Invited Feature Article pubs.acs.org/Langmuir
Self-Assembled Nanogels of Cholesterol-Bearing Hydroxypropyl Cellulose: A Thermoresponsive Building Block for Nanogel Tectonic Materials Yoshiro Tahara,†,‡ Mizuki Sakiyama,† Shigeo Takeda,† Tomoki Nishimura,†,‡ Sada-atsu Mukai,†,‡ Shin-ichi Sawada,†,‡ Yoshihiro Sasaki,† and Kazunari Akiyoshi*,†,‡ †
Department of Polymer Chemistry, Graduate School of Engineering and ‡ERATO Bio-nanotransporter Project, Japan Science and Technology Agency (JST), Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan S Supporting Information *
ABSTRACT: Hydroxypropyl cellulose (HPC) is a fascinating polysaccharide to use in developing a nanogel to be a thermoresponsive building unit for nanogel tectonic materials. Cholesterol-bearing HPC (Ch-HPC) self-assembled to form nanogels through hydrophobic interactions of the cholesteryl groups in water. Ch-HPC nanogels had a lower critical solution temperature in line with that of native HPC. The particle size of Ch-HPC nanogels was reversibly controlled by the temperature and salting-out effect. The thermoresponsive property was also observed in Ch-HPC nanogel-cross-linked macrogels. These results suggest that a Ch-HPC nanogel is an attractive building block for thermoresponsive nanogel tectonic materials.
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INTRODUCTION Hydrogels have been used as biomaterials for drug delivery systems and tissue engineering.1 Hydrogels localize drugs in their aqueous micro/nanospaces, which provide a hydrophilic and biocompatible environment, and release drugs through a controlled diffusion rate or degradation of cross-linking networks. Hydrogels are also developed as alternative materials for natural tissue because of their favorable mechanical properties as scaffolds for cell adhesions required for application in regenerative medicine. Cross-linking networks of hydrogels for biomaterials are generally consisted with synthetic, biocompatible polymers such as poly(lactic acid) and poly(ethylene glycol) (PEG) and naturally derived polymers such as gelatin and polysaccharide. In general, these hydrogels were prepared by polymerization with cross-linking reagents or the chemical/physical cross-linking of polymers. The design of hydrogels with a well-controlled nanostructure remains challenging. We proposed nanogel tectonic hydrogels as a new type of hydrogel in which nanogels are used as tectons, which are constituent units for controlling the nanoscopic structures of macroscale gels.2−4 Nanogels have been of great interest as nanocarriers in drug delivery systems.5,6 A nanogel tectonic system (nanogel cross-linking system), for example, is able to control the release of nanogel carriers by the hydrolysis of chemical bonds of the biodegradable cross-linkers. In this system, drug-loaded nanocarriers were gradually released, although in common polymer gels, naked drugs were directly released following the degradation of the polymers. Cholesterol-bearing pullulan (CHP) is one of the most studied materials © XXXX American Chemical Society
for developing self-assembled nanogels and is used as building blocks of nanogel tectonic hydrogels. In CHP nanogels, hydrophilic pullulan chains are self-assembled in water through the hydrophobic interactions of clustering cholesteryl groups.7 CHP nanogels can trap proteins inside the physically crosslinking nanometer-sized network and exhibited a chaperonelike function for protein folding. Several clinical trials have been conducted using CHP nanogels as immunological nanocarriers, and the safety of the repeated administration of self-assembled nanogels has been confirmed.8 For further application of nanogel-based materials, nanogel tectonic hydrogels were developed. In a typical procedure, for example, acryloylgroup-modified CHP nanogels were cross-linked using PEG bearing four branched terminal thiol groups via the Michael addition reaction,2,3 and this nanogel-cross-linked (NanoClik) macrogel was used for guided bone regeneration or bone formation as artificial extracellular matrixes in vivo. On the basis of this cross-linking approach, several types of macrogels have been prepared, such as porous macrosized gels2 and microspheres.3 From this research, nanogel-derived structures and nanocarrier-derived functions were maintained in the nanogelbased materials after cross-linking. Special Issue: Tribute to Toyoki Kunitake, Pioneer in Molecular Assembly Received: June 30, 2016 Revised: August 27, 2016
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DOI: 10.1021/acs.langmuir.6b02406 Langmuir XXXX, XXX, XXX−XXX
Langmuir
Invited Feature Article
