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Glucosamine-carrying temperature- and pH-sensitive microgels: Preparation, characterization, and in vitro drug release studies
Journal of Colloid and Interface Science 322 (2008) 333–341 www.elsevier.com/locate/jcis
Glucosamine-carrying temperature- and pH-sensitive microgels: Preparation, characterization, and in vitro drug release studies Dayong Teng, Jingli Hou, Xinge Zhang ∗ , Xin Wang, Zhen Wang, Chaoxing Li ∗ The Key Laboratory of Functional Polymer Materials of Ministry Education, Institute of Polymer Chemistry, Nankai University, 94# Weijin Road, Tianjin 300071, China Received 22 December 2007; accepted 12 March 2008 Available online 15 April 2008
Abstract Glucosamine-carrying temperature- and pH-sensitive microgels with an average diameter of about 100 nm were successfully prepared by free radical precipitation polymerization. The thermo- and pH-responsive properties of the microgels were designed by the incorporation of Nisopropylacrylamide (NIPAM) and acrylic acid (AAc) to copolymerize with acrylamido-2-deoxyglucose (AADG). The stimuli sensitivity of the microgels was studied by the measurement of their sizes and volume phase transition temperature (VPTT) under different surrounding conditions. The results showed that the microgels were responsive to temperature, pH, and ionic strength, and could have a desired VPTT by modifying AADG and AAc contents. The effect of temperature and pH on insulin release from the microgels was also investigated. The release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions. A preliminary in vitro cell study showed that the glucosamine-carrying microgels are more biocompatible to mouse fibroblast cells, compared to the microgels without glucosamine. These glucosamine-carrying dual-sensitive microgels may be promising carriers for targeted drug delivery to tumors. © 2008 Elsevier Inc. All rights reserved. Keywords: Glucosamine; Target delivery vehicles; Microgels; Stimuli-sensitive polymers
1. Introduction Microgels are cross-linked polymer particles with average diameters ranging from 50 nm to 5 µm [1]. Microgels, smaller than 200 nm, with hydrophilic surfaces are better target delivery vehicles particularly for solid tumors [2]. Using these microgels, drug targeting to solid tumors can be achieved by an enhanced permeation and retention effect (EPR effect) [3], and in contrast, the microgel particles with a larger diameter or hydrophobic surface lead to a rapid uptake by the reticuloendothelial systems (RES) [4–6]. D -Glucosamine (GA) is a monosaccharide, which can be obtained by either chemical or enzymatic hydrolysis of the natural polysaccharide—chitin and chitosan [7]. The existence of reactive hydroxyl and amino groups in GA offers many possibilities for obtaining new polymers. These derivatives have a * Corresponding authors. Fax: +86 22 2350 5598.
E-mail addresses: [email protected] (X. Zhang), [email protected] (C. Li). 0021-9797/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2008.03.014
range of biological activities [8–10], and GA in a certain concentration range could kill tumor cells without influencing normal cells [11]. Therefore, polymeric nanoparticles composed of a functional GA, acrylamido-2-deoxyglucose (AADG), have been prepared in a reverse micellar system recently [12]. The particles obtained were of >85 nm in diameter and were highly monodisperse. In addition, it is known that the interior temperature of tumors (T ∼ 42 ◦ C) is higher than that of normal tissues (T ∼ 37 ◦ C) [13], and most solid tumors have an acidic extracellular pH (6.85–6.95), while normal blood pH remains at 7.4 [14]. Until now, many studies were focused on enhancing the passive targeting ability by using a polymer sensitive to the surrounding temperature [15] or pH [16,17]. Only few reports were related to the application of temperature and pH dual-sensitive microgels in a tumor-targeted delivery system. To explore the potential of glucosamine-carrying temperature- and pH-sensitive microgels as tumor delivery vehicles, the microgels of AADG with N -isopropylacrylamide (NIPAM) and acrylic acid (AAc) as the comonomer were prepared by
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Scheme 1. Preparation of AADG monomer.
the method of a free radical precipitation polymerization. These microgels were characterized using FTIR, SEM, and DLS. We further studied the VPTT of the microgels as a function of pH and salt concentration to examine the microgels’ stimuli sensitivity and colloidal stability. We chose insulin as a model protein drug because tumor suppressor proteins have been increasingly used in anticancer gene therapy recently [18]. The in vitro release of insulin was investigated and preliminary cell viability was also evaluated. 2. Materials and methods 2.1. Materials NIPAM, EDC (1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride), HOBT (1-hydroxybenzotrizole), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from J&K-Acros Chemical Ltd. (USA) and used as supplied. AAc was freshly distilled before use. Pure crystalline porcine insulin (with a nominal activity of 28 IU/mg) that was used without further purification was obtained from the Xuzhou Wanbang Biochemical Co., Ltd. (Jiangsu, China). All other chemicals were of analytical grade and were used without further purification. Distilled and deionized water was used throughout the work.
2.2. Preparation of acrylamido-2-deoxyglucose monomer Typically, AAc (3.6 g, 50 mmol), EDC (11.2 g, 60 mmol), and HOBT (8.1 g, 60 mmol) were dissolved in 400 mL of DMF and the solution was stirred at 0 ◦ C for 1 h. Then a solution of glucosamine hydrochloride (21.5 g, 100 mmol) in DMF (200 mL) was added, followed by the addition of 200 mL of 1.2 mmol/mL NaHCO3 solution (Scheme 1). After being stirred for 24 h at room temperature, the solvents were evaporated under reduced pressure to give a solid that was purified by column chromatography on silica gel using methanol/ethyl acetate (1/4, v/v) as the eluent to obtain 6.5 g of AADG (yield 56%). 1 H NMR (D O) δ (ppm): 6.4 (1H, m, COCH=), 6.3 (1H, m, 2 CH=CHCO, anti), 5.8 (1H, m, CH=CHCO, syn), 5.2 (1H, m, CH), 3.4–4.0 (5H, m, CH). 2.3. Preparation and purification of microgels PNIPAM/AADG/AAc microgels were prepared by a free radical, precipitation polymerization reaction using MBA (N ,N -methylenebisacrylamide) as the crosslinking vehicle, APS (ammonium persulfate) as the initiator, and SDS (sodium dodecyl sulfate) as the stabilizer (Scheme 2). A detailed description of this polymerization method may be found elsewhere [1]. Different monomer molar ratios were used to prepare
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Scheme 2. Preparation of microgels.
