Self-assembly of well-defined fatty acid based amphiphilic thermoresponsive random copolymers

Abstract

A series of amphiphilic random copolymers, consisting of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and 2-(methacryloyloxy)ethyl stearate (SAMA), were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization. The compositions of SAMA and PEGMA in the copolymers were determined by proton nuclear magnetic resonance (¹H NMR) spectroscopy, and these were further used to calculate reactivity ratios of the SAMA and PEGMA monomers using the extended Kelen–Tüdös method at high monomer conversions. Crystallinity was observed in the copolymers with high (30% or higher) fatty acid content, studied by differential scanning calorimetry (DSC), X-ray diffraction (XRD) measurements and polarized optical microscopic (POM) study. The copolymers showed reversible phase transition in response to temperature cycles in aqueous medium with lower critical solution temperatures (LCST) between 42 and 65 °C, as determined by UV-vis spectrophotometer. The LCST values decreased with the increase in hydrophobic SAMA content in the copolymer. Self-assembly behavior of the copolymers to micellar structures in aqueous solution was investigated by measurement of critical micelle concentration using fluorescence spectroscopy, dynamic light scattering (DLS) and scanning electron microscopy (SEM). The in vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction (MTT) assay established no cytotoxic response in the presence of fatty acid based copolymers.

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Introduction

Recently, several strategies have been developed to synthesize renewable resource based monomers and polymers. In this regard, vegetable oils, fatty acids and their derivatives are the most attractive materials due to their low cost, renewability, low toxicity and biodegradability.^1,2 To date, plant oils or fatty acid derivatives have been widely utilized as raw materials in applications such as plastics,³ thermoplastic elastomers,^4,5 coatings,⁶ resins,⁷ and various thermosetting composites.^8,9 The long alkyl side chains in the fatty acids provide flexibility, lipophilicity and their hydrophobic nature makes them useful in various applications including oil absorbency agents, viscosity modifiers, and oil-soluble drag reducers.¹⁰

Step growth polymerization technique has been extensively applied to prepare fatty acid-based polymer.^11,12 Quinzler and Mecking reported novel semicrystalline polyesters with long-chain hydrocarbon segments based on complete linear incorporation of oleic acid and erucic acid through polycondensation reactions.¹³ Cádiz and coworkers reviewed the recent developments of thermostable resins derived from vegetable oils.¹⁴ There are very few reports on the use of controlled radical polymerization (CRP) techniques to synthesize polymer from fatty acids with targeted molecular weights, narrow dispersity (Đ) and specific chain end structures. However, atom transfer radical polymerization (ATRP) of fatty acid containing methacrylates has been reported to prepare side-chain fatty acid containing polymers with controlled molecular weights and narrow Đ.¹⁵ Reversible addition–fragmentation chain transfer (RAFT) method was used to polymerize lauryl methacrylate.^16,17 Recently, we have studied RAFT polymerization of methacrylate monomers having side-chain fatty acids, such as caprylic, capric, lauric, mysritic, palmitic, stearic and oleic acid.^18,19 We have shown that homopolymers with fatty acid pendants displayed crystalline behavior depending on the chain length of the fatty acids. Realizing that the stearic acid (SA) has potential to induce crystallinity, we sought to develop the synthesis of well-defined SA containing water soluble copolymers, in which stearate moiety will provide crystallinity and hydrophobicity to the copolymer structure.

