Therapeutic Applications of Self-assembled Indole-3-butanoyl-polyethylenimine Nanostructures

Abstract

This study demonstrates the therapeutic potential of indole-3-butanoyl-polyethylenimine (IBP) nanostructures formed via self-assembly in aqueous system. Dynamic light scattering (DLS) analysis confirmed the formation of the nanostructures in the size range of ~ 194–331 nm. These nanostructures showed commendable antimicrobial activity against wide range of microbes including multi-drug resistant bacteria. Besides, appreciable antioxidant and anti-inflammatory activities were also observed. Results of cytotoxicity studies, performed on normal transformed human embryonic kidney (HEK 293) cells and human red blood cells (hRBCs), revealed almost non-toxic behavior of these nanostructures, however, remarkable toxicity on human breast cancer cells (MCF-7), human osteosarcoma cells (Mg63) and human liver cancer cells (HepG2) was observed. The pre-apoptotic and anti-proliferative activity of IBP nanostructures were confirmed by acridine orange/propidium iodide dual staining assay followed by confocal microscopy and scratch assay on Mg63 cells. Taken together, these results advocate the promising potential of the synthesized IBP nanostructures in the therapeutic applications.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01015-y.

Introduction

Tremendous advancements have been shown in area of nanotechnology for their potential biotechnology applications [1–5]. Especially, cationic polymers are playing an important role as multifunctional materials [6, 7]. These have not only acted as efficient carriers by complexing with oppositely charged molecules (i.e. nucleic acids) but also exhibited antioxidant, anti-inflammatory, antimicrobial, and antitumor activities [8, 9]. Besides, reactive functionalities, present in their backbone, make them amenable to modifications for further yielding therapeutically important biomaterials. Polyethylenimines (PEIs) are one of the most extensively used class of polymers with a wide variety of applications [10, 11]. Due to high cationic charge density, the higher molecular weight polymers display high toxicity and low hemocompatibility, which have limited their biological applications [12]. Polyethylenimines also exhibit inherent antimicrobial activity due to their ability to interact with the cell wall electrostatically and permeabilize it [13]. Currently, pathogenic or microbial infections, a major cause of morbidity and mortality, have become a serious global concern [14–17]. Further, the increasing concerns about biofilm-associated infections and multidrug resistant bacterial strains demand development of new bacteriostatic or bactericidal agents. To address these issues, cationic polymeric materials, associated with antimicrobial properties, have been utilized as potential candidates in biomedical and other fields. Although the exact mechanism of their action is yet to be fully understood, the possible one could be via contact-killing by inducing permeability in the bacterial membrane followed by disruption in the cytoplasmic membrane or blocking of the transport of the nutrients leading to cell lysis [18]. By taking the advantage of PEI as a permeabilizer, uptake of the antibiotics has been enhanced with concomitant reduction in the minimum inhibitory concentrations (MICs) [19]. Modifications in PEIs, viz., alkylation, quaternization, tethering of therapeutic ligands, etc., have also resulted in an improvement in the antimicrobial action [20, 21]. Infection-mediated compromised immune system leads to development of other diseases including cancers [22]. Recently, microbial infections along with cancers have become the leading cause of mortality worldwide [23–26]. Therefore, development of newer multifunctional therapeutics that can take care of multiple disease conditions is the need of the hour. Also, the synthesis of materials or bio-composites has proven to be beneficial for human health and environmental applications [27–31].

Here, in the present study, we have explored the potential of amphiphilic derivatives of non-toxic polyethylenimine (10 kDa) after conjugating it with varying amounts of indole-3-butyric acid, which is basically a plant hormone that plays an important role in the development of a plant. Being hydrophobic in nature, it incorporated amphiphilicity in the hydrophilic polymer, here, hydrophilic polyethylenimine. The resulting indole-3-butanoyl-polyethylenimine (IBP) polymers, on self-assembly in an aqueous medium, generated IBP nanostructures with positively charged hydrophilic polyethylenimine on their surface and hydrophobic indole-3-butanoyl moieties residing inside the core. The amphiphilic IBP polymeric nanostructures, mimicking the design of antimicrobial peptides, were then evaluated for their activity against microbial infections including resistant clinical and Cutibacterium acnes (C. acnes) isolates. Besides, anticancer activity on mammalian cancer cells, antioxidant and anti-inflammatory activity were also examined.

