Postpolymerization modification of polyvinylpyrrolidone and evaluation of anti-quorum-sensing,antimicrobial,and antibiofilm activities with molecular docking and absorption,distribution,metabolism,and excretion studies

Abstract

Antibiotic resistance is a major health risk, and biocompatible polymers like polyvinylpyrrolidone and their derivatives are used in developing suitable antimicrobials. In this study, polyvinylpyrrolidone was reacted with potassium hydroxide and later coupled with benzoyl chloride (C₇H₅ClO) to produce polyvinylpyrrolidone 2. Polyvinylpyrrolidone 2 was reacted with 3-(dimethylamino)-1-propylamine to obtain polyvinylpyrrolidone 3. The polyvinylpyrrolidone derivatives were characterized using nuclear magnetic resonance and Fourier transform infrared spectroscopy. Polyvinylpyrrolidone 2 and polyvinylpyrrolidone 3 inhibited the production of violacein in Chromobacterium violaceum CV12472 and mutant strain C. violaceum CV026, indicating their potential anti-quorum-sensing properties. Polyvinylpyrrolidone 2 and polyvinylpyrrolidone 3 inhibited flagella-dependent swarming and swimming against Pseudomonas aeruginosa PA01 at minimal inhibitory concentration and sub-minimal inhibitory concentration concentrations in a dose-dependent manner. Polyvinylpyrrolidone 3 was more active than polyvinylpyrrolidone 2, which could be attributed to the introduction of the amide function. Minimal inhibitory concentration values were 0.3125 mg/mL for polyvinylpyrrolidone 3 against C. albicans and S. aureus, while those for polyvinylpyrrolidone 2 were 0.625 mg/mL against C. albicans and S. aureus. Minimal inhibitory concentration values were 1.25 mg/mL for both polymer derivatives against E. coli. Polyvinylpyrrolidone 2 and polyvinylpyrrolidone 3 inhibited biofilms against E. coli, S. aureus, and C. albicans. Polyvinylpyrrolidone 3 exhibited the highest concentration-dependent biofilm inhibition of 45.86 ± 0.15% at minimal inhibitory concentration, which reduced to 9.02 ± 0.25% at minimal inhibitory concentration/8 against S. aureus. Molecular docking indicated suitable interactions between polyvinylpyrrolidone 2 and polyvinylpyrrolidone 3 derivatives and binding sites of pathogen receptor proteins with negative binding energies, and drug-likeness predicted using Swiss ADME was appropriate. The results indicate that polyvinylpyrrolidone 2 and polyvinylpyrrolidone 3 can inhibit biofilm formation and mitigate the emergence and spread of resistant strains.

Keywords

antimicrobial polymers anti-quorum-sensing biofilm inhibition molecular docking polyvinylpyrrolidone postpolymerization

Introduction

Functionalized monomers undergo polymerization, while reactive polymers can be subjected to postpolymerization modification. Both processes are used to create chemically diverse and bioactive polymer libraries, with scientists concentrating on the first strategy and little attention being paid to the latter strategy.¹ Postpolymerization has proven to be more effective for the production of macromolecules with improved properties. Polymers can undergo postpolymerization modifications through ring-opening reactions to give access to various derivatives with grafted functionalities unto the original scaffold.^2,3 Such postpolymerization modifications can be achieved either by copolymerization or by subsequently converting the functional groups and side chains of the original polymer, which are chemo-selective or inert toward the conditions of polymerization.^4,5 Various properties of polymers can be improved through postpolymerization hemi-synthesis, and this approach imparts characteristic properties that are difficult to achieve during polymerization by adding functionalities that confer the desired properties.^6,7 In addition to techniques of controlled polymerization, postpolymerization modification of already synthesized polymers leads to the preparation of a library of macromolecules with diverse structures and functions from a single polymer scaffold.^8
–10 Polymer functionalization via postpolymerization provides an easy and efficient means of introducing new groups or chemical units into the polymer skeleton to offer desired properties. The original polymer main chain is not usually affected by postpolymerization modification, indicating that modified polymers only differ in the side chains or functional groups introduced, giving room for investigating their influence on the polymer properties.¹¹

It is necessary to avoid unwanted reactions during the functionalization and imparting of defects in the polymer backbone by selecting or using selective and efficient modification reactions. Poly(N-vinylpyrrolidone), also known as polyvinylpyrrolidone or povidone (PVP), is obtained by radical polymerization of N-vinylpyrrolidone and usually modified chemically by graft copolymerization or other techniques. PVP and most of its derivatives are pH-stable, temperature-resistant, biodegradable, biocompatible, physiologically inactive, nontoxic, and chemically stable with many desirable properties and are often used in delivery systems of nutraceutical, biomedical, and pharmaceutical products.^12
–14 Its chemical surface modification prevents nonspecific protein adsorption, making its derivatives promising antifouling surface modifiers.¹⁵ PVP and its derivatives have the advantage of being soluble in water and a variety of organic solvents, including butanol, chloroform, and dichloromethane. PVP and its derivatives offer remarkable advantages with a range of chemical properties, including enhanced solubility, increased bioavailability, and even the introduction of the desired swelling tract for control or sustained release.¹⁶ By altering the methyl iodide amounts, N-methyl quaternized PVP derivatives were produced in high amounts with variable quaternization degrees, providing compounds with unexplored optical and solvation properties.¹⁷ Postpolymerization of PVP in previous studies afforded novel macromolecular complex systems with thio-semicarbazone and thiourea sites through polymer-analogous transformation.¹⁸