Amphiphilic polymers are known to self-assemble in selective solvents into a wide variety of morphologies, such as spheres, rods, vesicles, micelles, nanofibers, and nanotubes, and find several applications such as drug delivery systems,²⁰ nanoreactors, surface coating, nano-templates.²¹ There are only a very few reports describing the synthesis and self-assembly of fatty acid based polymer.²² Amphiphilic copolymers composed of ethylene glycol and stearyl methacrylate were reported previously by other groups.²³ Amiel and co-workers designed polyoxazoline end-capped with fatty acids and described the self-aggregation in micelles of these macromolecules, exhibiting dissymmetry in size of blocks.²⁴ Recently, the same group also reported micellar structures of amphiphilic diblock copolymer in selective solvents, poly(isobutylvinylether-b-2-methyl-2-oxazoline), and investigated the micellar characteristics of a monoalkyl (C₁₂ or C₁₈) endcapped poly(2-methyl-2-oxazoline) (POXZ-C_n). These amphiphilic polymers self-assembled to form micelles in aqueous media.²⁵ Holder et al. prepared poly(ethylene oxide)-block-poly(octadecyl methacrylate) (PEO-b-PODMA) and showed the aggregation behavior in aqueous media.²⁶ Morandi et al. reported the synthesis of an amphiphilic copolymer from linseed oils using ATRP technique and its self-assembly property in response to pH.²⁷ Recently amphiphilic graft polymers based on renewable resources were synthesized via combination of two polymerization techniques namely step-wise polymerization and RAFT technique.²⁸ These amphiphilic graft copolymers formed micelles in water and also stable in a water–decane mixture. With these features in mind, herein we have reported the synthesis of SA based copolymers having different hydrophilic/hydrophobic ratios via RAFT polymerization from poly(ethylene glycol) methyl ether methacrylate (PEGMA) and 2-(methacryloyloxy)ethyl stearate (SAMA). Note that similar studies can be done using commercially available stearyl methacrylate. We have chosen PEGMA because of its hydrophilicity, biocompatibility, nontoxicity, as well as thermo-sensitivity.²⁹ Self-assembly in aqueous medium of a single copolymer molecule bearing hydrophilic and hydrophobic segments is currently a topic of great interest,³⁰ because random copolymer based nano-assembly are advantageous over amphiphilic block copolymers since they can be synthesized in a single polymerization step unlike block copolymers, which are generally synthesized in two steps. Therefore, the amphiphilic nature of the fatty acid based copolymers encourages us to investigate their solution self-assembly in aqueous medium. In addition, in vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction (MTT) assay was carried out to establish non-toxic nature of these fatty acid based copolymers.

Experimental section

Materials

Dicyclohexylcarbodiimide (DCC, 99%), 4-dimethylaminopyridine (DMAP, 99%), SA (98%), 2-hydroxyethyl methacrylate (HEMA, 97%) and pyrene (98%) were purchased from Sigma and used without any further purification. PEGMA (300 g mol⁻¹, Aldrich, 99%) was passed through a basic alumina column prior to polymerization. 2,2′-Azobisisobutyronitrile (AIBN, Sigma, 98%) was recrystallized from methanol. 4-Cyano-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid (CDP) was synthesized following the standard literature procedure.³¹ Side-chain SA containing methacrylate monomer, SAMA, was synthesized as reported previously.¹⁸ CDCl₃ (99.8% D) and D₂O (99.9% D) were obtained from Cambridge Isotope Laboratories, Inc., USA for NMR study. Dialysis membranes (Spectra/Por® 7, molecular weight cut-off (MWCO): 2000 Da) were purchased from Spectrum Laboratories, Inc. The solvents dichloromethane (DCM), tetrahydrofuran (THF), methanol (MeOH), ethyl acetate (EtOAc) and hexanes (mixture of isomers) were purified by following general procedure. For cytotoxicity test, HeLa cells were grown on Dulbecco's modified eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Hyclone Labs, Thermo Scientific) and penicillin–streptomycin (Hyclone Labs, Thermo Scientific) solution. Formazan crystals produced from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, USB Corporation) treatment of the cells were dissolved in DMSO (99.9%, cell culture grade, Amresco).

