Article pubs.acs.org/IECR
Reduction-Controlled Release of Organic Nanoparticles from Disulfide Cross-linked Porous Polymer Neil Grant,† Hong Wu,‡ and Haifei Zhang*,† †
Department of Chemistry, University of Liverpool, Oxford Street, Liverpool, L69 7ZD, United Kingdom Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
‡
S Supporting Information *
ABSTRACT: Reduction-controlled release is favored for many applications. The cleavage of disulfide bonds is known to be sensitive to reducing agents. Here, a cross-linker containing a disulfide bond is prepared and then used to prepare cross-linked porous polymer via an emulsion templating approach. Oil-in-water (O/W) emulsions are first formed where an organic dye is dissolved in the oil droplet phase and monomer/cross-linker/surfactant are added into the continuous aqueous phase. By polymerizing the O/W emulsion followed by freeze-drying, organic nanoparticles are formed in situ within the disulfide-crosslinked porous polymer. The release of organic nanoparticles in water is demonstrated and can be tuned by the presence of reducing agents such as dithiothreitol and tris(2-carboxyethyl)phospine. This approach has the potential to be used for the reduction-controlled release of poorly water-soluble drug nanoparticles from porous polymers or hydrogels.
1. INTRODUCTION Biodegradable polymers and hydrogels are used in a variety of applications including tissue engineering1 and drug release.2−4 Hydrogels are chemically or physically cross-linked hydrophilic polymers, which are insoluble in water but swell. Biocompatibility, biodegradability, and easily tuned mechanical stability have made hydrogels highly attractive in biological and biomedical applications. For example, hydrogels have been extensively investigated for targeted drug delivery and controlled release.5,6 Among the chemically cross-linked hydrogels, disulfide crosslinkers have been widely used.7−10 Disulfide matrices play an important role in pharmaceutical and biological applications due to their stability under normal conditions and degrading in a reductive environment.11−13 The disulfide bond can be cleaved in aqueous media by reducing agents such as dithiothreitol (DTT), glutathione (biologically available) and tris(2-carboxyethyl)phosphine (TCEP),11−14 and others.15,16 Disulfide cross-linked hydrogels, capsules, and micelles are mostly investigated.8,17−25 Various studies have shown the enhanced release by the addition of a reducing agent or within cells.17−28 Drug solubility in water is a major issue because over 40% of drugs in development pipelines are classed as poorly soluble.29 This can lead to issues such as low bioavailability, erratic absorption profiles, and reduced patient compliance.29,30 One approach to addressing this issue is to form drug nanoparticles.30,31 Not only can the drug dissolution rate increase significantly with reduced particle sizes, aqueous drug nanodispersion may be administrated directly.30−33 We have developed a new emulsion-freeze-drying approach to form organic/drug nanoparticles in situ within porous polymers.34,35 The nanoparticles can be released simply by dissolution34,35 or via a temperature trigger to produce stable aqueous nanodispersion.36 Although reduction-controlled release via disulfide © 2013 American Chemical Society
cleavage has been widely used for hydrogel, capsule, and micelle systems, all the studies have been focused on the release of soluble molecules in aqueous systems.17−28 Considering the large number of poorly water-soluble drugs and the intensive studies on drug nanoparticles, an investigation on the reduction-controlled release of poorly water-soluble drug nanoparticles would be highly appealing. Here, using an organic dye as a model compound, we report a study on the formation of organic nanoparticles within a disulfide crosslinked emulsion-templated porous polymer and the reductioncontrolled release of the organic nanoparticles into water.
