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. 2020 Oct 20;5(43):28375–28381. doi: 10.1021/acsomega.0c04395

Amphiphilic Small-Molecule Assemblies to Enhance the Solubility and Stability of Hydrophobic Drugs

Yogesh M Gangarde †, Sajeev T K ‡, Nihar R Panigrahi †, Ram K Mishra ‡, Ishu Saraogi †,‡,*
PMCID: PMC7643322  PMID: 33163821

Abstract

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Amphiphilic assemblies made from diverse synthetic building blocks are well known for their biomedical applications. Here, we report the synthesis of gemini-type amphiphilic molecules that form stable assemblies in water. The assembly property of molecule M2 in aqueous solutions was first inferred from peak broadening observed in the proton NMR spectrum. This was supported by dynamic light scattering and transmission electron microscopy analysis. The assembly formed from M2 (M2agg) was used to solubilize the hydrophobic drugs curcumin and doxorubicin at physiological pH. M2agg was able to effectively solubilize curcumin as well as protect it from degradation under UV irradiation. Upon solubilization in M2agg, curcumin showed excellent cell permeability and higher toxicity to cancer cells over normal cells, probably because of enhanced cellular uptake and increased stability. M2agg also showed pH-dependent release of doxorubicin, resulting in controlled toxicity on cancer cell lines, making it a promising candidate for the selective delivery of drugs to cancer cells.

Introduction

Living systems are composed of a variety of small amphiphilic building blocks that polymerize to form highly ordered assemblies, for example, DNA, RNA, and proteins. These polymers are responsible for performing diverse functions in the cell.1,2 Inspired from biological macromolecules, researchers have developed a variety of functional synthetic molecules based on amphiphilic building blocks.3−6 The design of such molecules mainly involves a linear hydrophobic tail tethered to ionic head groups that can form micellar assemblies in aqueous medium.7−9 Such assemblies are used in various applications such as surfactants, vesicles, and as transporters of biological cargo.10−15 Several new classes of amphiphiles have been reported in an attempt to increase the stability and efficiency of the amphiphilic assemblies.16,17 It is well reported that assemblies from dimeric amphiphiles are generated more readily with a lower critical micelle concentration (cmc) and higher stability in aqueous medium than its monomeric counterparts.18−20 Assemblies formed from amphiphiles with an aromatic core have also been reported. These amphiphiles assemble readily through π–π stacking interactions with a relatively low cmc.12,21,22

Here, we have synthesized two small amphiphilic molecules based on a benzamide scaffold, decorated with hydrophobic N-alkyl groups (iso-pentyl or decyl) on one face and hydroxyl groups on another face (Figure 1). A terminal carboxylic acid group ensures the aqueous solubility of the molecules. One of the molecules (M2) showed strong assembly formation in aqueous medium probably because of a combination of π-stacking, hydrophobic and hydrogen bonding interactions. The assembly formed from M2 (M2agg) was utilized to encapsulate the water-insoluble drug curcumin and to enhance its stability under UV irradiation. M2agg also efficiently encapsulated the anticancer agent doxorubicin and allowed sustained release of doxorubicin from the assembly at physiological pH.

Figure 1.

Figure 1

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Schematic representation of M2-assisted enhanced encapsulation and controlled release of hydrophobic molecules in water.

Results and Discussion

Synthesis and Characterization of Molecules and Their Assemblies

The molecules M1 and M2 (Figure 2A) were synthesized from 3-hydroxy 4-nitro benzoic acid using reported protocols with some modifications (described in detail in the Supporting Information).

Figure 2.

Figure 2

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(A) Structure of the molecules used in this study. (B) NMR spectrum of M1 (∼25 mM) in DMSO-d6 and D2O at 298 K. (C) NMR spectrum of M2 (∼25 mM) in DMSO-d6 at 298 K (bottom) and variable temperature NMR of M2 (∼25 mM) in D2O. (D) DLS analysis of M1 and M2 aggregates (1 mM) in water. (E,F) TEM images of M1 and M2 aggregates (500 μM in water), respectively.

