Synthesis of tailor-made bile acid sequestrants by supplemental activator and reducing agent atom transfer radical polymerization

Abstract

This work reports the synthesis of tailor-made polymeric bile acid sequestrants (BAS) by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP) using ecofriendly conditions. The new materials were based on amphiphilic poly(methyl acrylate)-b-poly((3-acrylamidopropyl)trimethylammonium chloride) (PMA-b-PAMPTMA) star block copolymers and cationic hydrogels (PAMPTMA). The in vitro sequestration ability of the polymers was investigated in simulated intestinal fluid using sodium cholate (NaCA) as the bile salt model molecule. Both polymeric structures investigated showed higher affinity towards NaCA micelles than unimers. The cationic hydrogels proved to be attractive BAS candidates, with binding parameters similar to those of the most effective commercial BAS: Colesevelam hydrochloride. Several polymer features were investigated for the star block copolymers in order to understand the structure/performance relationship. It was found that the binding parameters can be tuned by targeting different compositions of the block copolymers and, typically, longer cationic arms led to enhanced binding capacity.

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Introduction

Polymeric bile acid sequestrants (BAS) are polymers used as therapeutic agents for the treatment of hypercholesterolemia.¹ These polymers act in the gastrointestinal (GI) tract, by selectively binding and removing bile salts from the enterohepatic circulation, which will ultimately lead to the reduction of the plasma cholesterol level.² It is worth mentioning that the first line treatment of hypercholesterolemia is based on statins, which act directly on the cholesterol synthesis by inhibiting the HMG-CoA reductase.^3,4 However, by inhibiting the cholesterol production, statins are also compromising the synthesis of products from branches of HMG-CoA reductase pathway (e.g., ubiquinone and dolichol), that play important roles in vital metabolic processes.^5–7 Therefore, there are several advantages associated with the use of BAS over statins, such as the absence of complications in the liver by long-term administration and the fact that BAS can be safer for pregnant women or patients with hepatic dysfunction.⁸ Currently, there are a few BAS available on the market, being Colesevelam hydrochloride (Welchol® or Cholestagel®) the most recent and effective one.⁹ However, the therapeutic efficacy of BAS is still rather low in comparison to statins. This output is mainly due to the low selectivity of BAS for trihydroxylic bile acids, as well as the fast dissociation of the BAS–bile acid complex in the presence of the active bile acids reuptake transporter system of the GI tract.³ Due to that, high doses of BAS (16–24 g per day) are usually required to produce desirable therapeutic effects, which most of the times results in poor patient compliance.

There are several reports in the literature dedicated to the preparation of BAS.^10–16 Cationic hydrogels based on (meth)acrylates, vinyl polymers, allyl polymers, poly(meth)acrylamides and polyethers are the most common used polymeric structures. Also, polymers based on polystyrene backbones (as a hydrophobic segment), containing amine/ammonium pendant groups (as a cationic hydrophilic segment) have been employed.^2,3,17 It is known that the binding process is mainly ruled by both electrostatic and hydrophobic interactions.¹⁸ For this reason, an appropriate balance between the charged groups and the hydrophobic segments of the polymer is required in order to achieve high maximum binding capacity. In addition, the degree of crosslinking of the hydrogel (swelling characteristics) should be adjusted to allow the diffusion of bile acids through the polymer network. Despite the findings on the importance of controlling the BAS structure, these materials have been prepared mainly by conventional polymerization techniques, which hamper any fine control over the polymeric structures. To the best of our knowledge, there is only one work^11,19 which describes the controlled synthesis of polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) block copolymers by anionic polymerization, as new BAS candidates in the form of micelles. However, the reaction conditions employed were very strict (e.g., T = −78 °C) and it was necessary to perform post-polymerization reactions to obtain the cationic segment. Reversible deactivation radical polymerization (RDRP) techniques could be a very useful tool for the design of tailor-made BAS. By this means, it could be also possible to deepen the understanding of the polymer structure–performance relationship.

The aim of this work was to prepare alternative structures to the commercial hydrogels, namely amphiphilic star block copolymers, using an ecofriendly supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP) system to afford a fine control over the polymers structure and molecular weight. To the best of our knowledge, this is the first time that such polymer structures are studied as potential BAS. Controlled poly((3-acrylamidopropyl)trimethylammonium chloride) (PAMPTMA)-based cationic hydrogels were also synthesized by SARA ATRP to assess the influence of the architecture on the performance of the materials. The binding capacity of the synthesized polymers was evaluated by in vitro equilibrium binding experiments with sodium cholate (NaCA).

Experimental

Materials

Acetonitrile (high-performance liquid chromatography (HPLC) grade, Fisher Chemical), ((3-acrylamidopropyl)trimethylammonium chloride) (AMPTMA, solution 75 wt% in H₂O, Aldrich), alumina (basic, Fisher Scientific), 1,4 butanediol diacrylate (BDDA, 99+%, Alfa Aesar), CuBr₂ (Acros, 99+% extra pure, anhydrous), CuCl₂ (99% Sigma Aldrich), NaCA (99% Acros Organics), deuterated chloroform (CDCl₃, 99.8% Cambridge Isotope Laboratories), deuterium oxide (99.9%, Cambridge Isotope Laboratories), dimethylformamide (DMF, Sigma-Aldrich, +99.8%), dipotassium hydrogen phosphate (Merck), ethanol (99.5%, Panreac), ethyl 2-chloropropionate (ECP, 97%, Aldrich), hydrochloric acid (37%, Fisher Scientific), N-phenyl-1-naphthylamine (NPN) (Merck), pancreatin from porcine pancreas (Sigma), pepsin (Sigma Aldrich), phosphoric acid (85% Fisher Scientific), potassium dihydrogen phosphate (Merck), sodium chloride (99.5%, Acros Organics), tetrabutylammonium hydroxide 30-hydrate (≥99.0%, Sigma-Aldrich) and tetra(ethylene glycol)diacrylate (TEGDA, Sigma Aldrich) were used as received.

Colesevelam hydrochloride was kindly supplied by Bluepharma and it was used as received.