Sometimes, postpolymerization modification is used to confer biodegradability on the polymers and improve their mechanical, optical, and biological activities.^19,20 Antimicrobial properties are easily introduced in polymers through postpolymerization functionalization.²¹ The surface groups of PVP may be modified chemically and the resulting materials utilized to create antibacterial surfaces.²² The antimicrobial property of polymers can be an intrinsic feature, but biocide activity can be imparted on polymers by grafting active substances and functionalization of the polymers.^23,24 Antimicrobial polymers are a very promising class of medicines for treating microbial illnesses with a greater ability to successfully combat bacteria than traditional antibiotics.²⁵ In a variety of approaches including antifouling, anti-quorum-sensing (QS), efflux pumps, and biofilm inhibitors, the antimicrobial effects of polymers are described either as independent antibiotic substances or in combination with established active components.^26
–28 Unchecked use of antibiotics results in multidrug-resistant bacteria, which is among the principal human health concerns. Antibiotic-resistant bacteria, fungi, viruses, and parasites are defined by specific alterations that enable them to withstand the effects of antibiotics.²⁹ Finding novel antimicrobial treatments is suitable but not sufficient, because microbial resistance against them can arise; therefore, new modes of action can complement this, for example, targeting virulence factors. Currently, the majority of antibacterial substances target the physiological processes in bacteria to kill them. This high selective pressure on bacteria encourages various mutations and the formation of widespread drug-resistant forms.³⁰ It has been demonstrated that the QS mechanism of interbacterial communication, which is regulated by signaling molecules known as "auto-inducers," governs several functions between bacterial colonies.³¹ Individual cells can communicate with one another through the QS regulatory process, which also aids in coordinating collective behavior.³² QS network mediates virulence gene expression, establishment of biofilm, bacterial motility, and resistance to antibiotics.^33,34 The majority of pathogenic gene expression, antibiotic efflux pumps, swarming motility, toxin synthesis, and biofilm formation are examples of microbial virulence and resistance mechanisms.³⁵ Therefore, looking for quorum-quenching compounds that can stop microbiological resistance and employing them to combat infections resistant to drugs is a good strategy.

Most commercial polymers are biologically inert and require postpolymerization surface functionalization to render them bioactive and increase their applications. This work involves the postpolymerization chemical modification of commercial PVP, commonly called polyvidone or povidone. The antimicrobial, anti-QS, and antibiofilm effects of the obtained derivatives are also reported.

Results and discussion

The synthetic route for the PVP derivatives is presented in Figure 1. In this study, PVP was reacted with potassium hydroxide (KOH) and later esterified with benzoyl chloride (C₇H₅ClO) to produce PVP2. PVP2 was reacted with 3-(dimethylamino)-1-propylamine to obtain PVP3. The characterization of the derivatives of PVP obtained from postpolymerization was done through Fourier transform infrared (FTIR) spectroscopy and ¹H nuclear magnetic resonance (NMR) experiments. The schematic FTIR spectra are presented in Figure 2, while detailed annotated FTIR spectra are provided in supplementary material as Figure S1 (PVP), Figure S2 (PVP1), Figure S3 (PVP2), and Figure S4 (PVP3). The appearance of a broad band at 3584.28 cm^-1 and the modification of the nature of the peak at 1650.50 cm^-1 on the FTIR confirm the formation of PVP1 from PVP. This was also substantiated by the appearance of the N–H proton signal on the ¹H NMR at δ_H 5.20 ppm. The successful transformation of PVP1 to PVP2 is revealed by the appearance of an additional carbonyl absorption band at 1707.62 cm^-1 corresponding to the amide bond formed and the introduction of aromatic bands at 1602.17 cm^-1 and 1583.52 cm^-1. The disappearance of the N–H stretching peak on the FTIR spectrum is also observed. The ¹H NMR data showed aromatic proton shifts at δ_H ppm 7.93–8.05 (br, m), 7.49 (t, J = 7.4 Hz), and 7.35 (t, J = 7.5 Hz). Modification of PVP2 into PVP3 was made evident by the appearance on the FTIR spectrum of a signal at 1661.30 cm^-1 indicating the presence of an additional amide signal followed by C–N stretching bands at 1380 and 1350 cm^-1. The additional peaks of methylene protons (CH₂) and methyl protons (CH₃) were observed between δ_H 0.90 and 1.58 ppm. The ¹H NMR (400 MHz, CDCl₃) spectra of the PVP derivatives are provided in the supplementary material (Figures S5-S7).

Figure 1.

Scheme for the chemical modification of PVP into PVP2 and PVP3.

Figure 2.

FTIR spectra of the synthesized PVP derivatives.

QS and violacein production inhibition effects

Chromobacterium violaceum is an opportunistic Gram-negative bacterium that synthesizes purple violacein pigment, triggered by a cell–cell signaling process, which is easily measurable and represents QS. This is suitably measured at low concentrations, usually below the minimal inhibitory concentration (MIC). Values of MIC for the polymer derivatives PVP2 and PVP3 against C. violaceum CV12472 were measured and are presented in Table 1. An MIC value of 0.625 mg/mL with percentage inhibitions of violacein varying from 100% (MIC) to 14.5±0.8 (MIC/8) was observed for PVP2. An MIC value of 1.25 mg/mL against C. violaceum CV12472 was observed for PVP3 with the percentage of violacein inhibition ranging from 100% at MIC to 11.7±0.45% at MIC/8. MIC values, as presented in Table 2 against the C. violaceum CV026 mutant strain, were 0.312 mg/mL (PVP2) and 0.625 mg/mL (PVP3). Both derivatives inhibited QS with diameters of QS inhibition zones at MIC being 17.5±0.82 mm (PVP2) and 10.5±0.23 mm (PVP3). This reduced to 9.5±0.50 mm (PVP2) and 7.0±0.00 mm (PVP3) at MIC/2. Inhibition of QS and violacein production were both concentration-dependent, revealed by the decrease in the pigment with decreasing concentrations of the polymer derivatives (p<0.05).

Table 1.

Violacein inhibition against C. violaceum CV12472 by polymer derivatives.