Table 1 Samplea
NIPAM AADG (mmol) (mmol)
AAc (mmol)
NGA0 NGAa NGAb NGAc NGA1 NGA2 NGA3
8.8 8.8 8.8 8.8 8.8 8.8 8.8
2.20 2.20 2.20 2.20 1.65 1.10 0.55
0 0.55 1.10 2.20 1.10 1.10 1.10
MBA (mmol)
0.132
APS (mmol)
0.22
SDS (mmol)
0.176
Water (mL)
100
spectrometer (FTS-6000, Bio-Rad Co.) with a KBr tablet containing microgel powders. 2.4.2. SEM The SEM micrographs of the microgels were obtained with a scanning electron microscope (SS-550, Shimadzu). The sample was prepared by placing a drop of a highly diluted suspension of the microgels in a sample holder and drying before being sputtered with gold.
a All reactions were carried out at 75 ◦ C for 5 h.
the microgels with different compositions (Table 1). All microgels were purified via dialysis (cutoff: 8000–12,000 Da) against frequent changes of stirring water for a week at room temperature. 2.4. Characterization of the microgels 2.4.1. FTIR spectroscopy The purified microgel dispersions were lyophilized before analysis. The chemical structure was analyzed with an FTIR
2.4.3. DLS The hydrodynamic diameter (DH ) and size distribution of the microgels were determined in 150 mM NaCl aqueous solution as a function of temperature and pH by DLS (Brookhaven, INNDVO300/BI900AT) at a scattering angle of 90◦ . The scattered light of a polarized argon laser beam with a wavelength of 636 nm was used. The temperature of the dispersions was maintained by refrigerated circulator baths, and the samples were equilibrated at each temperature for a minimum of 30 min to arrive at swelling/deswelling equilibrium in water before data collection. Data were analyzed with the Brookhaven software
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provided by the manufacturer. DH under each temperature was the mean DH of three effective measurements. 2.4.4. Volume phase transition temperature The volume phase transition temperature (VPTT) of the microgels was determined by cloud point measurement as a function of pH and salt concentration with an UV–Vis spectrophotometer (Shimadzu, UV2550). The temperature was raised from 20 to 80 ◦ C in 0.5 ◦ C increments every 10 min and the transmittance was measured at 550 nm. The VPTT was determined as the temperature at the inflection point in the curve of dispersion transmittance versus temperature. 2.5. Loading of insulin Insulin loading was accomplished by equilibrium partitioning of insulin into the microgels. In a typical loading study, 3 mL of insulin solution (0.6 mg/mL) was added to 3 mL of the microgels dispersion (2.0 mg/mL) to form the dispersion. The dispersion was cooled to 4 ◦ C, stored for 24 h, and heated quickly to 37 ◦ C. The association efficiency of insulin was determined on separation of microgels from the dispersion containing free insulin by centrifugation (12,000 rpm, 30 min, 20 ◦ C). Before centrifugation, a small amount of 0.1 N HCl was added to the dispersion to enhance the hydrophobicity of the microgels, which made the microgels convenient for separation from the aqueous media. The amount of free insulin was analyzed in the supernatant by the Bradford method [19], using UV–Vis spectroscopy at 595 nm. Insulin entrapment efficiency (E%) and the loading efficiency (L%) were calculated as follows: total insulin − free insulin × 100%, total insulin total insulin − free insulin L% = × 100%. microgels weight All measurements were performed in triplicate and averaged. E% =
2.6. In vitro release studies Insulin release was determined by incubating the microgels in 2 mL phosphate buffer, respectively, at a constant temperature, with horizontal shaking. At predetermined time intervals, samples were centrifuged and the supernatant was taken and replenished by fresh buffer. The free insulin was determined by the Bradford method, and a calibration curve was made using nonloaded microgels to correct for the intrinsic absorption of the polymer. The release kinetics was studied in pH 6.9 and 7.4 buffers each at 37 and 41 ◦ C, using NaCl to adjust the salt concentration to 150 mM. In each experiment, the samples were analyzed in triplicate and the error bars represent the standard deviation. 2.7. Cell viability Cell viability was evaluated by using L-929 mouse fibroblast cells. The cell line was cultured in Dulbecco’s modified Eagles
Fig. 1. FTIR spectra of (A) AADG, (B) NGA0, and (C) NGA2.
medium (DMEM) in a humidified atmosphere (5% CO2 /95% O2 ). The cells were seeded into 96-well plates at 10,000 cells per well. The plates were then returned to the incubator and the cells were allowed to grow to confluence for 24 h. NGA0 and NGA2 microgel dispersions were prepared with culture medium. There dispersions were diluted to give a final range of microgel concentrations from 25 to 1000 mg/L. Then the media in the wells were replaced with the preprepared culture medium–sample mixture (200 µL). The plates were then returned to the incubator and maintained in 5% CO2 at 37 ◦ C for 48 h. Each sample was tested in six replicates per plate. After incubation culture medium and 20 µL of MTT solutions were used to replace the mixture in each well. The plates were then returned to the incubator and incubated for a further 4 h in 5% CO2 at 37 ◦ C. Then, the culture medium and MTT were removed. Isopropanol (100 µL) was then added to each well to dissolve the formazane crystals. The plate was placed in 5% CO2 at 37 ◦ C for 10 min and for 15 min at 6 ◦ C before measurement. The optical density was read on a microplate reader at 570 nm. Cell viability was determined as a percentage of the negative control (untreated cells). 3. Results and discussion 3.1. FTIR analysis Fig. 1 exhibits the FTIR spectra of (A) AADG monomer, (B) NGA0 (NIPAM/AAc = 80/20, mol/mol) microgels, and (C) NGA2 (NIPAM/AADG/AAc = 80/10/10, mol/mol) microgels. As shown in the figure, (B) and (C) present typical bands of PNIPAM (3300 cm−1 for N–H stretching; 2973, 2934, and 2876 cm−1 for C–H stretching; 1650 cm−1 for the amide I band assigned to C=O stretching; 1548 cm−1 for the amide II band assigned to N–H in-plane bending vibrations) and PAAc (1459 cm−1 for C–O stretching). Furthermore, (A) and (C) present typical bands of glucosamine (1100 cm−1 for C–O–C stretching), but (B) did not present this band, indicating that AADG had taken part in the polymerization.