Dipentaerythritol hexakis(2-bromoisobutyrate) (6f-BiB) (yield = 11%),²⁰ pentaerythritol tetrakis(2-bromoisobutyrate) (4f-BiB)²⁰ (yield = 20%) and tris[2-(dimethylamino)ethyl]amine (Me₆TREN)²¹ (yield = 90%) were synthesized according the procedures described in the literature. Both ¹H and ¹³C NMR spectra of 6f-BiB, 4f-BiB, and Me₆TREN are provided in the ESI (Fig. S1–S3,† respectively).

Metallic copper (Cu(0), d = 1 mm, Sigma Aldrich) was washed with HCl in methanol and subsequently rinsed with methanol and dried under a stream of nitrogen following the literature procedure.²²

Methyl acrylate (MA) (Acros, 99% stabilized), was passed through a sand/alumina column before use in order to remove the radical inhibitor.

Purified water (Milli-Q®, Millipore, resistivity > 18 MΩ cm) was obtained by reverse osmosis.

Simulated gastric fluid (SGF) was prepared by dissolving sodium chloride (100 mg) and pepsin (160 mg) in hydrochloric acid (350 μL). The volume was adjusted to 50 mL with water to give a solution with pH = 1.2.

Simulated intestinal fluid (SIF) was prepared by dissolving dipotassium hydrogen phosphate (203 mg), potassium dihydrogen phosphate (182 mg) and pancreatin (500 mg) in water (40 mL). The pH was adjusted with 0.2 N NaOH or 0.2 N HCl to 6.8 and the volume was completed to 50 mL with water.

Techniques

PAMPTMA samples were analyzed by a size exclusion chromatography (SEC) system equipped with an online degasser, a refractive index (RI) detector and a set of columns: Shodex OHpak SB-G guard column, OHpak SB-804HQ and OHpak SB-804HQ columns. The polymers were eluted at a flow rate of 0.5 mL min⁻¹ with 0.1 M Na₂SO₄ (aq)/1 wt% acetic acid/0.02% NaN₃ at 40 °C. Before the injection (50 μL), the samples were filtered through a polytetrafluoroethylene (PTFE) membrane with 0.45 μm pore. The system was calibrated with five narrow poly(ethylene glycol) standards and the molecular weights (M^SEC_n) and Đ (M_w/M_n) were determined by conventional calibration using the Clarity software version 2.8.2.648.

PMA samples were analyzed by a high performance size exclusion chromatography HPSEC; Viscotek (Viscotek TDAmax) with a differential viscometer (DV), right-angle laser-light scattering (RALLS, Viscotek), low-angle laser-light scattering (LALLS, Viscotek) and RI detectors. The column set consisted of a PL 10 mm guard column (50 × 7.5 mm²) followed by one Viscotek T200 column (6 μm), one MIXED-E PLgel column (3 μm) and one MIXED-C PLgel column (5 μm). HPLC dual piston pump was set with a flow rate of 1 mL min⁻¹. The eluent (THF) was previously filtered through a 0.2 μm filter. The system was also equipped with an on-line degasser. The tests were done at 30 °C using an Elder CH-150 heater. Before the injection (100 μL), the samples were filtered through a PTFE membrane with 0.2 μm pore. The system was calibrated with narrow polystyrene standards. The dn/dc was determined as 0.063 for PMA. Molecular weight (M^SEC_n) and Đ of the synthesized polymers were determined by Multidetectors calibration using OmniSEC software version: 4.6.1.354.

400 MHz ¹H NMR spectra of reaction mixture samples were recorded on a Bruker Avance III 400 MHz spectrometer, with a 5 mm TIX triple resonance detection probe, in D₂O or CDCl₃. Conversion of monomers was determined by integration of monomer and polymer NMR signals using MestRenova software version: 6.0.2-5475.

Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy was performed using a Jasco, model 4000 UK spectrometer. The samples were analyzed with 64 scans and 4 cm⁻¹ resolution, between 500 and 3500 cm⁻¹.

The critical micellar concentration (CMC) of NaCA in SIF at both pH = 6.8 and pH = 7.6 at 37 °C was determined using a method described in the literature,²³ based on the increase in fluorescence quantum yield and blue shift of the fluorescence emission spectra of NPN upon association with micelles (Fig. S4 and S5†). The total concentration of NPN was 1 μM, and the concentrations of NaCA were investigated between 0 and 40 mM. The fluorescent emission of NPN was followed using an excitation wavelength of 356 nm on a Cary Eclipse fluorescence spectrophotometer from Varian (Victoria, Australia) equipped with a thermostated multi-cell holder accessory. The CMC was determined to be 6.7 and 6.0 for pH = 6.8 and pH = 7.6, respectively. Based on the asymptotic statistical theory,²⁴ for a 95% confidence level, the confidence intervals obtained for the determination of the CMC values were [6.2–7.1] (CMC = 6.7) and [5.8–6.3] (CMC = 6.0).

The concentration of free NaCA remaining after the equilibrium binding experiments was determined by HPLC. The analyses were conducted at 25 °C using an Agilent Zorbax ODS, 5 μm, 4.6 × 250 mm column, under isocratic elution. The mobile phase used was 20 mM tetrabutylammonium hydroxide 30-hydrate (pH adjusted to 7.5 with phosphoric acid)/acetonitrile = 55/45 (v/v), with a flow rate of 0.7 mL min⁻¹. The NaCA was detected by ultraviolet light at 210 nm. A NaCA standard calibration curve (R² > 0.99) was constructed for the quantification of the NaCA present in the injected sample. For the quantification of low concentrations (0.1–3 mM), a 200 μL injection loop was used. For concentrations above 3 mM, a 20 μL injection loop was used. To check the system stability, one NaCA standard solution was injected after every 10 sample injections.

Isothermal titration calorimetry (ITC) analyses were performed on a VP-ITC instrument from MicroCal (Northampton, MA) with a reaction cell volume of 1410.9 μL, containing an amphiphilic star block copolymer solution, at 37 °C. A 5 mM solution of NaCA was injected at a speed of 0.5 μL s⁻¹, stirring speed was 459 rpm, and the reference power was 10 μcal s⁻¹. As recommended by the manufacturer, a first injection of 4 μL was performed before the experiment was considered to start in order to account for diffusion from/into the syringe during the equilibration period. The titration proceeded with additions of 10 μL per injection. All solutions were previously degassed for 15 min under reduced pressure.