Sample code	MIC (mg/mL)	Inhibition of violacein (%)
Sample code	MIC (mg/mL)	MIC	MIC/2	MIC/4	MIC/8	MIC/16
PVP2	0.625	100 ± 0.0	100 ± 0.0	75.1 ± 1.7	14.5 ± 0.8	-
PVP3	1.250	100 ± 0.0	100 ± 0.0	46.4 ± 0.5	11.7 ± 0.45	-

-: no inhibition.

Table 2.

Anti-quorum-sensing effects against C. violaceum CV026 by polymer derivatives.

Sample code	Quorum-sensing inhibition zone (mm)
Sample code	MIC (mg/mL)	MIC	MIC/2	MIC/4	MIC/8
PVP2	0.312	17.5 ± 0.82	9.5 ± 0.50	-	-
PVP3	0.625	10.5 ± 0.23	7.0 ± 0.00	-	-

-: No inhibition.

The purple violacein pigment is produced by the Gram-negative bacterium C. violaceum, and it is easily observable. This bis-indole alkaloid pigment is produced through a QS-mediated process and plays an antioxidant role that protects the membrane of bacterial cells from oxidative stress and also acts as an antibiotic against some Gram-positive pathogens.^36,37 The production of this pigment represents a QS process and it serves as a signal molecule in the bacterial intercolony cell-to-cell communication system for coordinative behavior. This implies that inhibiting the production of violacein could reduce a protective measure of the bacteria and also disrupts communication which weakens their virulence and pathogenicity. Both polymer derivatives (PVP2 and PVP3) prevented the synthesis of violacein pigment by C. violaceum CV12472 at MIC to sub-MIC concentrations, suggesting that PVP2 and PVP3 can suitably be used in reducing microbial virulence even without killing the bacteria, thus avoiding the emergence of resistance. The synthesis of violacein in normally growing C. violaceum CV12472 bacteria occurs spontaneously. But the C. violaceum CV026 mutant strain is unable to synthesize violacein except when an acyl-homoserine lactone (AHL) is supplied to it from an external source as a hormone to trigger the process. The quorum-sensing inhibition (QSI) by PVP2 and PVP3 against C. violaceum CV026 was revealed by cream or semitransparent zones whose diameters were measured in millimeters at MIC and sub-MIC concentrations despite the supply of external AHL. The inhibition of violacein in C. violaceum CV026 and C. violaceum CV12472 thus represents disruption of signal reception and signal emission, respectively, by the polymer derivatives.³⁸ Violacein production can also be influenced by variables such as temperature, pH, agitation rate, carbon source, and incubation period.³⁹

Inhibition of swarming and swimming motilities

Flagella-dependent motilities represent an important virulence factor of pathogenic bacteria, and they are controlled by QS processes. Pseudomonas aeruginosa PA01 is a model organism that is usually used for the assay of motilities. The polymer derivatives had MIC values of 0.625 mg/mL (PVP2) and 0.3125 mg/mL (PVP3) against P. aeruginosa PA01, as presented in Table 3. PVP3 exhibited the best antimotility effect with swarming varying from 75.1±1.95% (MIC) to 35.2±0.22% (MIC/4), while swimming inhibition reduced from 53.4±0.82% (MIC) to 16.5±0.14% (MIC/4).

Table 3.

Motility inhibition against P. aeruginosa PA01 by polymer derivatives.

Sample code	MIC(mg/mL)	Swarming inhibition (%)			Swimming inhibition (%)
Sample code	MIC(mg/mL)	MIC	MIC/2	MIC/4	MIC	MIC/2	MIC/4
PVP2	0.625	42.4±0.75	19.5±0.12	-	41.6±0.62	31.9±0.25	12.4±0.05
PVP3	0.3125	75.1±1.95	48.5±0.55	35.2±0.22	53.4±0.82	33.8±0.35	16.5±0.14

-: No inhibition

Swimming and swarming are flagellum-dependent motilities that are exhibited by bacteria such as Pseudomonas aeruginosa. Swimming is individual movement in low-viscosity or liquid media driven by flagella rotation, whereas swarming involves surface multicellular movement in solid or semisolid media propelled by revolving helical flagella.⁴⁰ In both models, the polymer derivatives were shown to be able to exercise biostatic effects against P. aeruginosa PA01 as they inhibited swarming and swimming movements varying in a concentration-dependent manner from MIC to sub-MIC. Inhibition of various coordinated movements in microbial colonies is highly advantageous and can reduce the spread of pathogens in environments and on various surfaces. Motilities are collective behaviors that have advantages over individual activity in that they enable the colonies to move toward sources of nutrients, establish biofilms, and colonize new niches as well as escape from damage, antibiotics, and competition.^41,42,43 The results suggest that the introduction of the amide function in PVP3 increases the antimotility effects of the polymer derivative.

Biofilm inhibition and antimicrobial activity

Antimicrobial (MIC values) and antibiofilm effects at subinhibitory concentrations of the polymer derivatives against C. albicans, E. coli, and S. aureus are reported in Table 4. Antimicrobial activity expressed as MIC was 0.3125 mg/mL for PVP3 against C. albicans and S. aureus, while that for PVP2 was 0.625 mg/mL against both C. albicans and S. aureus. MIC against E. coli for both polymer derivatives was 1.25 mg/mL. PVP3 exhibited the highest concentration-dependent biofilm inhibition of 45.86±0.15% and MIC, which reduced to 9.02±0.25% at MIC/8 against S. aureus. Against C. albicans, antibiofilm activity was 25.34±0.65% (MIC) and 10.24±0.25% (MIC/2) for PVP3 and 20.46±0.48% (MIC) and 7.15±0.12% (MIC/2) for PVP2. Biofilm inhibition was low against E. coli. Generally, PVP3 has better antibiofilm effects than PVP2.