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(A)
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associate. Following the structural change and consequent enhanced surface hydrophobicity, the microgels aggregated and formed larger particles. So when the particles began to aggregate, the microgel dispersion became turbid, as shown in Fig. 3B. The phenomenon was in good agreement with the experimental results described by Gupta et al. [20], who observed that the aggregation of the microgels accelerated the release of ketorolac at 37 ◦ C. However, according to the result reported by Lin et al. [21], the microgel did not aggregate with increasing temperature. They inferred that poly(NIPAM-co-AAc) microgels formed a slightly core–shell structure, and the hydrophobic groups in the core were packed by the AAc shell, that avoided the aggregation. Thus, the microgels shrank to a smaller one while the temperature increased obviously. The pH-responsive behavior of the microgels was investigated by DLS. As shown in Fig. 4, when pH < 5.0, the diameters of microgels were nearly invariable regardless of the change of pH, which was due to the protonation of the COO− groups. Indeed, the pKa of PAAc is around 4.7 in water, and at low pH it results in a protonation of the COO− groups to COOH [22]. Therefore, the microgels’ hydrophobicity is increasing. And when pH 5.0, the diameters became larger when the pH value was increased. The ionization of the COOH groups and consequent enhanced electrostatic repulsion caused the polymer chain stretching that made the particle size increase. 3.4. The effect of AADG and AAc on the VPTT of microgels
(B) Fig. 2. SEM image (A) and size distribution (B) of NGA2 microgel.
3.2. Size and morphology of microgels The morphology of the resulting microgels was investigated by SEM (Fig. 2A). It is evident that microgels are well dispersed as individual particles with spherical shapes. From Fig. 2A, it can be seen that the size of the microgels is around 50–60 nm in diameter in the solid state, which may be due to the collapse of the free hydrophilic segments of the polymer as well as the dehydration of the microgels. As shown in Fig. 2B, the microgels exhibit a narrow size distribution with an average diameter of around 100 nm. 3.3. Thermo- and pH-responsive structural changes of microgels DLS measurement is an efficient method for investigating the thermo-responsive properties of PNIPAM and related microgels. Fig. 3A shows the diameter change as a function of temperature. The results show that the size of the particles significantly increased with an increase in the temperature, which was correlated to the NIPAM segment in the microgels. As the temperature increased up to a certain point, the water contained in the microgels was expelled due to the disruption of hydrogen bonding between the water and the hydrophilic amide groups, and then the hydrophobic isopropyl groups began to
The cloud point method is a simple method for determining the VPTT. Table 2 shows the VPTT of microgels with various compositions as a function of pH value. 3.4.1. Effect of AADG NGA0, NGAa, NGAb, and NGAc were selected to research the effect of AADG on the VPTT of microgels. To provide a better overview of the observed effects, all VPTT at high ionic strength (150 mM) are plotted against the molar percentage of AADG to NIPAM at different pH (Fig. 5A). At pH 2.4 and 5.0, the VPTT was linearly increasing as the mol% of AADG content increased. At pH 5.0, the VPTT was obviously higher than that at pH 2.4, and NGAc exhibited no VPTT in the temperature range 20 to 80 ◦ C. The results indicate that the AADG segments could enhance the hydrophilicity of the microgels and then could increase the VPTT of the microgels. Moreover, the VPTTs of the microgels can be easily changed by the incorporation of AADG in the microgels and by considering the used pH. Glucosamine-based materials could not only enhance the hydrophilicity but also impart stability to bioactive molecules like proteins and enzymes [23]. These excellent qualities and the antitumor activity made glucosamine a useful material in the research of a tumor-targeted delivery system. 3.4.2. Effect of AAc NGAb, NGA1, NGA2, and NGA3 were selected to observe the VPTT change as a function of AAc content. The same as
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(A)
(B) Fig. 3. Thermo-responsive behaviors of NGA2 microgel at pH 5.0. (A) Diameter changes as a function of temperature. (B) Transmittance of the microgel on temperature change.
AADG, all VPTTs were plotted against the molar percentage of AAc to NIPAM at the studied pH in Fig. 5B. As seen, the pH dependence was clearly shown and at all pH it appears that the VPTTs nearly linearly ascended as the mol% AAc content increased. At pH 2.4, the VPTT shifted to a higher temperature when less AAc was incorporated into the microgels. On the contrary, at pH 5.0, the VPTT increased with an increase in the proportion of AAc in the microgel. The results indicate that the electrostatic repulsion was enhanced at pH 5.0, due to the ionization of the COOH groups, The increase of electrostatic repulsion reduced the association of hydrophobic groups and then led to a higher VPTT. At pH 7.4, the VPTT was higher than that at pH 5.0, and NGAb and NGA1 exhibited no VPTT in the studied temperature range from 20 to 80 ◦ C. The further ionization of the COOH groups made the VPTT increase further.