Instrumentation

Molecular weights and molecular weight distributions (dispersity (Đ)) of polymers were determined by gel permeation chromatography (GPC). The GPC instrument contains a Waters 515 HPLC pump, a Waters 2414 refractive index (RI) detector, one PolarGel-M guard column (50 × 7.5 mm) and two PolarGel-M analytical columns (300 × 7.5 mm), in THF at 30 °C at 1.0 mL min⁻¹ flow rate. Poly(methyl methacrylate) (PMMA) standards were used to calibrate the instrument. ¹H NMR spectra were acquired in a BrukerAvance^III 500 spectrometer operating at 500 MHz. UV-vis measurement was done on a Perkin-Elmer Lambda 35 UV-vis spectrophotometer. Fluorescence emission spectra were recorded on a Horiba JobinYvon (Fluoromax-3, Xe-150 W, 250–900 nm) fluorescence spectrometer. Differential scanning calorimetry (DSC) studies were carried out under nitrogen using a Mettler Toledo DSC1 STARe instrument at 10 °C min⁻¹ heating rate. Polymers were first cooled from room temperature to −70 °C, then heated to +120 °C and again cooled to −70 °C at 10 °C min⁻¹. The crystalline melting temperature (T_m) was taken from the third segment of the run (from +120 to −70 °C segment), as the remaining traces of solvents or other volatile impurities would have evaporated on heating from −70 to 120 °C. The thermogravimetric analysis (TGA) was performed on a Mettler Toledo TGA/SDTA 851e instrument at a heating rate of 10 °C min⁻¹ in nitrogen atmosphere. Rigaku Smart Lab powder diffractometer having Cu Kα = 1.54059 Å radiation was used for recording X-ray diffraction (XRD) spectra. Polarized optical microscopic (POM) studies were carried out using an Olympus CH 30 imaging microscope equipped with Image Pro Plus version 4.0 software. Scanning electron microscopy (SEM) images were recorded using Carl Ziess Sigma SEM instrument. Particle size in solution was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK) equipped with a 4.0 mW He-Ne laser beam operating at λ = 658 nm. All samples were measured in aqueous medium at room temperature at a scattering angle of 173°.

Copolymer synthesis

All polymerization reactions were carried out in the presence of AIBN as initiator and THF as solvent at 60 °C. A representative example is as follows: SAMA (0.29 mmol, 114 mg), PEGMA (0.29 mmol, 86 mg), AIBN (1.15 μmol, 0.19 mg, from stock solution), CDP (5.7 μmol, 2.3 mg) and THF (1.0 mL) were placed in a 20 mL reaction vial equipped with a magnetic stir bar. The vial was purged with dry N₂ for 20 min and placed in a preheated reaction block at 60 °C. The feed ratios of SAMA and PEGMA were varied to get copolymers of various compositions. After predetermined time, the polymerization reaction was stopped by cooling the vial in an ice-water bath and exposed to air. Finally, the copolymer was dialyzed against MeOH using a MWCO membrane of 2000 Da. The solvent was changed at least 8 times in 2 to 4 h interval. The resulting solution was evaporated under reduced pressure and finally the polymers were dried under high vacuum at 35 °C for 8 h.

Determination of LCST

Copolymer aqueous solutions (2.0 mg mL⁻¹) were prepared, filtered through a 0.45 μm membrane filter and transferred to a quartz cuvette. Then, turbidity of the solution was measured using a UV-vis spectrophotometer by monitoring the % transmittance (% T) changes at λ = 500 nm.^32,33 The solution was warmed from 30 to 70 °C. The solution was equilibrated for 5 min at the measurement temperature and the % T value at 500 nm was recorded. The LCST was defined as the temperature where a reduction of 50% T of the copolymer solution was noticed.

Determination of critical micelle concentration (CMC)

The CMC values of different water soluble copolymers were determined by fluorescence spectroscopy using pyrene as a hydrophobic probe.³⁴ 20 μL pyrene stock solution in acetone (10⁻⁴ mol L⁻¹) was taken in different glass vials and the solvent was removed by blowing nitrogen. Then copolymer aqueous solutions at different concentrations were separately added to each of these pyrene containing vials and the final volume was made up with water to get a series of solutions (from 1.0 mg mL⁻¹ to 1.0 mg L⁻¹) with constant final pyrene concentration in each vial, 10⁻⁶ mol L⁻¹. Each vial was sonicated for approximately 10 min and then allowed to stand for 4 h before the emission spectra were recorded (at excitation wavelength 337 nm, excitation band width 2.5 nm, emission band width 2.5 nm). The ratio of the emission intensities of the first (372 nm) and the third (384 nm) peaks were plotted against the concentration of polymer and CMC was determined from the inflection point observed in this plot.