Table 4.

MIC and biofilm inhibition activity results of polymer derivatives.

Microorganisms		Sample codes
		PVP2	PVP3
		MIC (mg/mL)
S. aureus		0.625	0.3125
E. coli		1.25	1.25
C. albicans		0.625	0.3125
Biofilm inhibition (%)
S. aureus	MIC	33.05±1.65	45.86±0.15
	MIC/2	10.82±0.31	31.57±0.80
	MIC/4	-	14.12±0.46
	MIC/8	-	9.02±0.25
E. coli	MIC	16.17±0.25	17.58±0.46
	MIC/2	3.58±0.10	5.90±0.20
	MIC/4	-	-
	MIC/8	-	-
C. albicans	MIC	20.46±0.48	25.34±0.65
	MIC/2	7.15±0.12	10.24±0.25
	MIC/4	-	-
	MIC/8	-	-

-: No inhibition

Motilities are crucial steps toward the passage from planktonic colonies to sessile ones and the establishment of biofilms. Biofilm is usually characterized as a cluster of cells of pathogenic microbes encased in a matrix predominantly composed of polysaccharides and usually adhering to a living or inert surface.^44,45 PVP2 and PVP3 inhibited growth and biofilm formation against C. albicans (fungi), E. coli (Gram-negative), and S. aureus (Gram-positive). The activities indicated that PVP3 was more potent than PVP2, and this is reflected by the binding energies from in silico studies. This may be due to the introduction of the amide and amine groups in PVP3 resulting from the reaction of PVP2 with 3-(dimethylamino)-1-propylamine. Biofilm can be controlled by preventing bacteria from switching to a biofilm lifestyle, and some amino amides have been shown to have this effect, most likely by changing metabolic activity and membrane permeabilization.⁴⁶ Some studies have shown that the introduction of an amide group significantly improves growth inhibition, biofilm disintegration, antivirulence, and bacteriostatic activity and also prevents expression of QS and biofilm-forming genes.⁴⁷ Bacterial biofilm communities are protected by the extracellular polymeric matrix, making them difficult to treat and making infections persistent.⁴⁸ Biofilm-forming pathogens represent a serious problem to global health concerns since they can show signs of growing resistance to traditional antibiotics and cause sickness through infections linked to devices, surfaces, or tissues.⁴⁹ For this reason, treating and inhibiting biofilm-associated illnesses requires early detection as well as a search for novel and alternative treatments that can eradicate biofilms or prevent their formation.⁵⁰ The results indicate the potential of the polymer derivatives to find applications as surface coatings in medical devices and high-touch surfaces, where they can reduce the spread and colonization by pathogens as well as prevent of resistant biofilms.

Molecular docking results

The observed growth inhibition of PVP2 and PVP3 against Candida albicans, Escherichia coli, and Staphylococcus aureus is given in Table 4. The activities were different, and PVP3 is an amide derivative of PVP2, in which OH is substituted by 3-(dimethylamino)-1-propylamine. In an attempt to explain the observed antimicrobial and antibiofilm effects of PVP2 and PVP3, molecular docking was employed to determine the binding modes between the monomeric repeating unit of PVP2 and PVP3 with the active residues of Candida albicans, Escherichia coli, and Staphylococcus aureus. Table 5 summarizes the number of hydrogen bonds, free binding energies and interactions within resulting complexes formed between PVP2 and PVP3 and the active residues of Candida albicans, Escherichia coli, and Staphylococcus aureus.

Table 5.

Hydrogen bonds, free binding energies, and number of closest residues to the docked compounds PVP2 and PVP3 into the binding sites Candida albicans, Escherichia coli, and Staphylococcus aureus along with their corresponding MIC values.

Compound	Free binding energy (kcal/mol)	H-Bonds(HBs)	Number of closest residues to the docked ligand in the active site/Number of interactions	MIC (mg/mL)
S. aureus
PVP2	-6.01	3	6/6	0.625
PVP3	-7.52	3	7/8	0.3125
E. coli
PVP2	-6.54	1	3/3	1.25
PVP3	-7.97	1	4/4	1.25
C. albicans
PVP2	-3.42	3	6/6	0.625
PVP3	-6.46	2	4/6	0.3125

From molecular docking results, PVP2 and PVP3 fit relatively well within the binding site of Candida albicans, Escherichia coli, and Staphylococcus aureus, with negative binding energies in the range of -6.01 to -7.52 kcal mol^-1, -6.54 to -7.97 kcal mol^-1, and -3.42 to -6.46 kcal mol^-1 and -6.85 kcal mol^-1, respectively, indicating the stability of the resulting complexes (Table 5). Those with negative binding energies more than -5.00 kcal mol^-1 may be considered as weak or inactive inhibitors, which is the case of the inhibitory potency of PVP2 toward Candida albicans. However, negative binding energies (< -5.00 kcal mol^-1) indicate that the polymer derivatives may have the potency to inhibit growth of Candida albicans, Escherichia coli, and Staphylococcus aureus. It also suggests that the inhibition process may be considered thermodynamically favorable (Table 5).