The 1H NMR spectra of both molecules in DMSO-d6 showed sharp and well-resolved proton resonances (Figure 2B,C). However, the 1H NMR spectrum of M2 in D2O showed broad proton resonances, probably because of assembly formation by M2, which exhibited slow rotational dynamics on the NMR timescale. This effect was much less pronounced for M1 having shorter side chains, indicating that the decyl chains in M2 strongly promoted assembly formation.

We next performed variable temperature NMR studies to study the assembly behavior of M2 at elevated temperatures. A sharpening of the NMR signals was observed as the temperature was gradually increased. This suggests partial dissociation and faster dynamics of the assembly, indicating noncovalent assembly formation by M2. Nonetheless, signal broadening still persisted at elevated temperatures (353 K) in D2O, indicating a relatively high thermal stability of the assembly (Figure 2C). The size of the assembly in aqueous medium was determined by dynamic light scattering (DLS) analysis. The average hydrodynamic diameter of the assembly was found to be 200 ± 88.5 nm with a polydispersity index (PDI) of 0.230 for M1 and 140.9 ± 106.7 nm with a PDI of 0.263 for M2 (Figure 2D). When the experiment was repeated in an aqueous solution containing 100 mM NaCl, assembly formation was still observed, indicating that electrostatic interactions did not play a major role in assembly formation (Figure S1).

The morphology and nature of the assembly were assessed by TEM, which showed uniform circular assemblies probably because of favorable hydrophobic interactions in water (Figures 2E,F and S2). The critical aggregation concentrations (CACs) of M1 and M2 were found to be 180 and 140 μM respectively, from fluorescence quenching at 370 nm as a result of aggregate formation (Figure S3). As the assemblies probably formed via hydrophobic interactions, we asked whether assembly formation would be affected in the presence of a nonionic surfactant Triton X-100 (0.05%).23,24 TEM images of the samples treated with Triton X-100 showed small and sparsely dispersed particles on the grid (Figure S4). The inability of M1 and M2 to form assemblies in the presence of Triton X-100 indicated that hydrophobic interactions played a major role in assembly formation.

The peak broadening in the NMR spectrum, hydrodynamic diameter measurement, and morphology analyses suggest that molecules M1 and M2 form submicrometer sized circular assemblies in aqueous medium.

Curcumin Encapsulation

Curcumin is a naturally occurring polyphenolic compound with a broad spectrum of pharmacological effects ranging from anti-inflammatory to anticancer activities. Although curcumin is readily available and has a high biosafety profile, it is not a successful clinical candidate because of poor water solubility, low stability at neutral pH, rapid degradation rate, and fast metabolism in the biological milieu.25,26

To address these problems, several nanomaterial-based drug delivery systems have been developed, many of which utilize additional triggers to release curcumin.7,14,15,27−31 Coordination-based self-assemblies of small organic ligands have shown enhanced solubility and stability of curcumin but suffer from biocompatibility issues because of metal toxicity.32 We tested the ability of our amphiphilic assemblies to solubilize curcumin. Curcumin powder was added to an aqueous solution of M1 or M2 (1 mM in PBS) and stirred at room temperature (rt) to facilitate assembly formation (Figure 3A). Unbound curcumin was removed by centrifugation and the absorbance of the curcumin-assembly solution was measured at 427 nm (Figure 3B). High encapsulation of curcumin (>25-fold) compared to PBS was observed in M2agg (∼1.35 mM curcumin in 1 mM M2agg), as determined from the reported molar absorptivity of 61,864 M–1 cm–1 at 427 nm for curcumin in ethanol. Curcumin did not precipitate from M2agg on dilution, indicating that it is strongly entrapped in the assembly (Figure 3C). Both M1 and M2 assemblies had an absorbance profile distinct from curcumin, allowing accurate determination of curcumin concentration in cur–M1agg or cur–M2agg (Figure S5). M1agg encapsulated twofold less curcumin (∼0.70 mM curcumin in 1 mM M1agg) compared to M2agg probably because it forms a less robust assembly.

Figure 3.