MTT assays

Cytotoxicity of copolymers was studied by MTT assay using HeLa cell line. In a 24 mm plate the cells were seeded at a density of (2 × 10⁴) cells per well in 1.0 mL (DMEM) containing 10% FBS and allowed to adhere for 24 h in an incubator (37 °C, 5% CO₂). Then copolymer solutions in phosphate buffer (10 mM, pH 7.4) of various concentrations were added. The cells were incubated for another 48 h. At the same time untreated control cells and (phosphate-buffered saline) PBS control cells were incubated for the same time period. MTT (100 μL from 5 mg mL⁻¹ in PBS) was added to each well of the 24 well plates and incubated for 4 h. Then culture media was removed and resulting purple formazan crystal was dissolved in 500 μL DMSO. After 10 min the absorbance was measured at 570 nm with 5 times dilution. The values were expressed as the mean from three independent experiments and the bars in the figures indicated standard deviation.

Results and discussion

RAFT polymerization

Copolymerizations of SAMA and PEGMA were carried out in THF at 60 °C by using CDP as the RAFT agent and AIBN as the radical source (Scheme 1). The CDP used in these copolymerization reactions has previously showed a well-controlled behavior in RAFT polymerizations of different fatty acids,¹⁸ and for PEGMA.³⁵ Co-monomer compositions were altered and several copolymers were prepared at [monomer] : [CDP] : [AIBN] = 100 : 1 : 0.2 ratio. The characterization results for these copolymerization reactions are presented in Table 1. Copolymers, P(SAMA-co-PEGMA), were named according to the following rules (Table 1): CP represents copolymer; 5, 10, 15, etc. numbers after CP stand for the % feed compositions of SAMA.

Scheme 1 Synthesis of P(SAMA-co-PEGMA) by RAFT polymerization.

Table 1 Experimental results from the RAFT copolymerization of SAMA and PEGMA in THF at 60 °C

Polymer^a	SAMA content in feed	Time (min)	Conv.^b (%)	SAMA content in copolymer^c	Mn,theo^d (g mol^−1)	Mn,GPC^e (g mol^−1)	Đ^e	Mn,NMR^f (g mol^−1)
a CP means copolymer.b Determined by gravimetric analysis on the basis of the amount of monomer feed.c Determined from ^1H NMR analysis.d Mn,theo = (([monomer]/[CDP] × average molecular weight (MW) of monomer × conversion) + (MW of CDP)).e Measured by GPC in THF.f Calculated by ^1H NMR from the integration ratio of the repeating unit protons to that of the polymer chain end protons.g Not determined.
PPEGMA	0	150	33	0	10 300	9200	1.26	7600
CP5	5	180	35	7.8	11 100	10 200	1.29	11 500
CP10	10	210	61	12.2	19 300	8800	1.31	ND^g
CP15	15	260	63	18	20 200	14 300	1.32	14 100
CP20	20	300	62	21.2	19 700	13 500	1.31	14 300
CP25	25	320	79	27	26 000	20 500	1.38	14 700
CP30	30	340	53	31	17 800	15 300	1.38	ND^g
CP35	35	360	51	34.8	17 400	14 400	1.32	16 900
CP40	40	400	65	39.8	22 400	21 800	1.29	14 000
CP50	50	420	71	45	25 100	24 900	1.26	ND^g
PSAMA	100	360	67	100	21 900	21 700	1.27	17 200