2. EXPERIMENTAL SECTION 2.1. Materials. Acrylamide (AM, 99%, Mw 71.08), N,N,N′,N′-tetramethylethylenediamine (TMEDA), Triton X405 (70% in water, density 1.096 g cm−3), oil red O (OR), cystamine dihydrochloride (96%), acryloyl chloride (97%), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Mw 286.65), DL-dithiothreitol (DTT, Mw 154.25), and all the other chemicals were purchased from Sigma-Aldrich and used as received. Chloroform, ethyl acetate, heptane, acetonitrile, and sodium hydroxide were all of analytical grade and used as received. Distilled water was used in each case. 2.2. Preparation of the Bisacryloylcystamine (BAC) Cross-Linker. N,N′-bis-acrylcystamine (Mw 204.35) was synthesized following the method reported previously:7 Briefly, 8 g of cystamine dihydrochloride was dissolved in 80 cm3 of 3.12 M NaOH solution, and then 9.6 g of acryloyl chloride in 20 cm3 of acetonitrile was added dropwise to the solution under vigorous stirring at 50 °C for 15 min or until Received: Revised: Accepted: Published: 246
September 11, 2013 November 15, 2013 December 12, 2013 December 12, 2013 dx.doi.org/10.1021/ie403001r | Ind. Eng. Chem. Res. 2014, 53, 246−252
Industrial & Engineering Chemistry Research
Article
where M is the normalized unit, At is the absorbance at time t, and A∞ is the highest final absorbance reading from the tests. The release rates were calculated using the Higuchi model for release of molecules from an insoluble matrix:
effervescence disappeared. The reaction product was extracted with hot chloroform at 50 °C. The extract was washed with 0.1 M HCl and saturated sodium chloride aqueous solution and dried on sodium sulfate for 1 day. After removal of the solvent under vacuum conditions, the residue was recrystallized using ethyl acetate/heptane (2:1) and identified by 1H NMR and microanalysis. 2.3. Preparation of Emulsion-Templated Polyacrylamide (PAM). Preparation of oil-in-water emulsions and nanoparticles/porous polymers using BAC as a cross-linker followed a similar procedure reported before.36 The molar ratios of AM/BAC were fixed at 20:1 and 40:1 in a 10 wt % monomer solution. BAC was dissolved in the monomer solution by sonication until fully dissolved. A total of 10 wt% aqueous ammonium persulphate solution (0.1 cm3) was added to the monomer/cross-linker solution (2 cm3) followed by Triton X-405 (0.3 cm3). A solution of OR in cyclohexane (0.02 wt/v%, 6 cm3) with TMEDA (30 μL) was added dropwise to the aqueous phase while stirring using a lab egg stirrer. The formed emulsion was left to stir for 5 min before transferring to an oven at 60 °C overnight. In the polymerized samples, 44 wt % was contributed from AM (others were mainly surfactant and cross-linker). For emulsions with an internal phase volume of 50%, the preformed emulsion was homogenized for 1.5 min at speed 5 using a Fisher Brand homogenizer. The polymerized emulsions were frozen and freeze-dried to remove the solvents, yielding porous material. All the OR in the original emulsions was transformed into nanoparticles within the emulsiontemplated macropores during freeze-drying.35,36 2.4. Release of OR Nanoparticles from Disulfide Cross-Linked Porous PAM. Release using DTT as the reducing agent: Solutions of DTT were prepared at the concentrations of 0.1, 0.2, and 1 wt % in pH 9 water and bubbled with nitrogen for 10 min. Prepared porous polymer with OR nanoparticles (0.05 g) were cut and placed in a vial with a septa and flushed with nitrogen for 10 min. DTT solution (5 cm3) was added via a syringe to the vial. The vial was placed into a water bath at 45 °C. Periodically, the aqueous medium was agitated five times to ensure uniform mixing of the suspensions, and 200 μL of clear red suspension was removed for UV analysis. The removed volume was replaced by a fresh volume of DTT. The collected samples were analyzed by a UV−vis plate reader at room temperature. Release using TCEP as the reducing agent: TCEP solutions in water at the concentrations of 0.02, 0.2, and 1 wt % were prepared. The prepared porous polymer with OR nanoparticles (0.05 g) was soaked in the TCEP solution (5.5 cm3) at room temperature at pH 7. Periodically, 200 μL of the aqueous medium was taken for UV analysis as described above. The removed volume was replaced with fresh TCEP solution. Monitoring of the release was performed using a UV plate reader (Quant, Bio-Tek Instrument Inc.). A total of 200 μL of the released aqueous suspension was pipetted into a well of a 96-well flat bottomed polypropylene plate. The absorbance was monitored by scanning from 200 to 800 nm in 2 nm steps (OR absorption at 514 nm was used). The height of the peak was subtracted from the baseline to give the absorption reading of each sample. The absorbance data were normalized using M=