Figures 3 –5 display the stable complexes showing 3D and 2D bonding interactions established between the docked PVP2 and PVP3 units within the binding site of S. aureus (Figure 3), E. coli (Figure 4), and C. albicans (Figure 5). The conversion of the acid function to amide enhances the binding affinity of PVP3 and leads to the formation of complexes with greater stability within the binding sites of S. aureus, E. coli, and C. albicans with relative binding energies of -7.52, -7.97, and -6.46 kcal mol^-1, respectively. MIC values are necessary for the optimization of antimicrobial substances, and it is proper to analyze MIC values together with pharmacokinetic parameters, which describe the fate of the compound in the host.⁵¹ In Table 5, the polymer derivatives can bind effectively to the active sites of pathogen enzymes as revealed by the binding energies, number of H-bondings, and good binding efficiencies, and this is likely to correlate with the MIC values. Proper binding efficiencies with receptor enzymes, and proteins of bacteria such as DNA-gyrase-B target protein, correlate with good antimicrobial activity of active compounds.⁵² Good binding affinities and a greater number of H-bonds indicate high binding interactions resulting from suitable orientation of the ligand molecule with the binding sites of the receptor proteins and this translates to higher antimicrobial activity.⁵³ However, antimicrobial compounds may bind with the medium, reducing the amounts of free molecules for interaction with bacteria. Greater binding efficiency to the receptor of the target organ or test medium reduces the amount of free active drug to interact with the pathogen and will require higher doses to be effective.⁵⁴ In all, the binding affinities of the polymer monomeric units together with experimental MIC values indicate their promising antimicrobial, antibiofilm, and anti-QS potentials.

Figure 3.

3D and 2D binding interactions of PVP2 and PVP3 into the binding site of Staphylococcus aureus.

Figure 4.

3D and 2D binding interactions of PVP2 and PVP3 into the binding site of Escherichia coli.

Figure 5.

3D and 2D binding interactions of PVP2 and PVP3 into the binding site of Candida albicans.

Predicted absorption, distribution, metabolism, and excretion (ADME) and drug-likeness

The predicted ADME and drug-likeness properties of the monomeric units of PVP2 and PVP3 are summarized in Figures 6 –8, with other details in supplementary material (Tables S1–S6). Both PVP2 and PVP3 respect the rule of five as described by Lipinski. Their lipophilicity values (M_logP) are below 4.15, the threshold value (Tables S2 and S5), suggesting their suitable drug-likeness. PVP2 and PVP3 have a topological polar surface area (TPSA) out of the range of 78–125 Å². Their bioavailability scores were 0.58 and 0.55 for PVP2 and PVP3, respectively, indicating that they are likely to be absorbed orally (Table S5). The bioavailability radars of PVP2 and PVP3 are displayed in Figure 6.

Figure 6.

Bioavailability radars of PVP2 and PVP3.

Figure 7.

Boiled-egg model of PVP2 and PVP3.

Figure 8.

Predicted biological targets of PVP2 and PVP3.

The bioavailability radar analysis on the polygon reveals that PVP2 is situated within the pink zone in all considered aspects, that is, flexibility, lipophilicity, size, polarity, insolubility, and insaturation (Figure 6). PVP3 has all aspects inside the pink portion as well, except that the flexibility is out of the pink zone of the polygon (Figure 6). This suggests that the oral bioavailability of PVP2 and PVP3 units may be good. Pharmacokinetic properties show that PVP2 and PVP3 can undergo blood–brain barrier (BBB) penetration and possess high gastrointestinal (GI) absorption (Table S4). The boiled-egg model, as shown in Figure 7, indicates good BBB penetration and good GI absorption. The pie chart generated in Figure 8 indicates predicted and probable biological targets of PVP2 and PVP3. The ADME predictions and drug-likeness of PVP2 and PVP3 were evaluated with the aid of the robust SwissADME web tool. The drug-likeness, physicochemical properties, medicinal chemistry friendliness, and pharmacokinetics provide insights into whether compounds will reach the target in sufficient concentration and exhibit expected biological activity.⁵⁵ Important physicochemical properties of the monomeric units of the polymer derivative such as molecular weights, TPSA, hydrogen bond donors, and acceptors, were computed and reported. As prescribed, the monomeric units of both PVP2 and PVP3 showed acceptable properties including physicochemical parameter ranges (MW ⩽ 500, log P ⩽ 5, H-bond donors ⩽ 5, H-bond acceptors ⩽ 10), which are observed for over 90% of oral drugs that have passed preliminary phases of clinical trials.⁵⁶ TPSA reflects the totality of the polar atoms in a molecule, and a TPSA value ⩽ 140 Å represents good oral bioavailability.⁵⁷ The monomeric units of PVP2 and PVP3 had TPSA values below 140 Å. The values of physicochemical parameters described are also associated with good lipophilicity and aqueous solubility of drugs. As concerns lipophilicity, log P ⩽ 5 is required, implying that all compounds demonstrated log P values within this range (Table S2). Drug molecules are expected to be soluble so that they can be absorbed into the bloodstream and also move across cell membranes. As concerns permeation, molecules are expected to possess optimal partition coefficient (LogP) and lipophilic groups, thereby requiring a balance between lipophilicity and hydrophilicity.⁵⁸ Estimated solubility (ESOL Log S) is a model that predicts the water solubility of a compound based on its molecular structure and when it falls in the range of -4 to -2, it means that the compound is soluble, and moderately soluble when between -6 and -4.⁵⁹ This means that PVP2 and PVP3 are overall, soluble as indicated in Table S3. Table S4 indicates that the compounds have high to low GI absorption, and none of them can cross the BBB. It also predicted that the polymer derivatives can use the P-glycoprotein (P-gp) transporter as a means of absorption and excretion. No prediction of the inhibition of cytochrome or impairment of enzymes that metabolize drugs in the intestines and liver. The skin permeability (Kp) measured as log Kp (cm/s), represents the rate of penetration of the membrane by molecules, and a negative log Kp value indicates reduced permeation.⁶⁰ PVP2 and PVP3 monomeric units showed negative values of log kp indicating their poor permeation. The computed drug-likeness of the monomeric units of the polymers is presented in Table S5. PVP2 and PVP3 showed no violation of all five filters (Muegge, Lipinski, Veber, Ghose, and Egan) used in the prediction. Bioavailability describes the amount of a drug or molecule that will be available completely to its intended biological target or the fraction of the active form of a drug that reaches the circulatory system unaltered.⁶¹ The bioavailability score is often expressed as a fraction or decimal (between 0 and 1). High values (closer to 1) indicate great oral bioavailability of the molecule or drug.⁶² PVP2 has higher bioavailability compared to PVP3 (Table S5). The alerts screening of PVP2 and PVP3 are provided in Table S6, indicating the absence of PAIN and BRENK alerts and suggesting that there are no unstable, toxic, or chemically reactive structural fragments.