Figure 3

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(A) Images showing the enhanced solubility of curcumin in M1agg and M2agg compared to PBS. A saturating amount of curcumin was added to 1 mM solution of M1 or M2 in PBS, and the images were recorded prior to removal of free curcumin. (B) UV–vis spectra of M1agg and M2agg (1 mM in PBS) saturated with curcumin. (C) Images of 10- and 20-fold diluted solutions of M2agg (1 mM in PBS) saturated with curcumin. (D) UV–vis spectra of curcumin retained in aqueous assembly of M1agg and M2agg (1 mM) after 24 and 48 h. The UV–vis spectra in (B,D) were recorded after dilution with ethanol (PBS/ethanol = 1:24) and are representative of three independent experiments.

Next, we assessed the spontaneous release of curcumin from the assemblies over time at rt. A small decrease (<20%) in curcumin absorbance at 427 nm was observed, indicating that curcumin is stably encapsulated in M2agg even after 48 h. The faster release of curcumin from M1agg showed its inability to retain curcumin as effectively as M2agg (Figure 3D). After 120 h at rt, a substantial amount (>50%) of curcumin was retained by M2agg, whereas M1agg exhibited complete release of curcumin (Figure S6).

To ascertain the effect of curcumin on the size and morphology of the assemblies, we measured the size of cur–M2agg and cur–M1agg by DLS. The average hydrodynamic diameter (Figure 4A) observed for cur–M1agg (193.2 ± 92.8 nm, PDI: 0.245) was similar to that obtained prior to curcumin entrapment. For cur–M2agg, ∼52% increase in the mean hydrodynamic diameter was observed (214.2 ± 113.5 nm, PDI: 0.196), indicating a significant increase in assembly size due to curcumin incorporation. TEM analyses of the curcumin-entrapped assemblies, in which cur–M2agg showed compact circular assemblies having a dense core, were consistent with this interpretation (Figures 4B,C and S7).

Figure 4.

Figure 4

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(A) DLS analysis of M1agg and M2agg (1 mM) saturated with curcumin. (B) TEM images of M1agg and (C) M2agg (500 μM each) saturated with curcumin. (D) UV–vis spectra of cur–M2agg solution in PBS and curcumin in 30% PBS/EtOH solution with and without irradiation under UV. The absorbance was measured after dilution with ethanol (PBS/ethanol = 1:24). All data are representative of three independent experiments.

Next, we checked whether encapsulation in M2agg could prevent curcumin degradation due to UV exposure. We irradiated cur–M2agg in PBS under a 450 W UV lamp for 2 h. Absorbance measurement indicated almost no degradation of M2-encapsulated curcumin (Figure 4D) analogous to a control curcumin solution in ethanol (Figure S8). Curcumin dissolved in 30% ethanol/PBS solution showed >30% degradation under the same conditions. Thus, M2agg rescued curcumin from UV degradation possibly because of its large aromatic cross-sectional area.

Encapsulation of Doxorubicin

Next, we explored the encapsulation of the anticancer agent doxorubicin by our assemblies. The solubility of doxorubicin in the presence of M1agg and M2agg (1 mM in PBS) was assessed using UV–vis spectroscopy. The increased absorbance at 485 nm for doxorubicin encapsulated in M2agg indicated enhanced solubility of doxorubicin in M2agg compared to M1agg or PBS alone (Figure 5A).

Figure 5.

Figure 5

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(A) UV–vis spectra of M1agg and M2agg (1 mM in PBS) saturated with doxorubicin. The absorbance was measured in a PBS/acetonitrile mixture (1:3). (B) pH-dependent release of doxorubicin from M2agg measured by the fluorescence emission of doxorubicin at 570 nm (λex. = 485 nm).

As doxorubicin is toxic to normal tissues,33 it is usually administered in various encapsulated forms including stimuli-responsive delivery systems.34−40 We reasoned that M2agg might be a good vehicle for delivery of doxorubicin to cancer cells, as the more acidic environment of cancer cells is expected to break down M2agg and release the drug. This is because at pH 7.4, M2agg predominantly exists in its carboxylate form, which allows for its aqueous solubility. Under acidic conditions, the equilibrium shifts toward the protonated form of the carboxylic acid, which decreases the aqueous solubility of M2, leading to precipitation of M2 and concomitant release of the encapsulated drug. To test this hypothesis, we encapsulated doxorubicin in M2agg and dialyzed this encapsulated solution against buffer solutions of pH 6.0 and 7.4. The quantification of the released doxorubicin by fluorescence spectroscopy indeed showed more efficient release of doxorubicin from M2agg at pH 6.0 (Figure 5B).