3.5. Colloidal stability of microgels At intermediate or high ionic strength, the electrostatic repulsion is weakened or screened out for the van der Waals forces, which caused the collapse of the microgel particles. NaCl is the most effective in the collapse of NIPAM-based microgel particles [24], and so we chose NaCl solutions with different concentrations to investigate the colloidal stability of the microgels. As seen in Fig. 6, at low salt concentration (<200 mM), the change tendency of VPTT was NGA0 > NGA1 > NGA2 > NGA3; the higher the mol% AAc in the microgel, the higher the VPTT. At high salt concentration (>400 mM), the change tendency of VPTT was NGA1 > NGA2 > NGA3 > NGA0; the higher the weight percentage of NIPAM segments in the microgel, the lower the VPTT. At the low salt concentration, the electrostatic stabilization is not
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(A) Fig. 4. Variation of diameters of NGA2 microgel against pH. The samples were analyzed in triplicate and the error bars in the plot were the standard deviation (mean ± SD, n = 3). Table 2 Sample
VPTTa (◦ C) pH 2.4
pH 5.0
pH 7.4
NGA0 NGAa NGAb NGAc NGA1 NGA2 NGA3
30 31 32 35 32.5 33.2 35
62.5 67 72 Nob 58 48 37
Nob Nob Nob Nob Nob 66 42
a VPTT determined by UV–Vis spectroscopy at 550 nm; concentration = 1 g/L in buffer solution, heating ramp = 0.5 ◦ C/10 min. b No phase transition observed in the temperature range 20–80 ◦ C.
(B)
completely eliminated and a significantly higher temperature is required to cause a volume phase transition. The increasing proportion of AAc led to a higher electrostatic repulsion, and then a higher VPTT. But at the high salt concentration, the high ionic strength can eliminate the electrostatic repulsion between the microgels and depress the stabilization of the microgels. Then the solution became a poorer solvent for PNIPAM with increasing NaCl concentration [25]; thus, a very low temperature was needed to cause a volume phase transition and aggregate, and the increasing proportion of the NIPAM segment led to a lower VPTT.
Fig. 5. Variation of VPTT of the microgels at different pH against mol% of (A) AADG and (B) AAc to NIPAM in the microgels.
3.6. In vitro release kinetics studies As a target delivery vehicle for tumors, the microgels surrounding tumor (pH 6.85–6.95) needed a certain VPTT which is higher than the temperature of normal tissue and lower than that of tumor. We used NGA3 as a model microgel; its VPTT is 42 ◦ C at pH 7.4 and 40.5 ◦ C at pH 6.9 (data not shown). The temperature sensitivity of the microgels was used for the insulin loading. The loading process was conducted at 4 ◦ C for 24 h and then the microgels were collapsed at 37 ◦ C. The temperature of 37 ◦ C was selected because it is the lower tem-
Fig. 6. The VPTT of microgel dispersion from turbidity measurements, as a function of (log) NaCl concentration.
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Fig. 7. Temperature- and pH-modulated insulin release from NGA3 microgels.
perature providing maximum volume swelling. Indeed a higher temperature could lead to the deformation and precipitation of the loaded microgels and induce a thermal denaturation of insulin in the loading process. The typical loading and entrapment efficiency were 5.5 and 17.8%, respectively. The low loading and entrapment efficiency were primarily due to the leakage in the centrifugation process, since insulin loading was accomplished by equilibrium partitioning of insulin into the microgels. In vitro insulin release from NGA3 microgels at different pH values (6.9, 7.4) and different temperatures (37 and 41 ◦ C) is shown in Fig. 7. The rate of insulin release from the microgels was initially rapid at 41 ◦ C as compared to that at 37 ◦ C. At 41 ◦ C, about 88% of drug was released within the initial 2 h at pH 6.9 and about 79% of drug was released within the initial 3 h at pH 7.4. At 37 ◦ C, there were no initial burst release and only about 79% of drug was released after 2 days. The results indicate that the release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions. But, as we can see, there is still a major leakage at 37 ◦ C; therefore, the necessary improvement should be made in further research to stop aggregation and prevent leakage at 37 ◦ C in order to apply this microgel. At pH 6.9 and 41 ◦ C, the VPTT of NGA3 microgels was low in comparison with the environmental temperature, the microgels shrunk and aggregated, and then the insulin was squeezed out [1], so that there was a burst release in the initial 2 h. At pH 7.4 and 41 ◦ C, different than expected, there was still an initial burst release, although the VPTT of NGA3 microgels was a little lower than the environmental temperature. The range of the volume phase transition was broad at a high pH value due to the ionization of the COOH groups [26]. The microgels may still shrink and squeeze out the loaded insulin, and the release rate was only slower than that at pH 6.9. At pH 6.9 and 37 ◦ C, as expected, there was no initial burst release. The VPTT of NGA3 microgels was lower than the surrounding temperature. The microgels may not become hydrophobic, so the loaded insulin just diffused out of the microgels. The diffusing process was slow and mild and only 79% of the drug was released after 2 days.
Fig. 8. Viability of cells after incubation as a function of microgel concentration by MTT assay, at 37 ◦ C for 48 h.
3.7. Cell viability NIPAM monomers were known to have cytotoxicity to some extent but the poly(NIPAM-co-AAc) microgels have little cytotoxicity [27]. In order to further evaluate the role of glucosamine in the microgels, the cells were exposed to NGA0 (no glucosamine) and NGA2 microgel dispersions with various concentrations and incubated for 48 h (Fig. 8). It was found that all the cell viabilities were nearly maintained over 80%. Furthermore, the viabilities of cells in NGA2 dispersions were all higher than that in NGA0 and maintained over 100%. The results suggested that glucosamine may enhance the biocompatibility of the microgels and the glucosamine-carrying microgels have potential for in vivo use. 4. Summary In this study, a novel series of glucosamine-carrying microgels incorporated by NIPAM and AAc as the thermo- and pH-responsive comonomer were prepared. The microgels exhibit an excellent temperature- and pH-responsive behavior and the VPTT of the microgels at a given pH value can be adjusted by modifying the amount of AADG or AAc. Therefore, insulin release was also temperature and pH sensitive, being more rapid under a high temperature and acidic environment. The cell viability study showed that the use of glucosamine may improve the biocompatibility of the microgels. In conclusion, the temperature- and pH-sensitive glucosamine-carrying microgels have the potential for application in a targeted drug delivery and can be further modulated to be used as efficient tumor delivery vehicles. Acknowledgments The authors gratefully acknowledge the help rendered by Dr. Zhongming Mu (Metabolic Diseases Hospital, Tianjin Medical University) for fibroblast cells and MTT assay. This work is supported by the Foundation Program for the Development of Science and Technology in Universities of Tianjin (No. 20070219).