Conclusion

The basis for copolymerization or postpolymerization of reactive polymer precursors is to obtain variable derivatives with different functional groups, properties, and applications. In this study, commercial PVP, which is a water-soluble, biocompatible, and biodegradable polymer, was reacted with KOH, leading to ring-opening, then esterified with benzoyl chloride to afford PVP2, and finally reacted with an amine to yield PVP3. PVP2 and PVP3 inhibited the growth of C. albicans, E. coli, and S. aureus and equally showed potential antibiofilm effects against the above pathogens. The polymer derivatives were able to inhibit violacein synthesis in C. violaceum CV12472 as well as in C. violaceum CV026 in the presence of AHL, indicating that they possess anti-QS effects capable of disrupting cell-to-cell networks for coordinated behavior within bacterial colonies. PVP2 and PVP3 were able to inhibit swimming and swarming motilities in P. aeruginosa PA01, suggesting that PVP2 and PVP3 can mitigate the spread and colonization of surfaces by pathogenic bacteria. In silico evaluations predicted suitable interactions between the polymer derivatives and receptor proteins of pathogenic bacteria as well as their drug-likeness. The produced polymer derivatives are able to inhibit certain microbial virulence factors, disrupt QS-mediated processes, and inhibit biofilms, suggesting that they can be used as new antimicrobials to circumvent antibiotic resistance.

Experimental Section

Chemicals and reagents

Chemical reagents such as anhydrous KOH (⩾99.95%), 4-(dimethylamino)pyridine (DMAP, ⩾99%), diisopropylcarbodiimide (DIC, 99%), and magnesium sulfate (anhydrous MgSO₄, ⩾99.5%) were procured from Sigma-Aldrich. Solvents including dichloromethane (DCM, ⩾99.8%, Merck), N-N-dimethylformamide (DMF, ⩾99.8%, Merck), and anhydrous methanol (MeOH, ⩾99.8%, Sigma-Aldrich) of analytical grade together with triethylamine (TEA, ⩾99.5%, Merck), benzoyl chloride (⩾99%, Merck), 3-(dimethylamino)-1-propylamine (99%, Sigma-Aldrich), and PVP (MW 40000, CALBIOCHEM, USA) were used for the synthetic modifications. Luria-Bertani broth (LBA), tryptic soy broth (TSB), nutrient broth, Mueller-Hinton broth (MHB), Sabouraud dextrose broth, and agar were purchased from Merck. Ethanol (Merck), D-(+)-glucose (Merck), glacial acetic acid (Merck), and crystal violet (Merck) were used for biofilm inhibition assays. N-Hexanoyl-DL-homoserine lactone (C6-HSL, ⩾97%, Sigma-Aldrich), tryptone (Sigma-Aldrich), D-(+)-glucose (⩾99.5%, Sigma-Aldrich), kanamycin sulfate (Sigma-Aldrich), and sodium chloride (Sigma-Aldrich) were used in QSI and motility inhibition assays.

¹H NMR and FTIR characterization

The polymer derivatives were synthesized as depicted in Figure 1. ¹H NMR (CDCl₃, 400 MHz) was recorded on a Bruker Avance 400 (AV400), and FTIR spectroscopy data were measured on a PerkinElmer FTIR spectrometer (UATR Spectrum II).

Chemical synthesis

Synthesis of PVP1

KOH (2.5 g) in 100 mL of distilled H₂O was mixed with water-soluble PVP (2.5 g) dissolved in a two-neck flask, stoppered with septa, and placed under nitrogen in a Schlenk line. It was refluxed at a surface temperature of 140 °C for 15 h, cooled under tap water followed by the addition of DMF (95 mL), and then the solvents removed under vacuum at 80 °C with the aid of a rotary evaporator.⁶³ The product was dissolved in 50 mL of distilled H₂O and extracted three times with 50 mL of DCM so that the polymer dissolves and leaves behind unreacted KOH and PVP in the aqueous phase. The organic layer was dried over anhydrous MgSO₄, filtered, and DCM was evaporated on a rotary evaporator to afford PVP1 (2.3 g).

PVP1: ¹ H NMR (400 MHz, CDCl₃) δ_H ppm 5.20 (br, s), 3.40–4.00 (br, m), 3.00–3.40 (br, t), 2.20–2.52 (br, t), 1.80–2.20 (br, d), 1.45–1.80 (br, d), and 1.20–1.40 (br, s). FTIR: 3584.28, 2922.66, 1650.50 (broadband), 1591.29, 1421.79, 1286.98 (broadband), 1272.98 cm^-1.

Synthesis of PVP2

PVP1 (2 g) was dissolved in 50 mL of DMF and mixed with TEA (10 mg) and DMAP (10 mg) and stirred for 2 h at 60 °C. 2.8 g of benzoyl chloride was added dropwise from a syringe for 5 min under nitrogen and the reaction mixture was kept for 16 h. The solvent was evaporated, and the product dissolved in distilled H₂O (50 mL), and extracted with DCM three times. The resulting product was dried over anhydrous MgSO₄, filtered, and DCM was evaporated on a rotary evaporator to afford PVP2 (1.8 g).

PVP2: ¹ H NMR (400 MHz, CDCl₃): δ_H ppm 7.93–8.05 (br, m), 7.49 (t, J = 7.4 Hz), 7.35 (t, J = 7.5 Hz), 3.48–3.73 (br, m), 3.00–3.31 (br, s), 2.14–2.49 (br, s), 1.82–2.05 (br, s). FTIR: 2955.02, 2779.37, 1707.62, 1630.94 (broadband), 1602.17, 1583.52, 1450.66, 1421.88, 1250.10 (large band), 1174.16, 709.18 cm^-1.