Cellular Toxicity of M2

We tested the cellular toxicity of M2 to assess its feasibility for potential in vivo applications. Cells were treated with increasing concentrations of M2 and incubated for 24 h at 37 °C. Cell viability, as judged by a trypan blue assay, suggested that M2 up to a concentration of 100 μM lacked any significant toxicity on HEK293T cells as well as HeLa and MDA-MB-231 cancer cell lines. However, MCF-7 cells showed sensitivity to M2, and ∼60% reduction in cell viability was observed at 100 μM (Figure 6A).

Figure 6.

Figure 6

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(A) Trypan blue assay to assess the toxicity of M2agg on HEK293T and cancer (HeLa, MDA-MB-231, and MCF-7) cell lines. (B) Concentration-dependent toxicity of cur–M2agg on normal and cancer cell lines (drug loading = 1.35 mM curcumin/1 mM M2agg). (C) Toxicity of 0.5 μM free doxorubicin on HEK293T and MCF-7 cell lines. (D) Toxicity of 0.5 μM doxorubicin encapsulated in M2agg (drug loading = 128 μM doxorubicin/1 mM M2agg) on HEK293T and MCF-7 cell lines by trypan blue assay. The error bars indicate standard deviations from three independent experiments.

Toxicity and Cellular Uptake of Curcumin and Doxorubicin from the Assemblies

We next tested the toxicity of cur–M2agg on the cells. All four cell lines were treated with an increasing concentration of cur–M2agg (drug loading = 1.35 mM curcumin/1 mM M2agg) for 48 h, and cell death was assessed by trypan blue assay. HEK293T cells did not show any significant toxicity with cur–M2agg (∼80% viability at 150 μM entrapped curcumin). In contrast, the cancer cell lines showed dose-dependent toxicity with ∼21 and 15% cell viability in HeLa and MDA-MB-231 cell lines, respectively, at 150 μM entrapped curcumin (Figure 6B). Control cells treated with curcumin in PBS did not show any toxicity, mainly because of the insolubility of curcumin (Figure S9).

Next, we treated HEK293T and MCF-7 cell lines with 0.5 μM doxorubicin (either in PBS or as dox–M2agg). The cells treated with free doxorubicin showed indiscriminate toxicity in both normal and cancer cell lines (∼28 and 15% viability of HEK293T and MCF-7 cell lines, respectively, at 36 h) (Figure 6C). Dox–M2agg showed reduced toxicity on both cell lines (∼43 and 32% viability of HEK293T and MCF-7 cell lines, respectively, at 36 h) (Figure 6D), probably because of slower release of doxorubicin from the assembly. Doxorubicin consistently showed higher toxicity on cancer cells compared to normal cells both with and without M2agg. However, dox–M2agg showed slower and more sustained drug release, resulting in lower toxicity for normal cells over 54 h. The consistent, albeit small, effect of dox–M2agg compared to free doxorubicin was probably due to enhanced release of doxorubicin at acidic pH from the assembly.

Uptake of Curcumin and Doxorubicin by Cells

To determine the uptake of curcumin from cur–M2agg by the cells, we treated HEK293T (Figure 7A) and HeLa (Figure 7B) cell lines with cur–M2agg and curcumin in PBS. The fluorescence observed inside the cell suggested efficient internalization of curcumin on treatment with cur–M2agg (Figure 7A,B), whereas cells treated with curcumin in PBS did not show any internalization (Figures S10 and S11). Thus, M2agg not only encapsulates curcumin but also releases it from the assembly, leading to the uptake of the curcumin by cells. Similar results were obtained when HeLa cells were treated with dox–M2agg (Figure 7C).

Figure 7.