The content of nitrogen in the amphiphilic star block copolymers was determined by elemental analysis using a Fisons – EA-1108 CHNS-O Element Analyzer.

Procedures

Typical procedure for the preparation of amphiphilic star block copolymers by SARA ATRP. A series of amphiphilic star block copolymers were synthesized by SARA ATRP, varying the number of arms of the star (using either 4f-BiB or 6f-BiB as the initiator) and the degree of polymerization (DP) of both MA and AMPTMA monomers. Generally, the SARA ATRP followed this procedure: a solution of CuBr₂ (1.2 mg, 5.5 μmol) and Me₆TREN (6.4 mg, 27.6 μmol) in water (0.2 mL) and a solution of 4f-BiB (40.4 mg, 55.2 μmol) in ethanol (0.8 mL) and MA (2.0 mL, 22.1 mmol), were added to a 10 mL Schlenk tube equipped with a magnetic stirrer bar. Next, Cu(0) wire (l = 5 cm; d = 1 mm) was added to the Schlenk tube, which was sealed with a glass stopper, deoxygenated with three freeze–vacuum–thaw cycles and purged with nitrogen. The reaction was allowed to proceed with stirring (700 rpm) at 30 °C. When MA reached high conversion, an aliquot was collected and analyzed by ¹H NMR spectroscopy in order to determine the monomer conversion, and by SEC to determine the M^SEC_n and Đ of the polymer. Then, the reaction mixture (Br–PMA macroinitiator) was transferred to a 25 mL Schlenk flask containing a degassed mixture of AMPTMA (3.8 mL, 13.8 mmol), CuBr₂ (12.3 mg, 55.2 μmol), Me₆TREN (7.6 mg, 33.1 μmol), Cu(0) wire (l = 10 cm; d = 1 mm), water (0.1 mL) and ethanol (5.2 mL), under nitrogen. The second monomer (AMPTMA) was allowed to react at 30 °C for 20 h. The product solution was purified through dialysis (cut-off 3500) against ethanol and then water and the amphiphilic star block copolymer was recovered after freeze drying. The polymer was analyzed by elemental analysis in order to estimate the DP of the AMPTMA.

Typical procedure for the preparation of hydrogels by SARA ATRP. Several PAMPTMA-based hydrogels were synthesized by SARA ATRP, using two different crosslinkers (BDDA or TEGDA). Generally, the SARA ATRP followed this procedure: a solution of CuCl₂ (5.4 mg, 40.0 μmol) and Me₆TREN (21.5 mg, 80.6 μmol) in water (760 μL) and a solution of ECP (11.3 mg, 80.6 μmol) in ethanol (1.26 mL), AMPTMA (2.0 mL, 8.05 mmol) and TEGDA (244 mg, 0.8 mmol), were added to a 10 mL Schlenk tube equipped with a magnetic stirrer bar. DMF (30 μL) was added to the mixture to serve as the internal standard for the determination of the monomers conversion by ¹H NMR spectroscopy. Next, Cu(0) wire (l = 5 cm; d = 1 mm) was added to the Schlenk tube, which was sealed with a glass stopper, deoxygenated with three freeze–vacuum–thaw cycles and purged with nitrogen. The reaction was allowed to proceed with stirring at room temperature during 24 h. Finally, the reaction mixture was washed several times with ethanol and water and the hydrogel was recovered by freeze drying.

Swelling tests. The hydrogel samples were soaked in SIF (without pancreatin) for 24 h at 37 °C (100 rpm), and then centrifuged sufficiently to pellet the swollen polymer. The supernatant was discarded and the remaining water was gently removed using filter paper. The swelling capacity of the hydrogels was determined from eqn (1):

In the eqn (1), w_s is the weight of the swollen sample after immersion and w_d is the weight of the dry sample before immersion. The samples were analyzed in duplicate.

In vitro degradation studies. The degradation of linear PAMPTMA and star-shaped PMA was evaluated in both SGF and SIF solutions. Briefly, 30 mg of purified polymer was immersed in 3 mL of each solution in 10 mL screw-cap glass tubes. The samples were incubated at 37 °C under continuous shaking at 100 rpm. After a predetermined incubation time (2 h for SGF and 3 h for SIF), the samples were analyzed by SEC and NMR to evaluate the extent of degradation. The experiments were done in triplicate.

NaCA equilibrium binding experiments. The equilibrium binding studies were performed by varying the initial concentration of NaCA ([NaCA]₀) in the SIF (without pancreatin). For each polymer, eight incubation flasks containing a 10 mg sample were set up. Next, 2 mL of SIF were added to the flasks and the polymers were allowed to soak at room temperature overnight. In the following day, predetermined volumes of SIF and 40 mM stock solution of NaCA were added to the flasks to make the final volume of the solvent mixture 10 mL with NaCA target concentrations of 0.1, 0.3, 1, 3, 7, 10, 20 and 30 mM. A blank incubation tube containing just 10 mL of SIF (control) was also prepared for each polymer. The nine flasks were incubated at 37 °C (100 rpm) for 24 h and then filtered. The hydrogel samples were filtered with polyvinylidene fluoride (PVDF) syringe filters (0.45 μm), while the amphiphilic star block copolymers were filtered using Macrosep® Advance Centrifugal Devices With Supor® Membrane 10 000 (Pall Corporation). The first 5 mL of filtrate were discarded and then an aliquot of 2.5 mL was collected for further analysis. The filtrates were analyzed by HPLC in order to determine the free concentration of NaCA. The amount of NaCA bonded to the polymers was calculated from the difference between the initial amount of NaCA introduced in the binding assays and the amount of free NaCA in the filtrates. The Hill equation (eqn (2)) was used to fit the experimental data, using the SigmaPlot10 software, in order to determine the binding parameters for each polymer.