Materials and Methods

Synthesis and Self-assembly of Indole-3-butanoyl-polyethylenimine (IBP) Polymers

A series of IBP polymers was obtained by the reaction of PEI (10 kDa) with varying amounts of indole-3-butanoic acid (IBA) in the presence of a condensing reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC), following the previously reported procedure [19]. Briefly, an aqueous solution of PEI (10 kDa, 2.0 mmol, dissolved in 10 ml of dd water) was mixed with a solution of indole-3-butyric acid (0.05 mmol, dissolved in 2 ml of THF, for 2.5% substitution). Then, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, 0.06 mmol) was added and the reaction mixture was stirred overnight at room temperature. Subsequently, the reaction mixture was concentrated in vacuo to obtain a syrupy residue, which was taken up in dd water and subjected to dialysis (MWCO 500 Da). The dialyzed solution was lyophilized to get IBP-1 polymer in ~ 78% yield. Similarly, IBP-2, IBP-3 and IBP-4 polymers with 5, 7.5 and 10% substitution of IBA were obtained in ~ 75–87% yield. These were characterized by ¹H-NMR and FTIR. Degree of substitution was also determined by the previously described method [19]. Self-assembly of IBP polymers was achieved following the reported procedure [19]. Typically, IBP-1 polymer (~ 5.0 mg) was taken up in methanol (1 ml) and dd water (9 ml) was added dropwise with continuous vortexing. After 2–3 h, the solution was lyophilized to get a syrupy residue, which was again dispersed in dd water by vortexing for 10–15 min. The resulting solution was used in all the experiments.

Results and Discussion

In the present study, a series of amphiphilic indole-3-butanoyl-polyethylenimine (IBP) polymers has been synthesized using branched polyethylenimine (bPEI, 10 kDa) and variable amounts of indole-3-butanoic acid (IBA) (Fig. 1) [19]. One of the conjugated polymers, viz., IBP-4, was characterized by FTIR. The band at 1630 cm⁻¹ due to amide stretching confirmed the formation of covalently linked conjugate. C=C stretching band at 1580 cm⁻¹ showed the presence of indole ring in IBP polymers. Other prominent bands at 2350, 2800, 3048 3237–3400 cm⁻¹ due to C–C, C–N and N–H stretching and vibrational stretching further confirmed the formation of IBP polymers. The series of compounds, IBP-1 to IBP-4, was then subjected to determination of the degree of substitution of IBA onto PEI by the standard 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. The substitution of IBA onto PEI was found to 2.5% (1.9%), 5% (3.4%), 7.5% (5.1%) and 10% (7.2%); values in the parenthesis corresponded to the actual percent substitution. These results revealed that IBA conjugation onto PEI ranged from ~ 68 to 76% of the attempted substitution (Table S1). By taking the advantage of the degree of amphiphilicity due to incorporation of IBA residues in IBP polymers, their ability to self-assemble in aqueous medium was explored. On self-assembly, these polymers formed micellar nanostructures of core–shell type with hydrophilic outer surface (positively charged polyethylenimine residues) and hydrophobic inner core (indole-3-butyric acid residues).

Fig. 1.

Preparation of indole-3-butanoyl-polyethylenimine polymers

Particle Size and Zeta Potential Measurements

The series of IBP nanostructures was analyzed for their size and zeta potential by Dynamic Light Scattering (DLS). Average size (hydrodynamic diameter) of IBP nanostructures was determined in dd H₂O which was found to be in the range of ~ 331–194 nm (Table S1). Size of self-assembled IBP nanostructures decreased on increasing the substitution of IBA onto PEI, i.e. an increase in the hydrophobicity resulted in a concomitant decrease in the size of the nanostructures which suggested more compactness in the resulted structures due to enhanced hydrophobic interactions among aromatic indole moieties. Similar trend was observed in zeta potential measurements which revealed a decrease in surface charge down the series from IBP-1 to IBP-4 (Table S1). This could be attributed to the capping of amine functionalities (reduction in amine group density) due to coupling of IBA on to PEI [5, 19]. Overall charge density on the formed micellar nanostructures remained positive.