Figure 7

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(A,B) Confocal microscopy images showing curcumin internalization in HEK293T (A) and HeLa (B) cell lines. (a,d) DAPI-stained nuclei, (b,e) internalized curcumin (20 μM) from cur–M2agg, and (c,f) merged panels (drug loading = 1.35 mM curcumin/1 mM M2agg). (C) Confocal microscopy images showing internalization of doxorubicin in HeLa cells: (a) cells stained with DAPI; (b) cells treated with doxorubicin (2 μM) from dox–M2agg (drug loading = 128 μM doxorubicin/1 mM M2agg); and (c) merged panels. The confocal images are representative of two independent experiments. (Scale bar: A and B-10 μm; C-20 μm.)

Conclusions

We have designed amphiphilic assemblies based on small gemini-type aromatic molecules, which showed assembly formation at a relatively low concentration in an aqueous medium. Assembly formation was supported by 1H NMR, DLS, and TEM analysis. M2agg was utilized to encapsulate and solubilize the pharmacologically relevant molecules curcumin and doxorubicin. M2agg showed enhanced encapsulation compared to M1agg, suggesting that the decyl chains of M2 were important for the formation of stable assembly and drug entrapment. M2agg also increased the stability of curcumin in aqueous medium at physiological pH. The nonselective anticancer agent doxorubicin showed sustained and higher toxicity for cancer cells over normal cells when it was encapsulated by M2agg. The low cytotoxicity and excellent drug release profile of M2agg along with its ability to enhance the solubility and stability of hydrophobic drugs makes it a suitable vehicle for drug delivery systems.

Experimental Section

Materials and Methods

Materials

Chemicals were purchased from commercial suppliers such as Sigma-Aldrich, Alfa Aesar, and Spectrochem and used without further purification. Curcumin powder (95%) and doxorubicin hydrochloride were purchased from Alfa Aesar and TCI Chemicals, respectively. Syntheses were performed in clean, oven-dry glassware. The progress of the reactions was monitored using a silica TLC plate coated with F254 for visualization under UV. NMR spectra were recorded on a Bruker 400 or 500 MHz instrument and chemical shifts were reported in ppm. The chemical shifts were calibrated to the residual proton and carbon resonances of the solvent: D2O (1H δ 4.79 ppm), CDCl3 (1H δ 7.26 ppm; 13C δ 77.16 ppm), and DMSO-d6 (1H δ 2.50 ppm; 13C δ 39.52 ppm). HRMS analysis was performed on a Bruker Daltonics micrOTOF-Q-II mass spectrometer. Melting points were recorded on a digital melting point apparatus. The assembly size was measured on a Delsa Nano (Beckman Coulter) using the CONTIN algorithm at 25 °C. TEM imaging was performed using an FEI Talos 200S system equipped with a 200 kV field emission gun (FEG). UV–vis spectra were recorded on an Agilent spectrophotometer with Cary Win software and fluorescence spectra were recorded on Horiba Fluorolog spectrofluorometer.

Aggregation by NMR

Molecule M1 or M2 was dissolved in 500 μL of D2O (∼25 mM each). A few drops of 1 M NaOH (in D2O) were added to ensure the complete solubilization of the molecule. Proton resonances were recorded on a 500 MHz NMR spectrometer at 298 K for M1 and from 298 to 353 K for M2.

CAC of the Molecules

A series of solutions (25–750 μM) of M1 and M2 were prepared in PBS (20 mM, 50 mM NaCl, pH 7.4) from 100 mM stock solution in DMSO. The fluorescence emission was recorded from 330 to 450 nm with excitation (λex.) at 310 nm.

Curcumin Encapsulation

To generate curcumin-containing assemblies, excess curcumin powder was stirred with a 1 mL solution of M1 or M2 (1 mM) in water. After stirring for 12 h at rt, the solutions were centrifuged at 5000 rpm for 10 min at 25 °C to obtain a clear yellow solution of the assembly with curcumin. These were designated as cur–M1agg or cur–M2agg.

