männedorf, Switzerland). Six replicates were performed for each concentration, and the experiment was repeated in triplicate. The relative cell viability was determined as percentage compared to cells incubated with only complete media (control). Blank values measured in wells with 2‐isopropanol and no cells, were in all cases subtracted.

Hemolysis study: Blood compatibility of PEI‐SO₃ ⁻ and parent PEI was assessed by hemolysis assay. Fresh human blood was drawn into plastic tube containing EDTA (2 mg/mL). Blood cells were centrifuged, and then plasma (supernatant) was discarded, while the erythrocytes pellet was washed with PBS three times and redispersed in PBS (pH 7.2 – 7.5) to a final cell concentration of 4 %. 250 μL of cell dispersion was mixed with 250 μL of various concentrations of PEI and PEI‐SO₃ ⁻ and the resulted dispersions were incubated for one hour at 37 °C. After centrifugation at 1,000 g for 10 min, 100 μL of the supernatant of each sample was transferred to 96‐well plate and the released hemoglobin was monitored at 540 nm using an Infinite M200 microplate reader (Tecan group Ltd., Männedorf, Switzerland). 250 μL of red blood cell dispersion were also mixed with 250 μL Triton X‐100 or 250 μL PBS as positive (100 % hemolysis) or negative (0 % hemolysis) control, respectively. The % percentage of hemolysis ratio was calculated using the following equation:

$${\displaystyle Hemolysisratio\left(\%\right)=\frac{ODs-ODnc}{ODpc-ODnc}\times 100}$$

where, ODs, ODnc and ODpc are the average absorbance of the tested samples, the negative control and the positive control, respectively. Each assay was performed in triplicate.

Statistical Analysis

Student's paired two‐tailed t‐tests was performed on the MTT cytotoxicity and hemolysis data obtained for both polymers to study the statistical significance of a difference between means. An independent t‐test was performed for comparison between the two polymers. Τhe statistical significance follows the assignment: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 and ns, not significant (p>0.05).

Supporting Information Summary

The Supporting Information includes Figures S1–S5 and Table S1.

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

Panagiotaki K. N., Lyra K.-M., Papavasiliou A., Stamatakis K., Sideratou Z., ChemPlusChem 2025, 90, e202400454. 10.1002/cplu.202400454

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.