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Copolymers were purified by dialysis and in the purified copolymers peaks corresponding to the vinyl protons of PEGMA and SAMA were completely disappeared (Fig. 1). In the ¹H NMR spectrum of poly(2-(methacryloyloxy)ethyl stearate) (PSAMA), the signals at 4.1–4.5 ppm are assigned to the side chain –O–CH₂–CH₂–O– protons (4H) (Fig. 1A). For the poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA), the typical methoxy protons (–OCH₃, 3H) resonate approximately at 3.38 ppm and methylene protons next to the ester functionality (–CH₂OCO, 2H) appear at 4.1 ppm (Fig. 1B). Compositions of copolymers were determined (Table 1) from their ¹H NMR spectra (Fig. 1C), from the integration of the signals at 4.1–4.5 ppm from –OCH₂–CH₂O– unit (after subtracting the peak area contributed by the PEGMA units in this region) and 3.38 ppm from –OCH₃ protons for PPEGMA. Incorporations of CDP at the end of the copolymer chains were observed by the characteristic resonance signals at 2.40–2.52 ppm from HOOC–CH₂–CH₂–C(CN)(CH₃)– chain ends. Comparison of the integration areas from the terminal group at 2.40–2.52 ppm and side chain protons at 3.38 ppm allowed the calculation of number average degree of polymerization (DP_n) of PEGMA unit in the homo- or copolymers. Similarly, from the ratio of integration areas at 2.40–2.52 ppm and at 4.1–4.5 ppm from –OCH₂–CH₂O– unit in SAMA (after subtracting the peak area contributed by the PEGMA units in this region), we have determined DP_n of SAMA unit in the copolymers. Finally, number average molecular weights of homo- and co-polymers were determined by NMR (M_n,NMR). For example, M_n,NMR values of the P(SAMA-co-PEGMA) copolymers (Table 1) were determined by using the formula: M_n,NMR = [(f_SAMA × DP_n,SAMA × M_SAMA) + (f_PEGMA × DP_n,PEGMA × M_PEGMA) + molecular weight of CDP], where f, DP_n and M are the mole fraction of monomer in the copolymer, number average degree of polymerization and molecular weight of monomer, respectively.

Fig. 1 ¹H NMR spectra of (A) PSAMA, (B) PPEGMA and (C) CP20 copolymer.

Fig. 2 shows that the GPC traces for the copolymers are symmetric and unimodal, indicating no chain transfer reactions and bimolecular coupling reactions during the copolymerization. The number average molecular weights (M_n,GPC) and Đ values for all polymers were determined from the GPC study and results are summarized in Table 1. Theoretical molecular weights (M_n,theo) were also calculated from stoichiometry and overall monomer conversion data by using the formula: M_n,theo= (([monomer]/[CDP] × average molecular weight (MW) of monomer × conversion) + (MW of CDP)). In Table 1, the M_n,theo values matches reasonably well with the corresponding M_n,GPC and M_n,NMR values. These results confirmed a high degree of chain end retention and controlled nature of the copolymerization reactions. The resulting copolymers were soluble in most of the organic solvents, except in hexanes, pet ether and diethyl ether. Only PPEGMA, CP5, CP10, CP20 and CP30 were soluble in water.

Fig. 2 GPC RI traces of P(SAMA-co-PEGMA) copolymers at different ratios.

For any copolymerization reactions knowledge of reactivity ratios is very helpful for tailoring copolymers with desired properties. Therefore, the reactivity ratios for the copolymerization of SAMA with PEGMA were calculated. For the living controlled copolymerization systems, generally greater than 20% overall monomer conversions should be reached to determine reactivity ratios,³⁶ and extended Kelen–Tüdös (extended K–T) method³⁷ is appropriate at relatively high monomer conversions. Hence, we have used extended K–T method to determine reactivity ratios for the copolymerization of SAMA with PEGMA by using monomer feed compositions, co-monomer contents in the copolymer and monomer conversions. The r_PEGMA = 0.609 and r_SAMA = 0.288 were obtained from Fig. 3, indicating random distribution of SAMA and PEGMA units in the copolymer chain and the copolymer is 2.12 times enriched with PEGMA units.³⁸ Although both the monomers are methacrylate derivatives, the r_PEGMA is almost two times more reactive towards copolymerization reactions compared to SAMA, probably due to the higher bulkyness of SAMA compared to PEGMA.