Size Measurement by DLS

A 1 mM solution of M1 or M2 was prepared by diluting a stock solution (100 mM in DMSO) with water. A few drops of 1 M NaOH were added to ensure the complete solubilization of the molecule. The pH of the solution was adjusted to ∼7 by adding 1 M HCl. The solution was sonicated for 10 min. The solutions were subjected to size measurement within 20–30 min on a DLS analyzer. Similarly, the sizes of cur–M1agg or cur–M2agg were also measured.

Preparation of Samples for TEM Imaging

A solution of M1 or M2 (1 mM) in water was prepared from a stock solution (100 mM in DMSO) at pH 7 and sonicated for 10 min. These solutions were diluted to 500, and 10 μL of the diluted solution was plated on a copper grid and allowed to adsorb for 5 min. Curcumin-containing assemblies (cur–M1agg or cur–M2agg) prepared as described above were also plated on TEM grids using a similar protocol. The excess liquid was removed using a tissue paper. The samples were stained with 10 μL of 0.3% phosphotungstic acid, dried in a desiccator, and imaged using an FEI Talos 200S system equipped with a 200 kV FEG.

Curcumin Encapsulation for UV–Vis Measurement

A solution of M1 or M2 (1 mM) in PBS (pH 7.4) was prepared from a stock solution (100 mM in DMSO) and sonicated for 10 min. Excess curcumin powder was stirred with a 1 mL solution of M1 or M2 (1 mM) in PBS. After stirring for 12 h at rt, the solutions were centrifuged at 1500 rpm for 10 min at 25 °C to settle down free curcumin. The curcumin entrapped suspensions were used for further study.

Curcumin Release

Cur–M1agg or cur–M2agg in PBS was prepared as described above. Curcumin retention by the assemblies was measured over time by aliquoting 10 μL of cur–M1agg or cur–M2agg (1 mM M1 or M2 in PBS) at 12 h intervals and diluting 25-fold with ethyl alcohol. This solution was subjected to UV–vis measurement and the concentration of the entrapped curcumin was determined by using molar absorptivity of 61,864 M–1 cm–1 at 427 nm for curcumin in ethanol.41

Preparation of Cur–M2agg Solution for UV Degradation Study

Excess curcumin powder was added to a 1 mL solution of M2agg (1 mM) in PBS or in 30% ethanol/PBS. These solutions were stirred at rt for 12 h to dissolve curcumin. The free curcumin was removed by centrifugation at 1500 rpm for 10 min. A curcumin solution in ethanol (∼1 mg/mL) was prepared as a control. For each of the three solutions, a 10 μL aliquot of the sample was diluted with 240 μL ethanol and immediately subjected to UV measurement. For the UV degradation study, the three solutions in glass vials were irradiated under a 450 W ultraviolet lamp. A 10 μL aliquot of each was kept under dark at rt for 2 h as control. After irradiation, the 10 μL solution was diluted 25-fold with ethanol and subjected to UV measurement.

Preparation of Cur–M2agg Solution for Toxicity Assay

Curcumin was encapsulated in 1 mM M2agg in PBS as described earlier. The concentration of curcumin in the assembly was measured by absorbance at 427 nm after 25-fold dilution with ethanol. A control sample of curcumin suspension in PBS was prepared by adding an equivalent amount of curcumin encapsulated in the assembly.

Doxorubicin Encapsulation

Doxorubicin hydrochloride powder (10 mg) was dissolved in 5 mL of Milli-Q water, and triethylamine (1.1 equiv) was added. The solution was stirred for 1 h followed by extraction thrice with chloroform and evaporation of solvent to yield free doxorubicin. The free doxorubicin (30 μL in DMSO, approx. 0.1 M) was added to a 1 mM solution of M1agg, M2agg (1 mL prepared in PBS), or PBS (1 mL). The solutions were stirred at rt for 12 h and centrifuged at 1500 rpm for 10 min at 25 °C. The supernatant (dox–M2agg) was collected, and the absorbance of doxorubicin was recorded at 485 nm with PBS/acetonitrile (1:3) as the solvent. The amount of doxorubicin entrapped in M2agg was determined by using the molar absorptivity of doxorubicin in water (11,500 M–1 cm–1 at 485 nm), and it was found to be 128 μM doxorubicin/1 mM M2agg.42,43

pH-Triggered Release of Doxorubicin

Dox–M2agg in PBS (pH 7.4) was prepared as described earlier. Dox–M2agg solution (800 μL) was added in two separate dialysis tubes (6–8 kDa) and dialyzed at 37 °C against 40 mL of PBS of pH 6.0 and 7.4, respectively. The dialysis buffer (400 μL) was aliquoted every 1 h. The dialysis solution was replenished with an equivalent amount of the buffer to keep the final volume constant. The doxorubicin released from the assembly was measured by fluorescence spectrophotometry with excitation at 485 nm and emission from 520 to 650 nm.