Recently, various intelligent functions including sensitivity to the thermal environment were added to these gels by designing the functional chains and cross-linking points with the stimuliresponsive unit.9 Thermoresponsive hydrogels are one of the most studied stimuli-responsive gels. Poly(N-isopropylacrylamide) (PNIPAAm) is a thermoresponsive polymer chain that is intensively investigated as a drug delivery carrier and for cell encapsulation and cell culture surfaces. Applications to drug delivery or cell attachment/detachment have been developed on the basis of its lower critical solution temperature (LCST) in the range of 32 °C, close to body temperature.10,11 Poly(N,N-diethylacrylamide) and poly(N-ethylmethacrylamide) had different LCST values at 25 and 58 °C, respectively.12 Triblock copolymers poly(ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPOPEO), known as pluronic, forms polymeric micelles, and highly concentrated pluronic aqueous solutions exhibit thermoreversible gel−sol transitions (melting points 27−56 °C).12 In pluronic hydrogels, spherical micelles are closely packed in a cubic lattice, and their conformation is changed to rodlike micelles packed in a hexagonal system at high temperature. Block copolymers containing oxyethylene side chains synthesized by living cationic polymerization were also used as the particulate core unit of thermoresponsive hydrogels.13,14 Poly(N-vinylcaprolactam) is another important thermoresponsive polymer that shows LCST behavior in water between 30 and 32 °C, and recently hydrogels have been designed for biomedical applications.15 Unlike these synthetic polymers, thermoresponsive hydroxypropyl cellulose (HPC) is a derivative of naturally occurring polysaccharide and has attractive characteristics including high biocompatibility, solubility in polar organic solvents, and thermal responses. For example, HPC has been used as a biocompatible material in ophthalmic inserts, which are an effective and safe treatment for dry eye syndrome.16 HPC also shows excellent solubility in both polar organic solvents and water.17 In addition, HPC has an LCST;18 HPC is transparently soluble below the LCST in water; its hydrophobicity increases, and it forms aggregates above the LCST in water.19 In the present study, thermoresponsive nanogels and their tectonic materials were prepared with an HPC-based nanogel as a new building block. HPC was modified with cholesteryl groups and used for the formation of a nanogel by the self-assembly of cholesterol-bearing HPC (Ch-HPC) in water (Figure 1). Next, the thermoresponsive property of Ch-HPC nanogels was investigated. Finally, ChHPC nanogels were cross-linked to prepare NanoClik macrogels. In this new type of hydrogel, thermoresponsive nanoparticles were cross-linked and built macroscale three-dimensional networks. The structural and morphological changes of NanoClik macrogels responding to temperature were investigated.
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Figure 1. (a) Structure of Ch-HPC. (b) Schematic illustration of the self-assembly of Ch-HPC to form nanogels. study.3,7 Five grams (12.9 mmol) of cholesterol (Ch) was reacted with 32.6 g (194 mmol) of 1,6-hexyldiisocyanate in dry pyridine (3 mL) and dry toluene (130 mL) for 48 h at 80 °C under an atmosphere of argon. After the removal of toluene and the addition of hexane at −30 °C, cholesteryl isocyanate (ChI) was crystallized. (Supporting Information, Figure S1a). HPC was vacuum-dried for at least 1 day at 70 °C. HPC (1.0 g), DBTDL (200 μL), and ChI (15.6, 23.5, 50.2 mg) were dissolved in anhydrous dimethyl sulfoxide (DMSO), and the resulting mixture was stirred in the absence of light for 24 h at 45 °C under an atmosphere of argon. The resulting solution was dialyzed against isopropanol for 2 days and deionized water for 1 week using a Spectra/Por 7 dialysis membrane with a molecular weight cutoff of 3500 Da (Spectrum Laboratories, Rancho Dominguez, CA, USA). After dialysis, the material was lyophilized to yield Ch-HPC. The modification of HPC with rhodamine and acryloyl groups was performed with a similar method using rhodamine isothiocyanate and AOI, respectively. Basic Characterization. Basic characterization of Ch-HPC was conducted as in previous studies.20 The degree of substitution (DS) of hydroxypropyl and cholesteryl groups by the glucose units of cellulose and HPC was determined by 1H NMR spectroscopy (Bruker, Billerica, MA, USA) in DMSO-d6. The hydrodynamic radius (RH) and diameter were determined using z averages measured by dynamic light scattering (DLS, Zetasizer Nano-ZS, Malvern, U.K.) at 25 °C after filtration (0.22 μm). Field-flow fractionation coupled with multiangle light scattering (FFF-MALS, Wyatt Technology, USA) was carried out as follows: The detector flow rate of 0.1 M NaCl aqueous solution was 0.5 mL/min, and the cross-flow rate decreased linearly from 2.0 to 0 mL/min in 20 min. The weight-average molecular weight (Mw) was determined using static light scattering methods (ASTRA analysis software via the Rayleigh scattering equation21), and the aggregation number of HPC per nanoparticle (NHPC) was calculated. From RH and Mw values, an average polymer density (ΦH) within a nanoparticle was calculated according to eq 1, where NA is Avogadro’s number.