Fig. 3 Extended Kelen–Tüdös plot for the RAFT copolymerization of SAMA and PEGMA at 60 °C in THF.

Thermal properties

The thermal behaviors of homo- and co-polymers have been studied by TGA and DSC. TGA thermogram (Fig. 4A) reveals that homopolymers of SAMA and PEGMA are comparatively little more stable than its copolymers (CP50 and CP10). It is also noted that there are two stages of weight loss for PPEGMA, CP50 and CP10. The first stage appeared between 175–265 °C, followed by the second stage of degradation above 265 °C. The first stage could be attributed to the decomposition of trithiocarbonate functionality in terminal groups. However, the weight loss in this stage is somewhat greater than that expected on the basis of loss of the end group alone. In the second stage, degradation of side-groups from repeating units and decomposition of residual chain occurs.³⁹

Fig. 4 TGA thermograms (A) and DSC curves (B) of various homo- and copolymers.

As PSAMA showed crystallinity and melting point (T_m) of 47 °C,¹⁸ thermal properties of copolymers were examined by DSC study. Fig. 4B shows DSC thermograms of various homo- and co-polymers, demonstrating T_m of 47, 34, 32, 29 and 26 °C for PSAMA, CP50, CP40, CP35 and CP30, respectively. Therefore, 30% or higher SAMA content in the copolymer is needed to observe T_m in the copolymer, which arises due to the crystallinity in the copolymer from the interactions of alkyl side chains from the SAMA units.⁴⁰ PPEGMA is amorphous and increasing PEGMA content into the copolymer increases the flexibility, thus T_m values decreased. We did not observe any T_m for CP25, C20, CP15 and CP10 due to the insufficient SAMA content in the copolymer, which is the major cause of induced crystallinity in the copolymer. Note that we have observed glass transition temperature (T_g) values for the CP25, CP20 and CP15 as 22, 20 and 16 °C, respectively.

Crystalline morphologies of polymers

The effect of stearate content in the copolymer on crystallinity is also studied by wide angle X-ray diffraction (WAXD) analysis (Fig. S1†). The peak around 2θ = 19.24° (d = 4.60 Å) in the WAXD plot for CP50 indicate the amorphous halo.⁴¹ In addition, we observed a small peak at 2θ = 21.27° (d = 4.12 Å), due to the crystallinity in the polymer. Interestingly, we did not observe the 2θ = 21.27° peak for CP40, CP30 and CP10 in Fig. S1† although these polymers showed T_m in the DSC study. This could be due to low crystallinity present in those copolymers because of increase in PEGMA content.⁴² Note that the 2θ = 21.27° peak in CP50 matches nicely with the PSAMA crystalline peak.¹⁸ Increasing fatty acid content helps crystalline domain formation due to the hydrophobic interaction among the large alkyl chains,⁴³ although the crystallinity was somewhat less compared to PSAMA homopolymer.⁴⁴ Furthermore, POM images of CP50 (Fig. 5) showed a birefringent texture distinctive of semicrystalline linear polymer chains, which melts and become isotropic when heated above their T_m value.⁴⁵ But, in case of CP10, CP30 and CP40 no birefringent texture was observed.

Fig. 5 POM images of CP50 at different temperatures, 10, 27 and 50 °C (left to right).