In the eqn (2), q_e is the amount of NaCA bonded to the polymer in mg g⁻¹ polymer, q_max is the apparent maximum amount of NaCA bonded to the polymer (binding capacity) in mg g⁻¹, K is the intrinsic binding constant (relative to the strength of binding) in L mg⁻¹, C_e is the free NaCA concentration in mg L⁻¹ and n is the cooperative parameter (measure of the cooperativity of binding). For n = 1, the eqn (2) corresponds to the Langmuir isotherm (noncooperative binding).

Results and discussion

Polymer synthesis and characterization

The potential of the SARA ATRP method to produce unique and controlled polymeric structures was explored for the preparation of novel BAS candidates based on star-shaped amphiphilic block copolymers. These copolymers were designed to have a PMA hydrophobic core, which could avoid the dissolution of the polymer in aqueous environment, and cationic PAMPTMA hydrophilic arms, that could be able to bind bile salts (Fig. 1). In opposition to the commercial BAS, in this case the binding of bile salts through electrostatic interactions should not be affected by the changes in the pH value along the intestine environment, due to the presence of permanent positive charges in the polymeric hydrophilic segment. It is also expected that the star-shaped architecture could provide high surface area, which could enhance the efficiency of the binding process. In addition, it is known that star-shaped amphiphilic polymers can form very stable nanoassemblies in solution in a vast range of concentrations, in opposition to micelles formed by the self-assembly of linear amphiphilic block copolymers.²⁵

Fig. 1 Generic structure of the amphiphilic star block copolymers (PMA-b-PAMPMTA) prepared by SARA ATRP.

Star-shaped polymeric architectures with controlled structure can only be prepared by advanced polymerization techniques, such as ATRP. Previous publications by our research group^26,27 reported the development of ecofriendly SARA ATRP systems for the synthesis of both PMA and PAMPTMA in ethanol/water mixtures near room temperature. In this work, the SARA ATRP method developed was extended to the preparation of the new star-shaped PMA-b-PAMPTMA block copolymers by “one-pot” polymerization in ethanol/water = 80/20 (v/v). It should be stressed that the direct copolymerization of MA and AMPTMA is a notable achievement, considering the very different nature of these two monomers. Due to solubility issues, it was not possible to find an appropriate solvent for the SEC analysis, in order to determine both block copolymers molecular weight and dispersity. Alternatively, the molecular weight of the block copolymers was estimated taking into account the molecular weight of the star-shaped PMA (first stage of the polymerization), which was determined by SEC, and the content of nitrogen from the PAMPTMA block on the final block copolymer. Additionally, the success of the copolymerization was confirmed by the appearance of the characteristic adsorption bands of both PMA and PAMPMTA blocks in the FTIR spectrum of the copolymers (Fig. 2): quaternary ammonium group at 1480 cm⁻¹ and amide C=O stretching, amide II band and amide I band at 1550 cm⁻¹ and 1650 cm⁻¹, respectively, from the PAMPTMA and ester C=O stretching at 1729 cm⁻¹ from the PMA.²⁸

Fig. 2 FTIR spectra of PMA, PAMPTMA and amphiphilic star block copolymer (PMA-b-PAMPTMA) samples prepared by SARA ATRP.

Block copolymers with different targeted properties, such as the number of arms, were prepared in order to evaluate the influence of the polymer structure/composition on the binding of bile acids. Table 1 summarizes the main features of the amphiphilic star block copolymers produced by SARA ATRP.

Table 1 Composition and molecular weight of the star-shaped PMA-b-PAMPTMA block copolymers synthesized by “one-pot” SARA ATRP in ethanol/water = 80/20 (v/v) at room temperature

Sample code	1^st block		2^nd block	Block copolymer
	MA DP/arm^a	Đ^b	AMPTMA DP/arm^a	Number of arms	Mn × 10^−3^c
a [Monomer]0/[initiator]0/number of arms.b Determined by SEC in THF at 30 °C.c The molecular weight of the second block (PAMPTMA) was estimated by elemental analysis.
4(PMA95-b-PAMPTMA50)	95	1.09	50	4	74.8
4(PMA94-b-PAMPTMA24)	94	1.13	24	4	52.9
4(PMA94-b-PAMPTMA16)	94	1.07	16	4	46.3
4(PMA199-b-PAMPTMA52)	199	1.08	52	4	112.2
6(PMA98-b-PAMPTMA52)	98	1.09	52	6	116.2
6(PMA106-b-PAMPTMA38)	106	1.09	38	6	103.0
6(PMA104-b-PAMPTMA18)	104	1.10	18	6	77.2

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The SARA ATRP method was also employed for the preparation of PAMPTMA-based hydrogels (Fig. 3), a common polymeric architecture used as BAS. It is expected that hydrogels prepared by ATRP present a more homogeneous structure that the ones obtained by conventional free radical polymerization techniques, due to the fast initiation and reversible deactivation reactions.^29–31

Fig. 3 Generic structure of the cationic hydrogels prepared by SARA ATRP.

ECP was chosen to initiate the polymerization since it was previously demonstrated that this is an effective initiator for the SARA ATRP of AMPTMA in ethanol/water mixtures.²⁷ The monomer concentration was maintained in all the polymerizations ([AMPTMA]₀ = 2 M) and two different diacrylate crosslinkers with distinct hydrophilicities were tested (BDDA and TEGDA). Different targeted DP values of both monomer and crosslinker were investigated and the main results are summarized in Table 2.