EXPERIMENTAL SECTION
Materials. HPC (HPC-SL, molecular weight 1 × 105 g/mol) was kindly provided by Nippon Soda Co., Ltd. (Tokyo, Japan). Cholesterol, 1,6-hexyldiisocyanate, di-n-butyltin(IV) dilaurate (DBTDL), rhodamine isothiocyanate, pyrene, and cetylpyridinium chloride (CPC) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2-Acryloyloxyethylisocyanate (AOI) was purchased from Showa Denko Co. (Tokyo, Japan). PEG bearing four branched terminal thiol groups (PEGSH, molecular weight 1.0 × 104 g/mol) was purchased from Nippon Oil and Fat Co. (Tokyo, Japan). Synthesis of Ch-HPC. Cholesterol was attached to the hydroxyl groups of HPC using a method similar to that described in a previous
ΦH =
−1 Mw ⎛ 4 3⎞ ⎜ πR ⎟ H ⎠ NA ⎝ 3
(1)
The mean aggregation number of associating cholesteryl groups (NCh) in one hydrophobic domain was estimated using a fluorescence quenching technique. The steady-state quenching data in a microheterogeneous system such as an aqueous micellar solution fit the quenching kinetics22 as eq 2, where I and I0 are the fluorescence intensities in the presence and absence of a quencher, respectively; [Q] is the bulk concentration of the quencher, and [M] is the concentration of the polymer self-aggregate. B
DOI: 10.1021/acs.langmuir.6b02406 Langmuir XXXX, XXX, XXX−XXX
Langmuir
Invited Feature Article
Table 1. Basic Characteristics of Ch-HPC Nanogels DS of Ch [/100 glu]a HPC Ch-HPC0.7 Ch-HPC1.5 Ch-HPC1.9
0.7 1.5 1.9
diameter [nm]
Mw [ × 106]
NHPC
ΦH [g/mL]
NCh
LCST [°C]
± ± ± ±
0.14 1.27 11.2 9.23
9.1 75.4 65.9
0.017 0.109 0.052
5.1 ± 0.3 7.3 ± 0.5 7.2 ± 0.5
51 49 49 49
29.5 61.2 68.9 82.6
2.7 1.1 0.9 3.1
a
Described as the number of substituted moieties per 100 glucose units. DS of Ch, degree of substitution of cholesterly groups; Mw, weight-average molecular weight; NHPC, aggregation number of HPC per nanoparticle; ΦH, average polymer density; NCh, aggregation number of associating cholesteryl groups; LCST, lower critical solution temperature.
Figure 2. (a) I1/I3 ratio of pyrene fluorescence in the presence of HPC and Ch-HPC at various concentrations. [Pyrene] = 1 × 10−6 M. I1 and I3 are the intensities of pyrene measured at 374 and 385 nm, respectively. (b) Natural logarithm of the I0/I ratio of pyrene fluorescence at various concentrations of quencher and Ch-HPC nanogels. I0 and I are the fluorescence intensities of pyrene in the absence and presence of a quencher, respectively. The concentrations of Ch-HPC nanogels were controlled at 3.0, 4.0, and 5.0 mg/mL.
(3)
upon repeated changes in the temperatures to 25 and 60 °C were determined by DLS. NanoClik macrogels consisting of Ch-HPC nanogels were prepared as follows:2,3 A rhodamine- and acryloylgroup-modified Ch-HPC (Ch-HPCOA-Rh) nanogel was swollen in PBS at a concentration of 30 mg/mL overnight. PEGSH was mixed with the Ch-HPCOA-Rh suspension in a glass capillary to form NanoClik macrogels. The molar ratio of the thiol to acryloyl groups was adjusted to 1:10. The NanoClik macrogels were incubated in 5 and 80 °C, and the sizes of the macrogels were measured.
Pyrene was used as a fluorescent probe in phosphate-buffered saline (PBS) containing various concentrations of HPC and Ch-HPC at 25 °C. CPC was added as a quencher at various concentrations. Fluorescence spectra were recorded using a fluorescence spectrophotometer equipped with a thermoregulated cell compartment. Pyrene was excited at 339 nm. Thermoresponsiveness and Preparation of NanoClik Macrogels. The LCSTs of HPC-based materials in PBS were determined by measuring the transmittance at 500 nm. The hydrodynamic diameters
RESULTS AND DISCUSSION Synthesis and Basic Characterization of Ch-HPC Nanogels. As the basic data, the degree of substitution (DS) of the hydroxypropyl groups to the glucose units of cellulose was determined by 1H NMR. The ratio of the integral values of the methyl group protons of hydroxypropyl groups (δ = 1.04 ppm) and the anomeric protons of glucose (δ = 4.4 ppm) were
⎛I ⎞ [Q] ln⎜ 0 ⎟ = ⎝I⎠ [M]
(2)
The plot of ln(I0/I) against [Q] shows a linear relationship with the slope corresponding to 1/[M]. Thus, NCh is given by eq 3, where [Ch] is the concentration of cholesteryl groups. NCh =
[Ch] [M]
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DOI: 10.1021/acs.langmuir.6b02406 Langmuir XXXX, XXX, XXX−XXX
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Invited Feature Article
Figure 3. (a) Transmittance of HPC and Ch-HPC nanogels at 500 nm. Hydrodynamic diameters of Ch-HPC nanogels upon repeated changes in temperatures in (b) PBS and (c) deionized water. The inset is expanded from 40 to 100 nm.
The persistence length of HPC and pullulan in water is reported to be 5−10 nm23 and