Thermo-responsiveness

Since PPEGMA is a thermoresponsive polymer with lower critical solution temperature (LCST) in water at 64 °C,⁴⁶ herein we studied thermo responsiveness of the water soluble copolymers (CP5 to CP30). The homopolymer PPEGMA showed LCST at 65 °C (Fig. 6), nicely matched with the reported LCST of 64 °C for this polymer. The CP10, CP15, CP20, CP25 and CP30 copolymers gave LCST of 61, 56, 52, 48 and 44 °C, respectively, indicating decrease of LCST values with the increase of molar fraction of hydrophobic SAMA content in the copolymer. The water solubility of the copolymers is based on hydrogen-bonding interactions of the pendant poly(ethylene oxide) (PEO) chains with the water molecules. The copolymer chains exist in random coil or micellar (see below) conformation at low temperature, and as the temperature increases to a critical value (LCST), polymer chains transferred to globular structures, leading to polymer precipitation. By increasing the hydrophilic content from CP30 to CP5, the solubility of the copolymers is improved. Therefore, more energy is required to break the hydrogen bonds to cause the aggregation and precipitation of the copolymer, leading to an increased LCST. It is important to mention that these phase transitions were reversible in nature. Nevertheless, these results indicate that the LCST can be tuned to a particular temperature by incorporating different molar factions of SAMA units in the copolymer chain.

Fig. 6 Variation of % T with temperature for the aqueous solutions (2.0 mg mL⁻¹) of P(SAMA-co-PEGMA) copolymers.

Self-assembly study of random copolymers

Self-assembly of polymers is usually regarded as an attractive method to produce various higher order structures, such as spheres, rods, vesicles, cylinders, etc. However, most of the self-assembly studies have been reported on block copolymers, because their morphologies can easily be tuned and even predicted by the molecular parameters, such as the molecular weight, length of the hydrophobic or hydrophilic block and the chemical nature of blocks. While random copolymers hardly self-assemble to predetermined morphologies because of their indistinct structure, arising from random distribution of hydrophilic and hydrophobic segments throughout the polymer chain. The balance between hydrophilic and hydrophobic segments, known as the hydrophilic–lipophilic balance (HLB) solely control the self-assembly.⁴⁷ Since our copolymers contain hydrophobic stearate moieties and hydrophilic PEO units in the side-chain of the copolymers, therefore we studied their aggregation behavior in aqueous medium.

To study the self-assembly behavior of copolymers, at first ¹H NMR spectroscopy was investigated in two different solvents: D₂O and CDCl₃. CDCl₃ is a good solvent for both the stearate and PEO units in the copolymer, whereas stearate moieties are insoluble in D₂O. The ¹H NMR spectrum of CP20 copolymer in CDCl₃ (Fig. 1C) shows peaks for all the protons from both segments, whereas characteristic resonance signals from the SAMA segment almost vanished when the NMR of CP20 was conducted in D₂O (Fig. S2†). This could be due the formation of self-assembled structure from the P(SAMA-co-PEGMA) copolymer through arrangement of micelle-like structure with the hydrophilic PEO units on the shells and stearate moieties in the core. As a result, the protons from stearate moieties in the core of the micelle did not appear in the spectrum. Similar results were obtained with CP10 and CP30 copolymers (data not shown here). Furthermore, the hydrodynamic diameter (D_h) of various copolymers was determined by DLS study at 25 °C to investigate the aggregation behavior in aqueous solution (Fig. 7). We observed D_h values of 34, 39 and 45 nm for the CP10, CP20 and CP30 copolymers, respectively, once again suggesting self-aggregated particles from these copolymers. To obtain a more accurate structure of the morphology from the self-assembled random copolymer particles, we recorded SEM images. Fig. 8 shows SEM images from CP10, CP20 and CP30 copolymers, indicating that aggregates are spherical in shape. From SEM images, we have determined average size of 57, 65 and 71 nm, for the CP10, CP20 and CP30 copolymers, respectively. Note that these sizes are somewhat higher compared to the DLS sizes, and this could be due to the differences in the measurement technique.

Fig. 7 DLS study of CP10, CP20 and CP30 at 25 °C in aqueous medium (concentration = 1 mg mL⁻¹).

Fig. 8 SEM images of CP10, CP20 and CP30 (from left to right). The scale bar corresponds to 200 nm. Copolymer solutions in de-ionized water (0.2 mg mL⁻¹) were deposited onto silicon wafer.