Synthesis of PVP3

PVP2 (1 g) in a flask was dissolved in DMF (40 mL), followed by the addition of a few drops of dilute HCl. 3-(Dimethylamino)-1-propylamine (4 mL) was introduced in the presence of DIC (200 µL) and reacted at 80 °C for 12 h. 20 mL of distilled H₂O was introduced to the resulting reaction mixture and evaporated to dryness. The crude product was dissolved in distilled H₂O (50 mL) and reextracted with DCM three times. The resulting product was dried over anhydrous MgSO₄, filtered, and DCM was evaporated on a rotary evaporator to afford PVP3 (700 mg).

PVP3: ¹ H NMR (400 MHz, CDCl₃): δ_H ppm 8.50 (s), 7.89–7.89 (m), 7.40–7.70 (br, s), 7.27–7.39 (m), 2.99–3.27 (m), 2.84–2.97 (m), 1.95–2.01 (m), 1.58–1.74 (m), 1.43–1.56 (m), 1.24–1.40 (m), 0.90–1.20 (m). FTIR: 2937.97, 2850.15 (broadband), 2209.17, 1661.30 (broadband), 1635.11, 1591.52, 1520.36, 1453.30, 1380.50 (broadband), 1069.51, 1043.01, 714.74 cm^-1.

Strains of pathogenic microbes

The following microorganisms were used: Candida albicans ATCC 10239, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa PA01, Escherichia coli ATCC 25922, Chromobacterium violaceum CV026, and Chromobacterium violaceum CV12472.

Measurement of MIC

Antimicrobial activity was expressed as MIC measured using the 96-well broth dilution method.⁶⁴ The MIC was considered as the lowest concentration of polymer derivatives at which no visible growth was observed. Fresh overnight cultures at 5×10⁵ colony-forming units (CFU)/mL and MHB medium were employed. Microbial cultures were inoculated in microtiter plate wells in the presence of polymer derivatives at varying final concentrations (5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953, 0.009765 mg/mL). The microplates were incubated for 24 h at 37 ºC and read for MIC determination.

Biofilm inhibition measurement

The effect of the polymers at MIC and sub-MIC concentrations on biofilm formation against test microorganisms was determined by the microplate biofilm assay.³² Overnight fresh bacteria cultures were seeded in TSB supplemented with glucose (0.25%) in the absence or presence of polymer derivatives at 37 ºC for 48 h. Wells without polymer derivatives (TSB+cells only) served as controls. Wells were rinsed with water to remove planktonic bacteria after incubation and stained for 10 min with 0.1% crystal violet solution. After removing the crystal violet, wells were filled with 200 μL of ethanol (for Gram-negative and Candida) or 33% glacial acetic acid (for Gram-positive) to dissolve the stained biofilms. 125 μL from each well was pipetted and transferred to sterile tubes, and the volume was adjusted to 1 mL with distilled water. Optical densities (ODs) were recorded at 550 nm (Thermo Scientific Multiskan FC, Vantaa, Finland). Percentage biofilm inhibition was deduced using the formula:

\begin{array}{l} Biofilm inhibition (%) \\ = \frac{O D 550_{C o n t r o l} - O D 550_{S a m p l e}}{O D 550_{C o n t r o l}} x 100 \end{array}

QSI against C. violaceum CV026

Determination of QSI was performed as described previously with little changes.³² 100 µL of overnight culture was added to 5 mL of warm molten Soft Top Agar (200 mL deionized H₂O, 1.3 g agar, 1.0 g NaCl, 2.0 g tryptone). 10 µL of kanamycin sulfate and 20 µL of C₆HSL AHL prepared at 100 µg/mL were introduced. The mixture was poured as an overlay on solidified LBA plates, and 5 mm diameter wells were made. Each well was filled with 50 µL of MIC and sub-MIC filter-sterilized polymer derivatives and incubated at 30°C for 3 days. A white or cream-colored halo around wells against a purple lawn of activated CV026 bacteria was an indication of QSI. Each experiment was done in triplicate, and the diameters of the QSI zones were measured.

Inhibition of violacein production against C. violaceum CV12472

The polymer derivatives were analyzed for their violacein inhibition potential against C. violaceum ATCC 12472.³² 10 µL of CV12472 overnight culture was added to 180 µL of LB broth in wells of sterile microtiter plates and incubated in the absence or presence of 20 µL of MIC and sub-MIC polymer derivatives. Wells without polymer derivatives served as controls. All plates were incubated for 24 h at 30°C and measured for reduction in violacein pigment amounts by determining absorbances at 585 nm. The inhibition of violacein percentage was deduced by the following formula:

\begin{array}{l} Violacein inhibition (%) \\ = \frac{O D 585 c o n t r o l - O D 585 s a m p l e}{O D 585 c o n t r o l} x 100 \end{array}

Motility inhibition against Pseudomonas aeruginosa PA01

Inhibition of swimming and swarming motilities was evaluated as described elsewhere with minor changes.^32,64 Swarming plates (0.5% of filter-sterilized D-glucose, 1% peptone, 0.5% agar, 0.5% NaCl) containing polymer derivatives (MIC, ½ MIC, ¼ MIC) were prepared and 5 µL of P. aeruginosa PA01 overnight cultures were point-inoculated at the center of the plates. Plates were incubated for 18 h in an upright position. The diameters of areas covered by growing swarm fronts were measured and used in calculating swarming inhibition. The same procedure was used for swimming motility except that swim plates were prepared differently (0.5% of filter-sterilized D-glucose, 1% peptone, 1.5% agar, 0.5% NaCl).