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Glucosamine-carrying temperature- and pH-sensitive microgels: Preparation, characterization, and in vitro drug release studies Dayong Teng, Jingli Hou, Xinge Zhang ∗ , Xin Wang, Zhen Wang, Chaoxing Li ∗ The Key Laboratory of Functional Polymer Materials of Ministry Education, Institute of Polymer Chemistry, Nankai University, 94# Weijin Road, Tianjin 300071, China Received 22 December 2007; accepted 12 March 2008 Available online 15 April 2008
Abstract Glucosamine-carrying temperature- and pH-sensitive microgels with an average diameter of about 100 nm were successfully prepared by free radical precipitation polymerization. The thermo- and pH-responsive properties of the microgels were designed by the incorporation of Nisopropylacrylamide (NIPAM) and acrylic acid (AAc) to copolymerize with acrylamido-2-deoxyglucose (AADG). The stimuli sensitivity of the microgels was studied by the measurement of their sizes and volume phase transition temperature (VPTT) under different surrounding conditions. The results showed that the microgels were responsive to temperature, pH, and ionic strength, and could have a desired VPTT by modifying AADG and AAc contents. The effect of temperature and pH on insulin release from the microgels was also investigated. The release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions. A preliminary in vitro cell study showed that the glucosamine-carrying microgels are more biocompatible to mouse fibroblast cells, compared to the microgels without glucosamine. These glucosamine-carrying dual-sensitive microgels may be promising carriers for targeted drug delivery to tumors. © 2008 Elsevier Inc. All rights reserved. Keywords: Glucosamine; Target delivery vehicles; Microgels; Stimuli-sensitive polymers
1. Introduction Microgels are cross-linked polymer particles with average diameters ranging from 50 nm to 5 µm [1]. Microgels, smaller than 200 nm, with hydrophilic surfaces are better target delivery vehicles particularly for solid tumors [2]. Using these microgels, drug targeting to solid tumors can be achieved by an enhanced permeation and retention effect (EPR effect) [3], and in contrast, the microgel particles with a larger diameter or hydrophobic surface lead to a rapid uptake by the reticuloendothelial systems (RES) [4–6]. D -Glucosamine (GA) is a monosaccharide, which can be obtained by either chemical or enzymatic hydrolysis of the natural polysaccharide—chitin and chitosan [7]. The existence of reactive hydroxyl and amino groups in GA offers many possibilities for obtaining new polymers. These derivatives have a * Corresponding authors. Fax: +86 22 2350 5598.
E-mail addresses: [email protected] (X. Zhang), [email protected] (C. Li). 0021-9797/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2008.03.014
range of biological activities [8–10], and GA in a certain concentration range could kill tumor cells without influencing normal cells [11]. Therefore, polymeric nanoparticles composed of a functional GA, acrylamido-2-deoxyglucose (AADG), have been prepared in a reverse micellar system recently [12]. The particles obtained were of >85 nm in diameter and were highly monodisperse. In addition, it is known that the interior temperature of tumors (T ∼ 42 ◦ C) is higher than that of normal tissues (T ∼ 37 ◦ C) [13], and most solid tumors have an acidic extracellular pH (6.85–6.95), while normal blood pH remains at 7.4 [14]. Until now, many studies were focused on enhancing the passive targeting ability by using a polymer sensitive to the surrounding temperature [15] or pH [16,17]. Only few reports were related to the application of temperature and pH dual-sensitive microgels in a tumor-targeted delivery system. To explore the potential of glucosamine-carrying temperature- and pH-sensitive microgels as tumor delivery vehicles, the microgels of AADG with N -isopropylacrylamide (NIPAM) and acrylic acid (AAc) as the comonomer were prepared by
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Scheme 1. Preparation of AADG monomer.
the method of a free radical precipitation polymerization. These microgels were characterized using FTIR, SEM, and DLS. We further studied the VPTT of the microgels as a function of pH and salt concentration to examine the microgels’ stimuli sensitivity and colloidal stability. We chose insulin as a model protein drug because tumor suppressor proteins have been increasingly used in anticancer gene therapy recently [18]. The in vitro release of insulin was investigated and preliminary cell viability was also evaluated. 2. Materials and methods 2.1. Materials NIPAM, EDC (1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride), HOBT (1-hydroxybenzotrizole), and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from J&K-Acros Chemical Ltd. (USA) and used as supplied. AAc was freshly distilled before use. Pure crystalline porcine insulin (with a nominal activity of 28 IU/mg) that was used without further purification was obtained from the Xuzhou Wanbang Biochemical Co., Ltd. (Jiangsu, China). All other chemicals were of analytical grade and were used without further purification. Distilled and deionized water was used throughout the work.
2.2. Preparation of acrylamido-2-deoxyglucose monomer Typically, AAc (3.6 g, 50 mmol), EDC (11.2 g, 60 mmol), and HOBT (8.1 g, 60 mmol) were dissolved in 400 mL of DMF and the solution was stirred at 0 ◦ C for 1 h. Then a solution of glucosamine hydrochloride (21.5 g, 100 mmol) in DMF (200 mL) was added, followed by the addition of 200 mL of 1.2 mmol/mL NaHCO3 solution (Scheme 1). After being stirred for 24 h at room temperature, the solvents were evaporated under reduced pressure to give a solid that was purified by column chromatography on silica gel using methanol/ethyl acetate (1/4, v/v) as the eluent to obtain 6.5 g of AADG (yield 56%). 1 H NMR (D O) δ (ppm): 6.4 (1H, m, COCH=), 6.3 (1H, m, 2 CH=CHCO, anti), 5.8 (1H, m, CH=CHCO, syn), 5.2 (1H, m, CH), 3.4–4.0 (5H, m, CH). 2.3. Preparation and purification of microgels PNIPAM/AADG/AAc microgels were prepared by a free radical, precipitation polymerization reaction using MBA (N ,N -methylenebisacrylamide) as the crosslinking vehicle, APS (ammonium persulfate) as the initiator, and SDS (sodium dodecyl sulfate) as the stabilizer (Scheme 2). A detailed description of this polymerization method may be found elsewhere [1]. Different monomer molar ratios were used to prepare
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Scheme 2. Preparation of microgels.