DPPH Assay

Antioxidant/free radical scavenging activity of IBP nanostructures was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, which is considered as a sensitive protocol. Compounds with antioxidant activity react with DPPH and convert it into a stable 2,2-diphenyl-1-picrylhydrazine of colorless to light yellow colored compound. Extent of change in color from violet to light yellow in the presence of IBP nanostructures indicated the potential of these nanostructures as antioxidant. All the nanostructures showed higher antioxidant potential than the native PEI in a concentration dependent manner (Fig. S1). Antioxidant potential increased from IBP-1 to IBP-4 nanostructures, which could be attributed to an increase in the lipophilicity down the series owing to incorporation of indole-3-butanoyl residues. The results were in complete agreement with the previous study [32]. The presence of abundant N–H protons as well as the existence of indole ring in the synthesized nanostructures could be among the factors for enhanced antioxidant activity of IBP nanostructures. As compared to the standard, ascorbic acid, which exhibited ~ 99% antioxidant activity at 200 µg, IBP-4 nanostructures showed ~ 80% activity at the same concentration.

Hemolytic Assay

In order to ascertain the safety of the synthesized IBP nanostructures on humans, hemolytic assay was performed on human red blood cells (hRBCs). The study was carried out at different concentrations of 50, 100, 150 and 200 µg/well. With increasing concentration of the synthesized IBP nanostructures including MIC and IC₅₀ values, the hemolysis was found to be less than ~ 30%. Considering the MIC value and IC₅₀ values, toxicity was found to be negligible or very less. Even at higher concentration (i.e. 200 µg), the hemolysis was less than 40% (Fig. S2). These results revealed that the projected IBP nanostructures were almost non-toxic and could be used safely on the human cells.

Anti-inflammatory Activity

Endorsement of bovine serum albumin—protein denaturation assay for the in vitro estimation of anti-inflammatory activity of IBP nanostructures bypasses the ethical issues linked with the utilization of the animals. However, denaturation of a protein has been reported as a pathological condition including loss of proteins’ native structure followed by loss of activity/functionality of the protein. Hence, BSA protein denaturation assay has become an ideal method for elucidation of the anti-inflammatory activity. From Fig. S3, it was observed that as compared to native PEI, IBP nanostructures showed increasing inhibition of protein denaturation which is proportional to the increasing substitution of indole-3-butyric acid on PEI. Aspirin was taken as positive control. This anti-inflammatory activity could be owing to the presence of indole moiety and abundant amine groups, which has also been reported in the literature and the results are in complete agreement [33, 34].

Antimicrobial Activity

Antimicrobial action of IBP nanostructures was investigated on clinical (i.e. PA and EC) and resistant strains (MDR-PA and MRSA, as well as resistant acne strains, SE and KP). Zone of inhibition (ZOI) assay for IBP nanostructures on these strains revealed the range of inhibition in the order of 10.0 ± 1.01–15.5 ± 1.09 mm (Table S2). As the substitution of indole-3-butanoyl moiety increased from IBP-1 to IBP-4, there was a gradual increase in the inhibition zone. The lowest ZOI was observed in native PEI, which was found to be totally ineffective against resistant acne strains. The highest ZOI was displayed by IBP-4 nanostructures against all the pathogens.

Further, the minimum inhibitory concentration (MIC), the least concentration at which IBP nanostructures demonstrated their antibacterial action, was determined. In the present study, methicillin, the antibiotic control, showed the least MIC on clinical strains, EC and PA, however, exhibited significantly higher MICs against resistant MDR-PA, MRSA and acne strains, SE and KP. Similarly, native PEI displayed higher MICs against all the strains. On the other hand, IBP nanostructures exhibited MICs in the range of 50–150 µg/ml against all the strains (Table 1). MICs of 68–100 µg/ml displayed by IBP nanostructures against clinical isolates were higher than the control, methicillin, however, against resistant strains, the MICs were significantly lower than the native PEI and methicillin. Amongst IBP nanostructures, IBP-4 exhibited the least MICs (50–120 µg/ml) against all the pathogens (Table 1). The promising antibacterial action of IBP-4 nanostructures against all the resistant bacteria might be due to disruption of cellular integrity after efficient interaction with the bacterial cellular structures leading to inhibited growth.