Molecular docking study

The binding affinities of the monomeric repeating units of PVP2 and PVP3 into the binding sites of Candida albicans, Escherichia coli, and Staphylococcus aureus were predicted with the aid of Autodock4 package.⁶⁵ Targeted E. coli, S. aureus, and C. albicans X-ray coordinates and their corresponding originally docked ligands were downloaded from the RCSB data bank website of PDB codes 1JIJ, 5FGN, and 1EAG, respectively.^66,67 Structural geometries of monomeric units of PVP2 and PVP3 were minimized using Merck molecular force field 94 (MMFF94) level44, and they were saved as PDB files. The molecular docking stepwise procedure can be found in our previously reported studies.⁶⁸ The binding interactions of the docked monomeric unit of PVP2 and PVP3 into the binding sites of E. coli, S. aureus, and C. albicans were visualized using the Discovery Studio Client (Discovery Studio Client is a product of Accelrys Inc., San Diego, CA, USA).

ADME and drug-likeness properties

The predictions of ADME, drug-likeness, pharmacokinetics, and physicochemical properties of the monomeric units of PVP2 and PVP3 were carried out using the Swiss ADME tool available at the https://www.swissadme.ch/ website. Further, the Swiss target prediction tool (https://www.swisstargetprediction.ch/) was employed to predict the probable targets of PVP2 and PVP3.

Statistical analysis

All experiments were repeated three times and results are mean values ± standard error of the means. The statistical differences between the test samples were analyzed through one-way ANOVA. Differences were considered statistically significant where p<0.05.

Supplemental Material

sj-docx-1-chl-10.1177_17475198251409909 – Supplemental material for Postpolymerization modification of polyvinylpyrrolidone and evaluation of anti-quorum-sensing, antimicrobial, and antibiofilm activities with molecular docking and absorption, distribution, metabolism, and excretion studies

Supplemental material, sj-docx-1-chl-10.1177_17475198251409909 for Postpolymerization modification of polyvinylpyrrolidone and evaluation of anti-quorum-sensing, antimicrobial, and antibiofilm activities with molecular docking and absorption, distribution, metabolism, and excretion studies by Alfred Ngenge Tamfu, Selahattin Bozkurt, Sameh Boudiba, Mehmet Kayhan, Ozgur Ceylan and El Hassane Anouar in Journal of Chemical Research

Footnotes

Acknowledgements

The Scientific and Technological Research Council of Turkiye (TUBITAK) is greatly acknowledged by A.N. Tamfu and S. Bozkurt for the grant (project code: 1059B212200092). The authors are grateful to the participating institutions, which are Usak University, Mugla Sitki Kocman University, Prince Sattam bin Abdulaziz University, and the University of Ngaoundere.

ORCID iDs

Alfred Ngenge Tamfu

Sameh Boudiba

Ethical Considerations

Ethical approval is not applicable for the paper.

Statement of informed consent

There are no human subjects in this paper, and informed consent is not applicable.

Consent to Participate

The authors confirm that all of them participated in the research work of this paper.

Consent for Publication

The authors confirm that they give their full consent for publishing this paper.

Author contribution

Alfred Ngenge Tamfu: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Resources, Software, Visualization, Writing—original draft, Writing—review & editing. Selahattin Bozkurt: Conceptualization, Formal analysis, Data curation, Supervision, Resources, Visualization, Writing—original draft, Writing—review & editing. Sameh Boudiba: Formal analysis, Data curation, Writing—original draft. Mehmet Kayhan: Formal analysis, Data curation, Visualization, Writing-original draft. Ozgur Ceylan: Methodology, Formal analysis, Data curation, Supervision, Resources, Visualization. El Hassane Anouar: Investigation, Formal analysis, Data curation, Resources, Software, Visualization, Writing—original draft, Writing—review & editing. In addition, Alfred Ngenge Tamfu and Ozgur Ceylan performed the biological assays. All authors read, corrected, and approved the final copy.

Funding

The authors received no financial support for the research, authorship, and/or publication of this paper.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this paper.

Data Availability Statement

The data generated in this study are available within the paper and its supplementary data files or upon request from the corresponding author.

Supplemental material

Supplemental material for this paper is available online.

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Supplementary Material

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Postpolymerization modification of polyvinylpyrrolidone and evaluation of anti-quorum-sensing,antimicrobial,and antibiofilm activities with molecular docking and absorption,distribution,metabolism,and excretion studies

Abstract

Keywords

Introduction

Results and discussion

QS and violacein production inhibition effects

Inhibition of swarming and swimming motilities

Biofilm inhibition and antimicrobial activity

Molecular docking results

Predicted absorption, distribution, metabolism, and excretion (ADME) and drug-likeness

Conclusion

Experimental Section

Chemicals and reagents

1H NMR and FTIR characterization

Chemical synthesis

Synthesis of PVP1

Synthesis of PVP2

Synthesis of PVP3

Strains of pathogenic microbes

Measurement of MIC

Biofilm inhibition measurement

QSI against C. violaceum CV026

Inhibition of violacein production against C. violaceum CV12472

Motility inhibition against Pseudomonas aeruginosa PA01

Molecular docking study

ADME and drug-likeness properties

Statistical analysis

Supplemental Material

sj-docx-1-chl-10.1177_17475198251409909 – Supplemental material for Postpolymerization modification of polyvinylpyrrolidone and evaluation of anti-quorum-sensing, antimicrobial, and antibiofilm activities with molecular docking and absorption, distribution, metabolism, and excretion studies

Footnotes

Acknowledgements

ORCID iDs

Ethical Considerations

Statement of informed consent

Consent to Participate

Consent for Publication

Author contribution

Funding

Declaration of conflicting interests

Data Availability Statement

Supplemental material

References

Supplementary Material

¹H NMR and FTIR characterization