Critical micellar concentration (CMC)

Since amphiphilic random copolymers formed micelles in water, herein we studied self-assembly characteristic of CP5, CP10, CP20 and CP30 in aqueous medium by fluorescence spectroscopy using, pyrene as fluorescent probe. Pyrene is a hydrophobic fluorescent molecule; its fluorescence behavior in the aqueous medium differs from that in the micelle core.⁴⁸ Therefore, emission spectra were recorded for different concentrations of each copolymer aqueous solution containing same concentration of pyrene.

Fig. 9A shows the fluorescence emission spectra of pyrene as a function of CP10 concentration in water. Intensity of fluorescence gradually increased with increasing CP10 concentration indicating solubilization of pyrene in the hydrophobic microdomains of the micelles. There are five electronic vibration peaks in the pyrene emission spectra. The ratio between intensities at two wavelengths, 372 nm (I₁) and 384 nm (I₃), was used to determine CMC because it varied with the micro polarity changes resulting from the micelle formation. The I₃₈₄/I₃₇₂ ratio of pyrene, encapsulated in aqueous solution of CP10, gradually changes with increasing polymer concentration (Fig. 9B). CMC values of 4.6, 2.4 and 2.3 mg L⁻¹ were determined respectively for the CP10, CP20 (Fig. S3†) and CP30 (Fig. S4†), indicating gradual increase in aggregation tendency with increasing SAMA contents into the copolymer chain. The CP5 did not increase the fluorescence intensity of pyrene with increasing polymer concentration, thus signifies the lack of aggregation to form hydrophobic cores due to the overall high hydrophilicity of CP5.

Fig. 9 (A) Emission spectra of pyrene at various concentrations of CP10 copolymer and (B) plot of intensity ratio I₃₈₄/I₃₇₂ versus concentrations of CP10 in water.

Cytotoxicity of polymers

Since side-chain PEO and stearate moieties are biocompatible, cytotoxicity of copolymers was evaluated by MTT assay, which is based on the conversion of pale-yellow MTT into dark purple formazan crystals by living cells, which determines mitochondrial activity.⁴⁹ Since for most cell populations the total mitochondrial activity is related to the number of live cells, this assay is generally used to estimate the in vitro cytotoxic effects of drugs on cell lines. The cell biocompatibility of the synthesized copolymers was tested using the MTT assay in HeLa cell, and the results are shown in Fig. 10 and S5,† where no obvious cytotoxicity against HeLa cells is observed even at high concentration of copolymer, 200 μg mL⁻¹. The cell viability of copolymer-treated wells is the same or greater when compared to the control group, thus indicating excellent biocompatibility of fatty acid based copolymers.

Fig. 10 Cytotoxicity of CP10 copolymer at different concentrations. For each concentration of CP10, the bar height shows the mean value of the three data sets and the error bar indicates the highest of the three values.

Conclusion

We have demonstrated the synthesis of well-defined biocompatible P(SAMA-co-PEGMA) copolymers from SA and PEGMA by using RAFT technique. Using copolymer compositions obtained from ¹H NMR analysis, the reactivity ratios was determined by the extended K–T method at high monomer conversion, which confirmed random distribution of SAMA and PEGMA units in the copolymer chain. These copolymers exhibited tunable thermoresponsive properties and with increasing hydrophobic/hydrophilic ratio in the copolymer the LCST values decreased continuously. DSC, POM and WAXD studies confirmed crystalline structures of the copolymers at ≥30% SAMA content in the copolymer. Self-aggregation of the copolymers was dependent on hydrophilic PEGMA to hydrophobic SAMA contents in the copolymers and the CP10, CP20 and CP30 random copolymers self-assembled to micellar type structures in aqueous solution with CMC values ranging between 4.6–2.3 mg L⁻¹. We believe that such biocompatible random copolymers may be attractive candidates for thermoresponsive drug delivery systems.^50,51

Acknowledgements

BM acknowledges UGC, New Delhi, India for the senior research fellowship.

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Footnote

† Electronic supplementary information (ESI) available: Additional figures as noted in the text. See DOI: 10.1039/c6ra00336b

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