Table 2 Targeted DP and conversion achieved of both monomer (AMPTMA) and crosslinker in the preparation of cationic hydrogels by SARA ATRP. Reaction conditions: [AMPTMA]₀ = 2 M; [ECP]₀/[CuCl₂]₀/[Me₆TREN]₀ = 1/0.5/1.0; Cu(0) wire: l = 5 cm and d = 1 mm; ethanol/water = 50/50; T = 25 °C; t = 24 h

Sample code	Crosslinker	AMPTMA DP^b	Crosslinker DP^b	AMPTMA conv. (%)	Crosslinker conv. (%)
a No macroscopic gelation was observed.b [AMPTMA or crosslinker]0/[ECP]0.c Data not available.
AT 100/10	TEGDA	100	10	50	49
AT 50/10	TEGDA	50	10	^c	^c
AT 50/3^a	TEGDA	50	3	^c	^c
AB 100/10	BDDA	100	10	35	35
AB 50/10	BDDA	50	10	61	42
AB 50/5	BDDA	50	5	61	63
AB 50/3^a	BDDA	50	3	^c	^c

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It was reported that the concentration of monomer influences the gelation point and ultimately it could prevent the formation of a crosslinked polymeric structure, due to the occurrence of intramolecular cyclization under dilute conditions.³² In this work, the AMPTMA concentration proved to be sufficient to allow macroscopic gelation (change from viscous liquid to elastic gel)²⁹ of the polymer, for [crosslinker]₀/[ECP]₀ = 5 or 10 (Table 2). For a targeted ratio [crosslinker]₀/[ECP]₀ = 3, the polymer remained soluble after the predetermined time of reaction (24 h) (AT 50/3 and AB 50/3 in Table 2). In this case, the system did not reach the gel point, probably due to a low conversion of the crosslinker achieved after 24 h (data not available). Macroscopic gelation should occur when, on an average basis, each primary chain contains at least one crosslink point, meaning that the ratio [reacted crosslinker]/[initiator] should be one, considering a high initiation efficiency (each molecule of initiator originates one primary polymer chain).³³

Regarding the AMPTMA conversion, the value achieved was not high as it was expected from the results obtained in the reported AMPTMA homopolymerization by SARA ATRP (conv. > 90% in less than 2 hours).²⁷ Similarly, both crosslinkers did not reach very high conversion. This observation could mean that the reaction should be kept longer to allow full monomer conversion, considering the diffusion limitations experienced by monomers and catalytic complex in the polymer network after the gelation point.³¹ A control SARA ATRP reaction was allowed to proceed during seven days and led to similar results (compared with a 24 h reaction) in terms of both monomer and crosslinker conversions. In order to prepare PAMPMTA-based hydrogels with targeted properties, such as crosslink density, further investigation of the gelation process is required. This includes kinetic studies and the determination of both the gelation points and the gel fractions for different hydrogels. Nevertheless, the results are reproducible and this work clearly demonstrates the potential of the SARA ATRP as versatile tool for the preparation of cationic hydrogels.

Sodium cholate equilibrium binding by cationic hydrogels

Bile acids are amphiphilic molecules that are usually conjugated with the amino acids taurine or glycine in the human bile.¹⁶ Under the small intestine conditions, these complexes are in their ionized form and for that reason they are also known as bile salts. In this work, NaCA was used as a model bile salt molecule for the equilibrium binding studies. Cholic acid-based bile salts represent a large part (30–40%) of the bile salts existing in the human bile.³⁴ Therefore, the results obtained wit NaCA could be a good indicator of the binding capacity of the new BAS candidates. The PAMPTMA-based hydrogels prepared by SARA ATRP exhibited sigmoidal binding curves (Fig. 4). These results suggest the existence of cooperative binding, meaning that the affinity of the hydrogel for the bile salts changes with the amount of bile salts already bonded. Therefore, the overall binding constant (K) and cooperative parameter (n) were determined from the Hill equation (corresponding R² values shown in Table 3) which is commonly used to describe cooperative binding processes.³⁵ This type of binding has been also observed for other hydrogel-based polymeric BAS.^13,14,35

Fig. 4 Isotherms for the binding of NaCA by the Colesevelam and the hydrogels prepared by SARA ATRP. Binding conditions: 50 mM phosphate buffer (pH = 6.8) at 37 °C. The lines represent the fitting to Langmuir or Hill models for the Colesevelam and hydrogels, respectively.

Table 3 Binding parameters of the cationic hydrogels prepared by SARA ATRP and the commercial BAS Colesevelam

Sample code	R^2	K (mM^−1)	n	qmax^a (mg g^−1) × 10^−3
a mg NaCA per g polymer.
Colesevelam	0.9771	0.260 ± 0.090	1.0	2.23 ± 0.24
AT 100/10	0.9949	0.17 ± 0.005	5.4 ± 0.9	1.63 ± 0.04
AT 50/10	0.9868	0.22 ± 0.011	7.3 ± 4.3	1.45 ± 0.10
AB 100/10	0.9988	0.22 ± 0.004	6.5 ± 0.8	1.73 ± 0.03
AB 50/10	0.9983	0.22 ± 0.004	5.9 ± 0.6	1.49 ± 0.02
AB 50/5	0.9962	0.17 ± 0.003	10.7 ± 2.3	1.82 ± 0.06

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In this work, Colesevelam was used as a reference material since it is the most effective commercial BAS present in the market. The equilibrium binding of NaCA by Colesevelam (black symbols in Fig. 4) followed a typical Langmuir isotherm (Hill equation with n = 1, meaning no cooperativity), as reported by other authors for the binding of conjugated bile salts.³⁶ The strength of binding of NaCA by the cationic hydrogels prepared was comparable to that of Colesevelam, as judged by the similar K values (Table 3). In addition, the cooperative parameter was higher than 1 for all the hydrogels, which indicates positive cooperativity in the binding. This parameter remained relatively constant, regardless the type of crosslinker used.

In order to assess the overall efficacy of the prepared polymers, it is important to evaluate not only the binding capacity (q_max), but also the dissociation constant (K_d = 1/K). This will provide information about the ability of the polymers to bind NaCA molecules, under the physiological conditions. K_d, which represents the concentration of bile salt at which the polymer is half-saturated, should be much lower than the concentration of bile salts in the intestine in order to provide an effective binding. In average, the total concentration of bile salts in the human intestine is above 15 mM.³⁷ Therefore, the PAMPTMA-based hydrogels revealed to be promising BAS candidates (K_d = [4.5–5.9] mM), with an affinity towards NaCA similar to the one of Colesevelam. In addition, the binding capacity of the hydrogels (Table 3) is at least two times higher than the one reported in the literature for other hydrogel-based BAS candidates, as well as for the commercial BAS Cholestyramine, under similar conditions.^13,15,35

As previously mentioned, due to the limited monomer conversions achieved, some of the hydrogel samples prepared by SARA ATRP presented similar composition (different from the one targeted) which did not allow the investigation of the polymer composition/binding parameters relationship. Besides the hydrogels composition, their swelling capacity can also influence the binding parameters. It was reported that a more elastic polymer network could promote the cooperativity of the binding.¹⁴ The swelling capacities of the hydrogels prepared by SARA ATRP were determined in duplicate and the results (Table 4) showed that the swelling capacity of the hydrogels prepared by SARA ATRP is at least two times higher than the one of the commercial BAS Colesevelam, regardless the crosslinker used. Considering the final application, one could expect that these new hydrogels could form a softer gel in the intestine environment, which can decisively contribute to better patient compliance (less constipation).