Table 1 Samplea
NIPAM AADG (mmol) (mmol)
AAc (mmol)
NGA0 NGAa NGAb NGAc NGA1 NGA2 NGA3
8.8 8.8 8.8 8.8 8.8 8.8 8.8
2.20 2.20 2.20 2.20 1.65 1.10 0.55
0 0.55 1.10 2.20 1.10 1.10 1.10
MBA (mmol)
0.132
APS (mmol)
0.22
SDS (mmol)
0.176
Water (mL)
100
spectrometer (FTS-6000, Bio-Rad Co.) with a KBr tablet containing microgel powders. 2.4.2. SEM The SEM micrographs of the microgels were obtained with a scanning electron microscope (SS-550, Shimadzu). The sample was prepared by placing a drop of a highly diluted suspension of the microgels in a sample holder and drying before being sputtered with gold.
a All reactions were carried out at 75 ◦ C for 5 h.
the microgels with different compositions (Table 1). All microgels were purified via dialysis (cutoff: 8000–12,000 Da) against frequent changes of stirring water for a week at room temperature. 2.4. Characterization of the microgels 2.4.1. FTIR spectroscopy The purified microgel dispersions were lyophilized before analysis. The chemical structure was analyzed with an FTIR
2.4.3. DLS The hydrodynamic diameter (DH ) and size distribution of the microgels were determined in 150 mM NaCl aqueous solution as a function of temperature and pH by DLS (Brookhaven, INNDVO300/BI900AT) at a scattering angle of 90◦ . The scattered light of a polarized argon laser beam with a wavelength of 636 nm was used. The temperature of the dispersions was maintained by refrigerated circulator baths, and the samples were equilibrated at each temperature for a minimum of 30 min to arrive at swelling/deswelling equilibrium in water before data collection. Data were analyzed with the Brookhaven software
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provided by the manufacturer. DH under each temperature was the mean DH of three effective measurements. 2.4.4. Volume phase transition temperature The volume phase transition temperature (VPTT) of the microgels was determined by cloud point measurement as a function of pH and salt concentration with an UV–Vis spectrophotometer (Shimadzu, UV2550). The temperature was raised from 20 to 80 ◦ C in 0.5 ◦ C increments every 10 min and the transmittance was measured at 550 nm. The VPTT was determined as the temperature at the inflection point in the curve of dispersion transmittance versus temperature. 2.5. Loading of insulin Insulin loading was accomplished by equilibrium partitioning of insulin into the microgels. In a typical loading study, 3 mL of insulin solution (0.6 mg/mL) was added to 3 mL of the microgels dispersion (2.0 mg/mL) to form the dispersion. The dispersion was cooled to 4 ◦ C, stored for 24 h, and heated quickly to 37 ◦ C. The association efficiency of insulin was determined on separation of microgels from the dispersion containing free insulin by centrifugation (12,000 rpm, 30 min, 20 ◦ C). Before centrifugation, a small amount of 0.1 N HCl was added to the dispersion to enhance the hydrophobicity of the microgels, which made the microgels convenient for separation from the aqueous media. The amount of free insulin was analyzed in the supernatant by the Bradford method [19], using UV–Vis spectroscopy at 595 nm. Insulin entrapment efficiency (E%) and the loading efficiency (L%) were calculated as follows: total insulin − free insulin × 100%, total insulin total insulin − free insulin L% = × 100%. microgels weight All measurements were performed in triplicate and averaged. E% =
2.6. In vitro release studies Insulin release was determined by incubating the microgels in 2 mL phosphate buffer, respectively, at a constant temperature, with horizontal shaking. At predetermined time intervals, samples were centrifuged and the supernatant was taken and replenished by fresh buffer. The free insulin was determined by the Bradford method, and a calibration curve was made using nonloaded microgels to correct for the intrinsic absorption of the polymer. The release kinetics was studied in pH 6.9 and 7.4 buffers each at 37 and 41 ◦ C, using NaCl to adjust the salt concentration to 150 mM. In each experiment, the samples were analyzed in triplicate and the error bars represent the standard deviation. 2.7. Cell viability Cell viability was evaluated by using L-929 mouse fibroblast cells. The cell line was cultured in Dulbecco’s modified Eagles
Fig. 1. FTIR spectra of (A) AADG, (B) NGA0, and (C) NGA2.
medium (DMEM) in a humidified atmosphere (5% CO2 /95% O2 ). The cells were seeded into 96-well plates at 10,000 cells per well. The plates were then returned to the incubator and the cells were allowed to grow to confluence for 24 h. NGA0 and NGA2 microgel dispersions were prepared with culture medium. There dispersions were diluted to give a final range of microgel concentrations from 25 to 1000 mg/L. Then the media in the wells were replaced with the preprepared culture medium–sample mixture (200 µL). The plates were then returned to the incubator and maintained in 5% CO2 at 37 ◦ C for 48 h. Each sample was tested in six replicates per plate. After incubation culture medium and 20 µL of MTT solutions were used to replace the mixture in each well. The plates were then returned to the incubator and incubated for a further 4 h in 5% CO2 at 37 ◦ C. Then, the culture medium and MTT were removed. Isopropanol (100 µL) was then added to each well to dissolve the formazane crystals. The plate was placed in 5% CO2 at 37 ◦ C for 10 min and for 15 min at 6 ◦ C before measurement. The optical density was read on a microplate reader at 570 nm. Cell viability was determined as a percentage of the negative control (untreated cells). 3. Results and discussion 3.1. FTIR analysis Fig. 1 exhibits the FTIR spectra of (A) AADG monomer, (B) NGA0 (NIPAM/AAc = 80/20, mol/mol) microgels, and (C) NGA2 (NIPAM/AADG/AAc = 80/10/10, mol/mol) microgels. As shown in the figure, (B) and (C) present typical bands of PNIPAM (3300 cm−1 for N–H stretching; 2973, 2934, and 2876 cm−1 for C–H stretching; 1650 cm−1 for the amide I band assigned to C=O stretching; 1548 cm−1 for the amide II band assigned to N–H in-plane bending vibrations) and PAAc (1459 cm−1 for C–O stretching). Furthermore, (A) and (C) present typical bands of glucosamine (1100 cm−1 for C–O–C stretching), but (B) did not present this band, indicating that AADG had taken part in the polymerization.