At A∞
M=
At = kt 1/2 A∞
(2)
where k is the rate constant and t is the release time. The regression function on Excel was used to calculate the R2 for each release curve. 2.5. Characterization. The dried materials were sectioned to reveal the internal porous structures. The samples were adhered to an aluminum stub using a silver colloidal suspension and allowed to dry. A sputter coater (EMITECH K550X) was used to coat the samples with gold at 30 mA for 3 min. A Hitachi S-4800 field emission SEM was used to reveal the pore structure at 3 kV. The pore sizes and pore volumes of the dried materials were examined using a Micromeritics Autopore IV 9500 porosimeter. Samples were subjected to a pressure cycle starting at approx 0.5 psi, increasing to 60000 psi in predefined steps. The OR nanoparticles were released into water in the presence of a reducing agent. The OR nanoparticles were released and an aqueous OR nanoparticle dispersion was formed. The OR nanodispersions were centrifuged for 15 min at 13 000 rpm using an Eppendorf Centrifuge 5415D, and the supernatant liquid was filtered through a 1 μm syringe to remove the degraded polymer. OR nanoparticles were not precipitated during centrifuging, as a clear red solution was still observed. The OR dispersions were analyzed at 25 °C by dynamic laser scattering (DLS) using a Malvern Zetasizer with a backscattering detection at 173°. The scattering intensity signal for the detector was passed through a correlator where these data were analyzed by the software and gave a size distribution. The size of the hydrated OR nanoparticles in water were obtained. Each measurement was repeated at least three times, and the average data were used to plot the DLS curves and obtain the particle size. A total of 10 μL of a diluted OR nanodispersion was dropped onto holey carbon filmed copper grids (400 mesh) and allowed to dry overnight. A scanning transmission electron microscopic (STEM) detector attached to the Hitachi S-4800 SEM was used to observe the dry OR nanoparticles at 30 KV. Breaking of the disulfide bonds and therefore the formation of thiols were performed using DTT or TCEP. To prove the formation of thiols, the TCEP Ellman’s stain method was used. The stain was prepared based on a reported procedure:37 5,5′dithiobis-(2-nitrobenzoic acid) (DTNB; 0.15 g, 0.034 mmol) in 1:1 ethanol and tris-HCl (150 mL total). Preparation of 1 M tris-HCl: Tris(hydroxymethyl)aminomethane (30.28 g) was dissolved in distilled water (150 cm3) and acidified to pH 7.4 with concentrated HCl. The solution was made up to 250 cm3 using distilled water. To prove the formation of the thiols, a drop of the released medium was placed onto a TLC plate and submerged in the stain solution. The formation of the thiols was observed by a bright yellow spot where the drop was placed.
3. RESULTS AND DISCUSSION N,N′-bis-acrylacystamine (BAC), the disulfide cross-linker, was synthesized following a procedure reported previously7 and confirmed by the NMR spectrum and the microanalysis (Figure
(1) 247
dx.doi.org/10.1021/ie403001r | Ind. Eng. Chem. Res. 2014, 53, 246−252
Industrial & Engineering Chemistry Research
Article
Figure 1. 1H NMR spectrum for the synthesized cross-linker BAC. 1H NMR data (400 MHz, CDCl3): δ 2.85 (m, 4H, environment a), δ 3.7 (m, 4H, environment b), δ 5.62 (m, 2H, environment f), δ 6.2−6.4 (m, 4H, trans-H on alkene, environments d and e), δ 6.7 (s, 2H, -NHc). Elemental percentage by microanalysis: %C = 45.64 (46% calculated), %H = 6.11% (6.2% calculated), %N = 10.28% (10.8% calculated).