Table 4 Composition and swelling capacity of the hydrogels prepared by SARA ATRP and Colesevelam

Sample code	Crosslinker	AMPTMA DP^a	Crosslinker DP^a	Swelling capacity × 10^−3 (%)
a Determined by ^1H NMR spectroscopy.b Data not available.
Colesevelam	—	—	—	0.64 ± 0.07
AT 100/10	TEGDA	50	4.9	2.11 ± 0.47
AT 50/10	TEGDA	^b	^b	1.77 ± 0.09
AB 100/10	BDDA	35	3.5	1.96 ± 0.08
AB 50/10	BDDA	30	6.1	2.00 ± 0.29
AB 50/5	BDDA	30	3.0	2.42 ± 0.33

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Sodium cholate equilibrium binding by amphiphilic star block copolymers

The equilibrium binding of NaCA by the amphiphilic star block copolymers was investigated using HPLC and the experiments were conducted under the same conditions used for the hydrogels. The Hill model fitted very well the experimental data for all the samples investigated (R² ≥ 0.98, Table 5) suggesting that the NaCA binding was cooperative, similarly to what was observed for the PAMPTMA-based hydrogels. Despite having K values (Table 5) similar to those of Colesevelam and PAMPTMA-based hydrogels, generally the star block copolymers presented lower NaCA binding capacity (compare Fig. 4 with 5).

Table 5 Binding parameters of the amphiphilic star block copolymers prepared by SARA ATRP and the commercial BAS Colesevelam

Sample code	R^2	K (mM^−1)	n	qmax^a (mg g^−1) × 10^−3
a mg NaCA per g polymer.
Colesevelam	0.9771	0.260 ± 0.090	1.0	2.23 ± 0.24
4(PMA95-b-PAMPTMA50)	0.9968	0.172 ± 0.003	7.0 ± 0.9	1.37 ± 0.03
4(PMA94-b-PAMPTMA24)	0.9997	0.129 ± 0.004	2.0 ± 0.1	0.95 ± 0.02
4(PMA94-b-PAMPTMA16)	0.9791	0.215 ± 0.025	2.6 ± 0.6	0.90 ± 0.07
4(PMA199-b-PAMPTMA52)	0.9971	0.301 ± 0.012	4.2 ± 0.5	0.80 ± 0.02
6(PMA98-b-PAMPTMA52)	0.9905	0.215 ± 0.013	3.4 ± 0.6	1.43 ± 0.08
6(PMA106-b-PAMPTMA38)	0.9896	0.172 ± 0.009	4.8 ± 1.1	1.36 ± 0.05
6(PMA104-b-PAMPTMA18)	0.9987	0.172 ± 0.013	1.6 ± 0.1	0.64 ± 0.02

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Fig. 5 Isotherms for the binding of NaCA by the Colesevelam and the amphiphilic star block copolymers prepared by SARA ATRP. Binding conditions: 50 mM phosphate buffer (pH = 6.8) at 37 °C. The lines represent the fitting to Langmuir or Hill models for the Colesevelam and amphiphilic star block copolymers, respectively.

In addition, the cooperative parameter was lower than the one observed for the hydrogels (compare Tables 3 and 5), suggesting that either larger polymer chains in solution or higher density of cationic charges (or a combination of both factors) could enhance the cooperativity of binding. In fact, it is interesting to note that even within the same set of star block copolymers, the cooperativity increases with the increase of both the size of the copolymer and the number of positive charges. Regarding the size of the polymers in solution, these results suggest that polymer networks could be more beneficial for the binding process, in comparison to some nanoassembled polymer structures, probably due to their high charge density and due to the fact that the swollen hydrogels could facilitate the diffusion of the bile salt molecules and their access to the polymer binding sites.

Nevertheless, the results also highlight the importance of fine-tuning the polymers composition, since it was possible to significantly increase the maximum binding capacity by changing, for instance, the size of the PMA hydrophobic core of the star (e.g., red and pink symbols in Fig. 5). Such control over the polymers features is only possible by using RDRP techniques, as the one investigated in this work (SARA ATRP).

As previously mentioned, the library of block copolymers synthesized in this work was designed to allow the investigation of the polymers structure/sequestration performance relationship. Fig. 6 shows the variation of the maximum binding capacity (q_max) with the increase of the hydrophilic cationic arm length (AMPTMA), for both 4-arm and 6-arm star block copolymers with a fixed core size (MA DP/arm = 100). Also, the concentration of free bile salt in solution at 95% of polymer saturation (C_e^0.95) was calculated from the binding parameters and it is shown in Fig. 6. This parameter, which depends on both K and n, provides a good estimate of the overall efficacy of the polymers under physiological conditions, for comparison purposes. Generally, it seems that there is a tendency of the star polymers to be more effective BAS for longer cationic segments (AMPTMA), regardless the number of arms of the star. This conclusion is supported by the increase of the cooperativity (considering similar K values; see details on Table 5), as well as by the decrease of the C_e^0.95 (black symbols in Fig. 6) to values below the physiological concentration of bile salts (C_e^0.95 < 15 mM). It is also worth to mention that the significant increase of the efficacy of the star polymers is observed for different lengths of the cationic segment, depending on the number of arms of the star. In addition, the increase of the hydrophilic cationic arm length led to an increase of the maximum binding capacity (red and green symbols in Fig. 6), regardless the number of arms of the stars. These results confirm that the electrostatic interactions play an important role in the binding process. The same trend was also observed by other authors¹¹ for BAS candidates based on cationic micelles.