Table 1.

Determination of minimum inhibitory concentration (μg/ml) of IBP nanostructures, native bPEI and antibiotic control (methicillin) against various clinical and multi-drug resistant strains

Samples	Minimum inhibitory concentration (μg/ml)
	Clinical strains		Resistant strains		Resistant acne strains
	EC	PA	MDR-PA	MRSA	SE	KP
IBP-1	100	70	90	75	80	150
IBP-2	99	70	90	73	80	140
IBP-3	99	69	89	72	60	130
IBP-4	97	68	85	70	50	120
bPEI10	> 170	> 100	> 100	> 120	> 100	> 180
Methicillin	~ 10	~ 1	> 120	> 250	> 290	> 300

Further, the effect of IBP nanostructures on the growth dynamics of various clinical and resistant strains was monitored by microbroth dilution assay (MDA). Figure 2 depicts the results of growth kinetics of all the strains in the presence of nanostructures and controls (native PEI and methicillin, 30 μg/ml). The assay was carried out at the minimum inhibitory concentration of IBP nanostructures, bPEI and methicillin. Consistently lower growth was observed for all IBP nanostructures treated bacteria at different time points (i.e. 0, 4, 8 and 12 h) representing appreciable antibacterial trait of the designed nanostructures.

Fig. 2.

Growth kinetics profiles of IBP nanostructures, PEI and methicillin (control) treated clinical and resistant strains. Strains used: EC, PA, MRSA, MDR-PA, KP and SE

Colony forming unit (CFU) assay results are depicted in Fig. S4 which were obtained after treatment of Staphylococcus epidermis strain with IBP-4 nanostructures. No active growth of the bacteria was observed which suggested efficient bacterial growth inhibition and potential antimicrobial activity of the designed nanostructures. Amphiphilic character of the nanostructures resembling the antimicrobial peptides might be the major factor for exhibiting the antimicrobial action that might have facilitated efficient interactions with the bacterial membrane followed by its disruption leading to cell death.

Anti-cancer Activity

Carcinogenic cells (Mg63, MCF-7, HepG2) and non-carcinogenic cells (HEK 293) were screened against IBP nanostructures for analyzing their cancer-specific toxicity profiles. Post-treatment with IBP nanostructures for 24 h at different concentrations (ranging from 5 to 80 μg/well), cells were incubated after adding MTT solution 2 h. Subsequently, solubilizing agent was added and the absorbance at 540 nm was recorded.

From the cell viability data, the anti-cancer activity of IBP nanostructures was established. The highest anti-cancer activity was observed in Mg63 cells followed by HepG2 and MCF-7 cells (Mg63 > HepG2 > MCF-7) (Fig. S5). The highest IC₅₀ values were obtained for MCF-7 cells and the lowest for Mg63 cells post-treatment with IBP nanostructures. Similarly, IC₅₀ values were also calculated post-screening on non-carcinogenic cells (HEK 293) to demonstrate cancer-specific toxicity induction by IBP nanostructures and found that the values were the highest among all the cells (Table S3). After evaluation of IC₅₀ value profiles on both cancerous and non-cancerous cells, it was confirmed that at lower concentration, the projected nanostructures conferred toxicity in cancerous cells, whereas normal cells were the least affected at that concentration, which ensured cancer-specific toxicity response of IBP nanostructures. However, post-screening of IBP-treated HEK 293 cells, IC₅₀ values increased corresponding to increasing substitution of IBA onto PEI, whereas, in case of carcinogenic cells, inverse relation was obtained, in which IC₅₀ values declined with increasing substitution. This might be attributed to the fact that the substitution of IBA onto PEI reduced the surface charge and charge-induced toxicity chiefly caused by the presence of abundant primary amine groups leading to an increase in the viability of non-cancerous cells (HEK 293) even at higher concentration. However, on the other hand, increased percent substitution of IBA onto PEI was found to be directly proportional to the toxicity in cancerous cells, which might be due to the presence of indole moiety in the nanostructures. The results are in agreement to the previous studies which have reported anti-cancer activity of indole derivatives. In these studies, indole derivatives have been shown to exhibit anti-cancer activity by inducing apoptosis or acting as inhibitors for protein kinases, DNA topoisomerases, microtubules, etc. [35–38].