Table 1. Preparation Conditions and Porosity of Disulfide Crosslinked Polyacrylamide sample
AM solution (cm3)
BAC (%)
OR-CH (cm3)
oil volume ratio (%)
PAM_X2.5_O50 PAM_X2.5_O75 PAM_X5_O50 PAM_X5_O75
4 2 4 2
2.5 2.5 5 5
4 6 4 6
50 75 50 75
pore volume (cm3 g−1)
peak pore size (μm)
± ± ± ±
3.9 26.0, 2.0 3.8 25.9, 1.5
7.0 8.5 6.9 7.2
0.4 0.4 0.4 0.3
DTT is not stable in water, and oxidation can occur when exposed to O2 or metal ions such as Fe3+.39 TCEP, however, is a nonvolatile solid and can be easily handled in the air.40 In aqueous solutions, TCEP is significantly more stable than DTT and is a stronger reductant than DTT.41 It was believed that the addition of a reducing agent such as DTT or TCEP could cleave the disulfide bonds, degrade the cross-linked polymer, and enhance the release of OR nanoparticles from the porous scaffolds. In our study, both DTT and TCEP were dissolved in water as reducing agents (0.05 g composite sample soaked in 5 cm3 aqueous solution) to tune the release of OR nanoparticles. Red color (from OR nanoparticles) was observed to diffuse out of the polymer into water, and clear red nanodispersions were formed. As the disulfide bonds in the polymer were cleaved, the size of the polymeric scaffolds reduced, and in some cases the scaffolds broke apart before being completely dissolved. Release by the addition of DTT was conducted in a water bath at pH 9 and 45 °C, although it was possible to perform a slow cleavage at 37 °C.19 DTT concentrations of 0.1, 0.2, and 1 wt % in water were studied for all four prepared composites (Table S1). The DTT solution was kept under nitrogen to ensure that the DTT did not self-oxidize. The number of moles of DTT was in excess of that of the disulfide bonds (3.24 × 10−5 mol [0.1 wt % DTT] to 7.77 × 10−6 mol [2.5% crosslinked polymer]). As a result, the rates of the disulfide cleavage reaction were not changed proportionally with the DTT
1). The organic dye OR was used as the model compound because the release can be easily observed and monitored.36 OR-cyclohexane (CH) solution (0.02 wt/v%) was emulsified into aqueous solution containing monomer acrylamide (AM, 10 wt %), cross-linker BAC, and surfactant Triton X-405 to form oil-in-water emulsions. The molar ratios of AM to BAC were 40:1 and 20:1. The oil to water ratios in the emulsions were 1:1 (50 v/v % oil phase) and 3:1 (75 v/v % oil phase). These emulsions were polymerized at 60 °C in an oven overnight and then frozen in liquid nitrogen and freeze-dried to produce dry porous cross-linked polyacrylamide (PAM) with in situ formed OR nanoparticles.35,36 The compositions of the formed emulsions and the porosity of the polymers (characterized by Hg porosimeter) are given in Table 1. Sample PAM_X2.5_O50 indicates the porous polymer prepared from the emulsion with 2.5% cross-linker (BAC/ AM = 1:40) and 50% oil phase. Highly interconnected porous structures are formed from the emulsions with 75% oil phase (Figure 2A and C). While for the emulsions with 50% oil phase, isolated cellular pores are still well connected by ice-templated pores, consistent with the previous studies.34,38 Hg intrusion porosimetry measurements show the distribution of macropores (Figure 2E and F). Bimodal pore size distributions are observed for the structures made from 75% oil-phase emulsion, indicating higher pore interconnectivity. When the OR/polymer composites were placed in water, a negligible release of OR nanoparticles was observed. Reducing agent DTT or TCEP was then added to enhance the release. 248
dx.doi.org/10.1021/ie403001r | Ind. Eng. Chem. Res. 2014, 53, 246−252
Industrial & Engineering Chemistry Research
Article
Figure 2. The micrographs show the pore morphology of sample (A) PAM_X2.5_O75, (B) PAM_X2.5_O50, (C) PAM_X5_O75, and (D) PAM_X5_O50 and the relevant pore size distributions (E and F).