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(A)
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associate. Following the structural change and consequent enhanced surface hydrophobicity, the microgels aggregated and formed larger particles. So when the particles began to aggregate, the microgel dispersion became turbid, as shown in Fig. 3B. The phenomenon was in good agreement with the experimental results described by Gupta et al. [20], who observed that the aggregation of the microgels accelerated the release of ketorolac at 37 ◦ C. However, according to the result reported by Lin et al. [21], the microgel did not aggregate with increasing temperature. They inferred that poly(NIPAM-co-AAc) microgels formed a slightly core–shell structure, and the hydrophobic groups in the core were packed by the AAc shell, that avoided the aggregation. Thus, the microgels shrank to a smaller one while the temperature increased obviously. The pH-responsive behavior of the microgels was investigated by DLS. As shown in Fig. 4, when pH < 5.0, the diameters of microgels were nearly invariable regardless of the change of pH, which was due to the protonation of the COO− groups. Indeed, the pKa of PAAc is around 4.7 in water, and at low pH it results in a protonation of the COO− groups to COOH [22]. Therefore, the microgels’ hydrophobicity is increasing. And when pH 5.0, the diameters became larger when the pH value was increased. The ionization of the COOH groups and consequent enhanced electrostatic repulsion caused the polymer chain stretching that made the particle size increase. 3.4. The effect of AADG and AAc on the VPTT of microgels
(B) Fig. 2. SEM image (A) and size distribution (B) of NGA2 microgel.
3.2. Size and morphology of microgels The morphology of the resulting microgels was investigated by SEM (Fig. 2A). It is evident that microgels are well dispersed as individual particles with spherical shapes. From Fig. 2A, it can be seen that the size of the microgels is around 50–60 nm in diameter in the solid state, which may be due to the collapse of the free hydrophilic segments of the polymer as well as the dehydration of the microgels. As shown in Fig. 2B, the microgels exhibit a narrow size distribution with an average diameter of around 100 nm. 3.3. Thermo- and pH-responsive structural changes of microgels DLS measurement is an efficient method for investigating the thermo-responsive properties of PNIPAM and related microgels. Fig. 3A shows the diameter change as a function of temperature. The results show that the size of the particles significantly increased with an increase in the temperature, which was correlated to the NIPAM segment in the microgels. As the temperature increased up to a certain point, the water contained in the microgels was expelled due to the disruption of hydrogen bonding between the water and the hydrophilic amide groups, and then the hydrophobic isopropyl groups began to
The cloud point method is a simple method for determining the VPTT. Table 2 shows the VPTT of microgels with various compositions as a function of pH value. 3.4.1. Effect of AADG NGA0, NGAa, NGAb, and NGAc were selected to research the effect of AADG on the VPTT of microgels. To provide a better overview of the observed effects, all VPTT at high ionic strength (150 mM) are plotted against the molar percentage of AADG to NIPAM at different pH (Fig. 5A). At pH 2.4 and 5.0, the VPTT was linearly increasing as the mol% of AADG content increased. At pH 5.0, the VPTT was obviously higher than that at pH 2.4, and NGAc exhibited no VPTT in the temperature range 20 to 80 ◦ C. The results indicate that the AADG segments could enhance the hydrophilicity of the microgels and then could increase the VPTT of the microgels. Moreover, the VPTTs of the microgels can be easily changed by the incorporation of AADG in the microgels and by considering the used pH. Glucosamine-based materials could not only enhance the hydrophilicity but also impart stability to bioactive molecules like proteins and enzymes [23]. These excellent qualities and the antitumor activity made glucosamine a useful material in the research of a tumor-targeted delivery system. 3.4.2. Effect of AAc NGAb, NGA1, NGA2, and NGA3 were selected to observe the VPTT change as a function of AAc content. The same as
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(A)
(B) Fig. 3. Thermo-responsive behaviors of NGA2 microgel at pH 5.0. (A) Diameter changes as a function of temperature. (B) Transmittance of the microgel on temperature change.
AADG, all VPTTs were plotted against the molar percentage of AAc to NIPAM at the studied pH in Fig. 5B. As seen, the pH dependence was clearly shown and at all pH it appears that the VPTTs nearly linearly ascended as the mol% AAc content increased. At pH 2.4, the VPTT shifted to a higher temperature when less AAc was incorporated into the microgels. On the contrary, at pH 5.0, the VPTT increased with an increase in the proportion of AAc in the microgel. The results indicate that the electrostatic repulsion was enhanced at pH 5.0, due to the ionization of the COOH groups, The increase of electrostatic repulsion reduced the association of hydrophobic groups and then led to a higher VPTT. At pH 7.4, the VPTT was higher than that at pH 5.0, and NGAb and NGA1 exhibited no VPTT in the studied temperature range from 20 to 80 ◦ C. The further ionization of the COOH groups made the VPTT increase further.