Fig. 6 Variation of q_max and C_e^0.95 with the increase in the cationic arm length of the amphiphilic star block copolymers, for a fixed hydrophobic core size (MA DP/arm = 100): (a) 4-arm star and (b) 6-arm star. The error bars were obtained directly from the best fit of the Hill equation (q_max) or from the upper and lower limits of the average and standard deviation of K and n (C_e^0.95).

The influence of the balance between the cationic charges (PAMPMTA) and the hydrophobic regions (PMA) of the star block copolymers on the binding capacity, was also investigated (Fig. 7). The increase of the ionic density of the polymers led to an increase of the maximum NaCA binding capacity, regardless the number of arms of the stars. These results suggest that electrostatic interactions are the major driving force for the association between the bile salt and the polymer at high bile salt concentrations. Nevertheless, the interpretation of the results should be done with caution, since the binding parameters could not have a linear variation with the polymers features.^12–14 However, the results presented in this work suggest that the cationic monomer (AMPTMA) binds NaCA with high affinity under conditions mimicking the intestine environment.

Fig. 7 Variation of q_max with the increase of the ionic/hydrophobic density within the star block copolymers. The ionic/hydrophobic ratio of each star block copolymer was obtained from the AMPTMA DP/PMA DP ratio.

This work represents the first approach towards the design of BAS based on new polymeric structures (amphiphilic star block copolymers) and shows that the advanced polymerization techniques could be valuable tools for the understanding of the binding process.

Insight into the binding mechanism

The results on the binding of NaCA by both hydrogels and amphiphilic star block copolymers prepared by SARA ATRP suggested the existence of cooperative binding. Nichifor and co-authors interpreted this type of binding as a two-step process.^13,14 In the proposed model, the binding of the first bile salt molecules to the cationic hydrogel is stabilized by electrostatic interaction and is characterized by the K₀ constant. The binding of additional bile salt molecules involving hydrophobic interactions with the hydrogel and previously bound bile salt, is characterized by the cooperative parameter (n). In the proposed model, the overall binding constant (K) and the cooperative parameter can be determined directly from the Hill equation and the K₀ value is given by the ratio between K and n.

In this work, ITC was used for the preliminary investigation of the binding thermodynamics between the NaCA molecules and the amphiphilic block copolymers. This technique has been employed for the study of the binding of bile salts by several receptors.^38–40 Contrary to the determination of the binding isotherms by HPLC, ITC can provide valuable information about the binding process, such as the binding enthalpy change.⁴¹ The preliminary ITC experiments were conducted using an initial concentration of NaCA below the CMC to avoid the formation of micelles, which could difficult the analysis of the titration results. However, in those conditions, all the star block copolymers showed to have low affinity towards the NaCA molecules (Fig. S6†). Therefore, the titration curves did not allow the determination of the binding parameters. Nevertheless, it was possible to observe that the enthalpy of binding was positive (endothermic process), suggesting that the binding process was mainly governed by hydrophobic interactions, for the low NaCA concentration investigated (below 1 mM). Additionally, the heat involved in the binding was strongly dependent on the buffer ionization enthalpy indicating changes on the ionization of the NaCA upon binding, corresponding to an increase in the NaCA neutral form (Fig. S7†). These results indicate the prevalence of hydrophobic interactions under the conditions investigated, contrary to what is proposed by the binding model reported in the literature.^13,14

It is worth to mention that the interpretation of the ITC results should be done with caution, since the NaCA concentration investigated corresponds to the low affinity region of the binding isotherm. Further analyses will be needed to clarify the binding mechanism. One of the parameters that could influence a cooperative binding process is the starting concentration of both polymer and NaCA, which ultimately will affect their conformation in solution (e.g., formation of micelles) and their affinity towards each other. To test this hypothesis, different initial concentrations of a selected hydrogel sample were used for the equilibrium binding assays (Fig. 8).

Fig. 8 Isotherms for the binding of NaCA by a PAMPTMA-based cationic hydrogel (AB 100/10) prepared by SARA ATRP, using different starting concentrations of hydrogel for the binding assays: (a) amount of NaCA bound as a function of the initial concentration of NaCA and (b) amount of NaCA bound as a function of the free NaCA in solution at equilibrium. Binding conditions: 50 mM phosphate buffer (pH = 6.8) at 37 °C. The lines represent the fitting to the Hill model.

The cooperative binding effect showed to be dependent on the concentration of polymer used in the binding assay (Fig. 8(a)). However, it was possible to observe that the abrupt increase of the binding capacity (q_e) of the hydrogel occurred for the same concentration of free NaCA in the solution (C_e) (Fig. 8(b)), irrespectively of the polymer concentration. The region of high affinity was observed for starting concentrations of NaCA above 7 mM (Fig. 8(a)), which is above its CMC (NaCA_CMC (pH = 6.8) = 6.7 mM).⁴² Therefore, the results obtained suggest that instead of the existence of a cooperative binding process, the behavior observed could be in fact the result of a stronger interaction between the PAMPTMA-based materials and NaCA micelles (electrostatic interaction), than with NaCA unimers (both hydrophobic and electrostatic interactions). This aspect could be advantageous considering BAS applications, since the bile salts are usually present in the intestine at concentrations above their CMC.³⁷ Based on these results, it seems that the cooperative mechanism proposed in the literature^13,14 is not the most suitable one to describe the binding of NaCA by the AMPTMA-based materials prepared by SARA ATRP. In this case, one might consider the existence of two different binding mechanisms corresponding to NaCA concentrations below and above the bile salt CMC.