this could result in the increased loading of OR nanoparticles in the porous polymer. Figure 5A shows the fast release of OR nanoparticles from the porous polymer prepared from the emulsion with a 75% oil phase. Both the highly interconnected porosity and increased OR loading contributed to this observation. To identify the effect of porosity, a diluted OR solution may be used to form the emulsions and then test the release in a further study. Another factor that affects the release is the degree of crosslinking. Generally for hydrogels, low cross-linking degree can result in a higher degree of swelling and often fast release. There is additional influence for disulfide cross-linked polymers in the presence of a reducing agent such as TCEP. Lower crosslinking degree (i.e., lower ratio of BCA to monomer AM) means a smaller number of disulfide bonds available for cleavage. That can lead to fast degradation of the polymer. As shown in Figure 5B, when the cross-linking ratio was changed from 2.5% to 5.0% while the other conditions were kept the same, fast release is observed for the polymer with 2.5% crosslinking ratio. The change in release rate is not to a larger degree. However, this has been consistent for the samples tested under other conditions (Table S1, Table S2). Figure 6 shows that the sizes of the released OR nanoparticles by the addition of DTT are around 200 nm, as characterized by transmission electron microscopy (TEM). A copper grid with holey carbon film was used for the TEM imaging. Therefore, the black spots are nanoparticles, while the white features are the holes in the carbon film in Figure 6. As expected, release by DTT or TCEP (Figure S1) had no influence on the OR nanoparticles. All the OR nanoparticle dispersions were examined by a dynamic laser scattering
concentrations, as demonstrated by the slowly increased release of OR nanoparticles (Figure 3). The intermediate in the DTT reduction is highly unstable. The reducing capabilities of DTT are limited due to the fact that only the negatively charged S-thiolate is the reactive species; the reaction is often limited to pH values above pH 7. We had thus chosen pH 9 for the release with DTT. TCEP, however, is a useful reductant over a much wider pH range (1.5−8.5) than DTT is.14 The release studies with TCEP were thus performed at room temperature and pH 7 (Table S2). With TCEP as a stronger reducing agent and more stable in solution, lower starting concentrations of TCEP (0.02, 0.2, 1 wt %) were used. The release profiles for PAM_X2.5_O50 are shown in Figure 4A. All the release data in this study are correlated using the classical Higuchi model where a linear relationship between the normalized absorbance and square root of releasing time is observed.42 The correlation parameter R2 is close to 1, indicating that the release profiles conform to the Higuchi model (Figures 3B and 4B). As shown in Figure 4B, the rate of OR release increases from 0.0181 to 0.0295 and 0.0437 (min−1/2) for concentrations of 0.02, 0.2, and 1 wt %, respectively. At 0.02 wt % TCEP, the number of moles of TCEP in solution is 3.48 × 10−6 mol, which is lower than that of disulfide bonds. As TCEP reaction with disulfide is a stoichiometric reaction,40,41 the cleavage rate of disulfide bonds may increase linearly until the TCEP (1 wt %) is in excess. Correspondingly, this results in the largely increased release of OR nanoparticles. The porosity of the polymer can be tuned by varying the internal phase volume of the original emulsion, as shown in Figure 2. When the same concentration of OR solution is used, 249
dx.doi.org/10.1021/ie403001r | Ind. Eng. Chem. Res. 2014, 53, 246−252
Industrial & Engineering Chemistry Research
Article
Figure 5. (A) Comparison of the release of OR nanoparticles from 50% and 75% emulsion-templated samples. (B) Comparison of the release for samples with different cross-linking ratios.
Figure 3. The release profiles (A) and the rate determination (B) for the release of OR nanoparticles based on the Higuchi model for sample PAM_X2.5_O50 under DTT concentrations of 0.1 wt % (▲), 0.2 wt % (■), and 1 wt % (◆).
Figure 6. TEM image of the OR nanoparticles released in the presence of DTT.
technique (Table S3). The hydrated diameter of the OR nanoparticles is on average 210 nm, which is consistent with the results from TEM imaging. The cross-linking ratio had little effect on particle size while higher porosity of the polymer slightly increased the particle size. However, all the measurements showed that the average particle sizes were below 300 nm, which suggests that stable aqueous nanoparticle dispersions could be readily formed.31,35
4. CONCLUSION In summary, a disulfide cross-linker is synthesized and used to prepare a reduction-responsive porous polymer. By combining emulsion templating and freeze-drying, organic nanoparticles are formed in situ within the porous polymer. Through the cleavage of disulfide bonds by reductants, the reductioncontrolled release of organic nanoparticles (