Cell Culture

The mammalian cells (HEK293T, HeLa, MCF-7, and MDA-MB-231) used in this study were obtained from American Type Culture Collection (ATCC). The cells were grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (100 units/mL penicillin and streptomycin) in a humidified incubator with 5% CO2 at 37 °C.

Curcumin and Doxorubicin Uptake Assay

HEK293T and HeLa cells (10,000 cells/well) were seeded on a glass coverslip in a six-well plate and cultured as above. After 24 h, the cells were treated with 20 μM curcumin (as cur–M2agg) or 2 μM doxorubicin (as dox–M2agg) for 12 h. Equivalent amounts of curcumin or doxorubicin in PBS (pH 7.4) were used as control. The cells were allowed to grow for another 12 h in the media. The cells were washed three times with PBS and fixed with 4% formaldehyde for 10 min at rt. The cells were further washed with PBS and incubated in rehydration buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Triton X-100) for 15 min on ice. The cells were washed with PBS and incubated with Hoechst-33258 [1:7000 dilution (v/v) of 10 mg/mL stock solution] for 10 min. Excess Hoechst-33258 was removed by washing with PBS, and the coverslips were mounted on the slides (VECTASHIELD H-1000). The cells were imaged under an Olympus Fluoview FV3000 live-cell confocal microscope. The curcumin signals were recorded after exciting samples with green laser and capturing the emitted light.

Trypan Blue Exclusion Assay

In a 24-well culture plate, 50,000 cells per well were seeded and allowed to grow for 24 h. The cells were treated with different concentrations of M2agg (50, 100 and 150 μM) for 24 h, and control cells were treated with the corresponding volume of PBS required to dissolve M2agg. In parallel, the cells were treated with 50, 100, and 150 μM curcumin encapsulated in M2agg, and for corresponding control, the volume of M2agg solution required for curcumin encapsulation was used. The cells were also treated with an equivalent amount of curcumin suspension in PBS for 48 h. For experiments with doxorubicin, the cells were treated with 0.5 μM doxorubicin in dox–M2agg and a solution of doxorubicin prepared in PBS. In control, cells were treated with the equimolar solution of M2agg used for encapsulation of doxorubicin. In this case, cells were collected at 18 h intervals until 72 h. Subsequently, the cells were washed with PBS and dislodged by trypsinization and were collected by centrifugation at 450g for 2 min. Cell pellets were dissolved in 1 mL of PBS and 10 μL of cell suspension was mixed with 10 μL of 0.4% trypan blue solution. The live and dead cells were counted on a hemocytometer using an inverted fluorescence microscope (Leica Microsystems).

Acknowledgments

We thank IISER Bhopal and SERB for financial support and DST-FIST facilities at IISER Bhopal for the TEM and Olympus live-cell confocal microscopy images. I.S. is a recipient of the Ramanujan Fellowship. Y.M.G. thanks UGC for a fellowship.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c04395.

  • Reaction scheme, synthetic procedure, and characterization of all compounds; hydrodynamic diameter of M1agg and M2agg; TEM images of assemblies of M1 and M2; concentration-dependent change in relative fluorescence emission intensity of M1 and M2 in PBS at 370 nm; TEM images of M1agg and M2agg; UV–vis absorption spectra of M1agg and M2agg; retention of curcumin in the assembly of M1agg and M2agg; toxicity of PBS–curcumin suspension; confocal microscopy images of curcumin uptake by HEK293T and HeLa cells; and NMR spectra of the compounds used in the study (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c04395_si_001.pdf (89.5MB, pdf)

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