As previously mentioned, the star block copolymers prepared by SARA ATRP present a PMA-based hydrophobic core and hydrophilic cationic arms, with high charge density (one charge per each AMPTMA repeating unit). The NaCA is an amphiphilic molecule that possesses both nonpolar (steroid skeleton) and polar (negatively charged) regions. In this case, one might consider that the NaCA unimers (concentration below the CMC) can interact either with the hydrophobic part of the polymer, which is less accessible, or/and with the cationic arms of the polymer by electrostatic interactions. On this matter, the ITC results suggested that the binding of NaCA unimers by the star block copolymers is relatively weak (low affinity constant K₁) and it is mainly governed by hydrophobic interactions with the nonpolar region of the polymer. This hypothesis was also confirmed by the low binding capacity obtained in the equilibrium binding experiments using [NaCA]₀ < NaCA_CMC (e.g., Fig. 8(a)). In addition, data reported in the literature¹² indicates that the binding of NaCA unimers can be suppressed in the presence of small electrolytes (e.g., NaCl). This fact might have also contributed for the low affinity showed by the PAMPTMA-based materials towards NaCA molecules below the CMC, due to the intrinsic high concentration of Cl⁻ anions from the PAMPTMA segment. On the other hand, for initial concentrations of NaCA above its CMC, the NaCA micelles/aggregates formed will be able to compete with small Cl⁻ anions and establish electrostatic interactions with the polymer, since the nonpolar segment of the NaCA will be in the micelles core. In this case the binding process will be characterized by an affinity constant K₂, higher than K₁, and could be described by the Langmuir isotherm. This hypothesis is currently under investigation.

During the course of this work, we have noticed that the NaCA precipitated from the SIF solution at pH = 6.8, after several hours, for [NaCA] ≥ 15 mM. In fact, it is known that the pK_a of bile salts increases when they are in polar media⁴³ or aggregated in the form of micelles.⁴⁴ Therefore, most probably, there was a small fraction of NaCA molecules in their neutral form at pH = 6.8. To confirm the validity of the results obtained at pH = 6.8 and to understand the influence of the ionization state of the NaCA on the binding mechanism, new binding experiments were conducted at pH = 7.6. At this pH value, there is a larger fraction of NaCA in the ionized form both as monomers and when associated in micelles, and the solutions are stable for several days in the range of concentrations investigated (0.1–30 mM). Colesevelam hydrochloride and the hydrogel showing the best performance (AB 50/5) were chosen as model polymers. The results (Fig. S8†) showed that the shape of the binding isotherms was the same as the one obtained at pH = 6.8, which confirms the validity of the results obtained. In addition, it is interesting to note that a slightly lower binding capacity was obtained at pH = 7.6. This supports the interpretation that the driving force for the association is not simply the electrostatic interaction between the bile salt and the polymer. This observation is in agreement with information found in the literature.¹⁸

In vitro degradation studies

Considering that polymeric BAS are administered orally, it is important to evaluate the stability of the synthesized materials under conditions that could simulate the GI tract environment. The determination of the molecular weight and the confirmation of the polymers chemical structure, after being subjected to the mentioned conditions, are effective tools to quantify any possible degradation. Due to the lack of proper solvents to analyze the amphiphilic star block copolymers by either SEC or ¹H NMR spectroscopy, the degradation of the materials was evaluated by analyzing each polymeric segment separately (linear PAMPMTA and star-shaped PMA). Therefore, it is assumed that the degradation mechanism of the block copolymer will be similar to that of the constituent parts. The degradation studies were carried out in triplicate at 37 °C in SGF at pH 1.2 for 2 h and in SIF at pH 6.8 for 3 h.^45,46 Table 6 summarizes the molecular weights and dispersity of the polymers before and after degradation.

Table 6 Molecular weight and dispersity of linear PAMPTMA and star-shaped PMA before and after exposure to the degradation solutions at 37 °C

Sample	Medium	Degradation time (h)	M^SECn × 10^−3	Đ
PAMPTMA	SGF (pH = 1.2)	0	19.6	1.09
		2	19.6 ± 0.1	1.09 ± 0.01
PMA	SGF (pH = 1.2)	0	51.7	1.07
		2	51.3 ± 0.7	1.07 ± 0.01
PAMPTMA	SIF (pH = 6.8)	0	19.6	1.09
		3	19.4 ± 0.1	1.08 ± 0.01
PMA	SIF (pH = 6.8)	0	51.7	1.07
		3	51.7 ± 0.8	1.08 ± 0.01

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The results obtained by SEC suggest that there was no degradation of the polymers backbones, since there was no variation on the molecular weight values. Nevertheless, one must be careful in the interpretation of the SEC results, since degradation of some polymer side chains (amide and ester linkages) could have minimal influence in the polymers molecular weight value. Therefore, additional ¹H NMR spectroscopy analyses were performed. The results showed that there was no degradation of the side chains of both PMA and PAMPTMA, since the ratio between the integral of the protons of the backbone and the protons of the side chains was constant for the samples studied (Fig. S9 and S10†).

Conclusions

The SARA ATRP method was used for the synthesis of BAS candidates based on novel PAMPMTA-based hydrogels and PMA-b-PAMPTMA amphiphilic star block copolymers. Both stars and hydrogels exhibited higher affinity towards NaCA micelles rather than NaCA unimers. This behavior was explained by the high density of cationic charges in the polymer and by the suppression of the NaCA binding, for low initial NaCA concentration, due to the presence of competing Cl⁻ anions from the AMPTMA monomer. The cationic hydrogels proved to be very promising materials with similar performance to the most effective commercial BAS (Colesevelam hydrochloride), concerning both the binding affinity and the maximum binding capacity. Regarding the amphiphilic star block copolymers, it was possible to conclude that the binding parameters could be adjusted by small changes on the polymers targeted composition. Typically, longer cationic hydrophilic arms lead to higher binding capacity. The polymers prepared proved to be stable in degradation solutions mimicking the GI tract conditions.

Acknowledgements

Patrícia V. Mendonça acknowledges FTC-MCTES for her Ph.D. scholarship (SFRH/BD/69152/2010). The ITC experiments were performed in the Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, Unidad Asociada BIFI-IQFR, CSIC, Zaragoza, Spain. The ¹H NMR data were obtained from Rede Nacional de RNM in the University of Coimbra, Portugal. The Coimbra Chemistry Centre (CQC) is supported by the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT) through the Project No. 007630 UID/QUI/00313/2013, co-funded by COMPETE2020-UE.

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Footnote

† Electronic supplementary information (ESI) available: ITC titration curves, binding parameters of the star block copolymers and ¹H NMR spectra. See DOI: 10.1039/c6ra06087k

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