In an attempt to further establish the anti-cancer effect of IBP nanostructures, dual staining assay was carried out i.e. acridine orange (AO)/propidium iodide (PI) assay. Mg63 cells were treated with IBP nanostructures at IC₅₀ values and allowed for 24 h of incubation at 37 °C under 5% CO₂ conditions. After AO/PI treatment, images captured are depicted in Fig. S6 (a–e). It was noted that appearance of red stained cells gradually increased with increasing substitution of IBA onto PEI (i.e. IBP-1 to IBP-4 nanostructures) and there was a concomitant reduction in green stained cells. As green fluorescence signifies live cells and red reflects dead cells, further quantitative evaluation was carried out using image-J software. Collected total cell fluorescence (CTCF) values for both live and dead cells were calculated and shown in Fig. S6, f. The results of these studies were found to be in agreement with IC₅₀ values suggesting the induction of cancer specific toxicity by IBP nanostructures.

Morphological Analysis Using Confocal Microscopy

Post-examination of IC₅₀ and CTCF values, IBP nanostructures were further explored to investigate the morphological alterations involved during cancer cell death after treatment, in which the labeling of IBP nanostructures was carried out using fluorescein isothiocynate (FITC). After treatment of Mg63 cells with FL-IBP-4 nanostructures at the corresponding IC₅₀ value followed by staining with DAPI, confocal images were captured and analyzed. The results, depicted in Fig. 3, clearly represent nuclear condensation and blebbing as indicated by arrows. Treated cells were observed with cell membrane damage. Overall damaged nucleus with condensed genetic material, followed by nuclear blebbing and cell membrane disorientation indicated the phenomenon of apoptotic cell death or programmed cell death. Efficient anti-cancer activity of IBP nanostructures was observed due to the presence of indole-3-butanoyl moiety. The alkyl chain (butanoyl) inhibits histone deacetylase (HDAC) enzyme and ultimately over acetylation of DNA results into growth inhibition followed by apoptotic cell death [39, 40]. It has been reported in the literature too that cell death is facilitated by action of caspases which stimulate pro-apoptotic molecules and induce tumor suppressor function [41, 42].

Fig. 3.

Confocal images of FL-IBP-4 treated Mg63 cells post-staining with DAPI (counter nuclear stain). White arrows indicate nuclear condensation followed by nuclear membrane blebbing (red arrows)

In Vitro Anti-proliferative Assay

The anti-proliferative activity of the projected IBP nanostructures was demonstrated on Mg63 cells. The cells were treated with IBP nanostructures at their IC₅₀ values. For determination of anti-proliferative activity using scratch assay, cell migration was carried out in response to the mechanical scratch which was formed both in the absence and presence of the nanostructures. It was observed that the anti-proliferative activity followed the trend IBP-4 > IBP-3 > IBP-2 > IBP-1 (cell migration and proliferation decreased with increasing substitution of IBA on to PEI) (Fig. S7). These results provide supportive conclusion for anticancer activity of IBP nanostructures [43].

Conclusion

Here, in this study, multifunctional activity of IBP micellar nanostructures has been explored as an alternative against multidrug resistant microbial infections. The synthesized nanostructures exhibited magnificent antimicrobial activity against differential pathogenic microorganisms. Besides, synthesized nanostructures also exhibited noteworthy anti-cancerous activity against mammalian cancer cells. When tested upon normal HEK 293 cells and hRBCs, the projected nanostructures showed almost non-toxic behavior. The nanostructures also showed good antioxidant and anti-inflammatory activity. Current study beholds the prospect that the projected nanostructures can play their role in biomedical applications as novel anti-cancer and antimicrobial agents.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ Contribution

RS and DJ, Performed research, analyzed data and wrote paper; HKG and UD, editing and data analysis; PK, designing, editing and supervising.

Declarations

Conflict of interest

Authors do not have any conflict of interest.