3.5. Colloidal stability of microgels At intermediate or high ionic strength, the electrostatic repulsion is weakened or screened out for the van der Waals forces, which caused the collapse of the microgel particles. NaCl is the most effective in the collapse of NIPAM-based microgel particles [24], and so we chose NaCl solutions with different concentrations to investigate the colloidal stability of the microgels. As seen in Fig. 6, at low salt concentration (<200 mM), the change tendency of VPTT was NGA0 > NGA1 > NGA2 > NGA3; the higher the mol% AAc in the microgel, the higher the VPTT. At high salt concentration (>400 mM), the change tendency of VPTT was NGA1 > NGA2 > NGA3 > NGA0; the higher the weight percentage of NIPAM segments in the microgel, the lower the VPTT. At the low salt concentration, the electrostatic stabilization is not
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(A) Fig. 4. Variation of diameters of NGA2 microgel against pH. The samples were analyzed in triplicate and the error bars in the plot were the standard deviation (mean ± SD, n = 3). Table 2 Sample
VPTTa (◦ C) pH 2.4
pH 5.0
pH 7.4
NGA0 NGAa NGAb NGAc NGA1 NGA2 NGA3
30 31 32 35 32.5 33.2 35
62.5 67 72 Nob 58 48 37
Nob Nob Nob Nob Nob 66 42
a VPTT determined by UV–Vis spectroscopy at 550 nm; concentration = 1 g/L in buffer solution, heating ramp = 0.5 ◦ C/10 min. b No phase transition observed in the temperature range 20–80 ◦ C.
(B)
completely eliminated and a significantly higher temperature is required to cause a volume phase transition. The increasing proportion of AAc led to a higher electrostatic repulsion, and then a higher VPTT. But at the high salt concentration, the high ionic strength can eliminate the electrostatic repulsion between the microgels and depress the stabilization of the microgels. Then the solution became a poorer solvent for PNIPAM with increasing NaCl concentration [25]; thus, a very low temperature was needed to cause a volume phase transition and aggregate, and the increasing proportion of the NIPAM segment led to a lower VPTT.
Fig. 5. Variation of VPTT of the microgels at different pH against mol% of (A) AADG and (B) AAc to NIPAM in the microgels.
3.6. In vitro release kinetics studies As a target delivery vehicle for tumors, the microgels surrounding tumor (pH 6.85–6.95) needed a certain VPTT which is higher than the temperature of normal tissue and lower than that of tumor. We used NGA3 as a model microgel; its VPTT is 42 ◦ C at pH 7.4 and 40.5 ◦ C at pH 6.9 (data not shown). The temperature sensitivity of the microgels was used for the insulin loading. The loading process was conducted at 4 ◦ C for 24 h and then the microgels were collapsed at 37 ◦ C. The temperature of 37 ◦ C was selected because it is the lower tem-
Fig. 6. The VPTT of microgel dispersion from turbidity measurements, as a function of (log) NaCl concentration.
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Fig. 7. Temperature- and pH-modulated insulin release from NGA3 microgels.
perature providing maximum volume swelling. Indeed a higher temperature could lead to the deformation and precipitation of the loaded microgels and induce a thermal denaturation of insulin in the loading process. The typical loading and entrapment efficiency were 5.5 and 17.8%, respectively. The low loading and entrapment efficiency were primarily due to the leakage in the centrifugation process, since insulin loading was accomplished by equilibrium partitioning of insulin into the microgels. In vitro insulin release from NGA3 microgels at different pH values (6.9, 7.4) and different temperatures (37 and 41 ◦ C) is shown in Fig. 7. The rate of insulin release from the microgels was initially rapid at 41 ◦ C as compared to that at 37 ◦ C. At 41 ◦ C, about 88% of drug was released within the initial 2 h at pH 6.9 and about 79% of drug was released within the initial 3 h at pH 7.4. At 37 ◦ C, there were no initial burst release and only about 79% of drug was released after 2 days. The results indicate that the release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions. But, as we can see, there is still a major leakage at 37 ◦ C; therefore, the necessary improvement should be made in further research to stop aggregation and prevent leakage at 37 ◦ C in order to apply this microgel. At pH 6.9 and 41 ◦ C, the VPTT of NGA3 microgels was low in comparison with the environmental temperature, the microgels shrunk and aggregated, and then the insulin was squeezed out [1], so that there was a burst release in the initial 2 h. At pH 7.4 and 41 ◦ C, different than expected, there was still an initial burst release, although the VPTT of NGA3 microgels was a little lower than the environmental temperature. The range of the volume phase transition was broad at a high pH value due to the ionization of the COOH groups [26]. The microgels may still shrink and squeeze out the loaded insulin, and the release rate was only slower than that at pH 6.9. At pH 6.9 and 37 ◦ C, as expected, there was no initial burst release. The VPTT of NGA3 microgels was lower than the surrounding temperature. The microgels may not become hydrophobic, so the loaded insulin just diffused out of the microgels. The diffusing process was slow and mild and only 79% of the drug was released after 2 days.
Fig. 8. Viability of cells after incubation as a function of microgel concentration by MTT assay, at 37 ◦ C for 48 h.
3.7. Cell viability NIPAM monomers were known to have cytotoxicity to some extent but the poly(NIPAM-co-AAc) microgels have little cytotoxicity [27]. In order to further evaluate the role of glucosamine in the microgels, the cells were exposed to NGA0 (no glucosamine) and NGA2 microgel dispersions with various concentrations and incubated for 48 h (Fig. 8). It was found that all the cell viabilities were nearly maintained over 80%. Furthermore, the viabilities of cells in NGA2 dispersions were all higher than that in NGA0 and maintained over 100%. The results suggested that glucosamine may enhance the biocompatibility of the microgels and the glucosamine-carrying microgels have potential for in vivo use. 4. Summary In this study, a novel series of glucosamine-carrying microgels incorporated by NIPAM and AAc as the thermo- and pH-responsive comonomer were prepared. The microgels exhibit an excellent temperature- and pH-responsive behavior and the VPTT of the microgels at a given pH value can be adjusted by modifying the amount of AADG or AAc. Therefore, insulin release was also temperature and pH sensitive, being more rapid under a high temperature and acidic environment. The cell viability study showed that the use of glucosamine may improve the biocompatibility of the microgels. In conclusion, the temperature- and pH-sensitive glucosamine-carrying microgels have the potential for application in a targeted drug delivery and can be further modulated to be used as efficient tumor delivery vehicles. Acknowledgments The authors gratefully acknowledge the help rendered by Dr. Zhongming Mu (Metabolic Diseases Hospital, Tianjin Medical University) for fibroblast cells and MTT assay. This work is supported by the Foundation Program for the Development of Science and Technology in Universities of Tianjin (No. 20070219).
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