Article pubs.acs.org/Langmuir
Fabrication of Biopolymeric Complex Coacervation Core Micelles for Efficient Tea Polyphenol Delivery via a Green Process Huihui Zhou,† Xiaoyi Sun,† Lili Zhang,† Pei Zhang,† Juan Li,*,†,‡,§ and You-Nian Liu*,†,§ †
College of Chemistry and Chemical Engineering, ‡State Key Laboratory of Powder Metallurgy, and §Key Laboratory of Resources Chemistry of Nonferrous Metals, Ministry of Education, Central South University, Changsha, Hunan 410083, P. R. China ABSTRACT: Nanoencapsulation is a promising method to improve the bioavailability of tea polyphenol (TPP). In this work, we adopted a green process to develop a new kind of complex coacervation core micelles (C3Ms) based on biopolymers for efficient tea polyphenol delivery. First, gelatin−dextran conjugate was synthesized using Maillard reaction. Then the C3Ms were produced by mixing gelatin−dextran conjugate with TPP. Variable factors on the self-assembly of the C3Ms were investigated. Under optimal conditions, the obtained C3Ms are of nanosize (average 86 nm in diameter) with narrow distribution. The formation of the C3Ms is attributed to hydrophobic interaction and hydrogen bonding instead of electrostatic interaction. Transmission electron microscope (TEM) and scanning electron microscope (SEM) results showed that C3Ms have a spherical shape with core−shell structure. ζ-Potential measurement suggested that the core is composed of gelatin with TPP, whereas the shell is composed of dextran segments. The encapsulation efficiency of the C3Ms is pH-independent, but the loading capacity is controllable and as high as 360 wt % (weight/weight of protein). In addition, the C3Ms show sustained release of TPP in vitro. MTT assay revealed that the C3Ms have comparable or even stronger cytotoxicity against MCF-7 cells than free TPP.
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INTRODUCTION Tea polyphenol (TPP) is the major polyphenolic substance found in green tea and believed to have benefits on human health. TPP plays important roles in the prevention and treatment of several diseases such as cancer and atherosclerosis.1−5 Unfortunately, the bioavailability of free TPP is poor in most tissues after administration due to inefficient delivery. Several studies have been focused on utilization of nanoscale vehicles to improve the bioavailability of TPP6−12 and some of them showed promising results for cancer control both in vitro and in vivo.7,8,10,11 For example, Mukhtar and co-workers encapsulated the major TPP component, (−)-epigallocatechin3-gallate (EGCG), in polylactic acid−polyethylene glycol (PLA-PEG) nanoparticles and found that the encapsulated EGCG has 10-fold dose advantage over nonencapsulated EGCG on proliferative ability in human PCa PC3 cells.11 Smith et al. found that the nanolipidic EGCG particles improves the neuronal α-secretase enhancing ability in vitro and the oral bioavailability in vivo.12 Gelatin, generally recognized as safe (GRAS) in pharmaceutical fields, is one of the most attractive polymers used as drug delivery vehicles.10,13−15 Specifically, TPP strongly binds with gelatin and often leads to precipitates due to the high proline contents in gelatin. Though several studies have reported the nanocomplexation of gelatin and TPP, 10,15 the broad size distribution and low loading capacity of the reported gelatinbased nanoparticles are still the big challenges in the application of TPP. © 2012 American Chemical Society
Recent studies has evidenced that the self-assembly strategies are useful tools to fabricate various nanostructured materials.16−18 Among the self-assembly strategies, complex coacervation, a phase separation process that frequently occurs when two oppositely charged polyelectrolyte solutions are mixed in their stoichiometric charge conditions,19−21 has gained a lot of interest in the fabrication of complex coacervation core micelles (C3Ms).22−27 Generally, C3Ms can be prepared by simply mixing double hydrophilic copolymers (having neutralionic hydrophilic chains) with oppositely charged species. Due to the facile preparation and nanosize with narrow distribution, various C3Ms are being explored for incorporation of hydrophilic bioactive molecules, such as DNA and proteins.22−24,27 However, among the reported C3Ms, most of the present double hydrophilic copolymers were synthesized via chemical processes involving the use and generation of toxic chemicals which are strictly prohibited in biomedical fields. Recently, we prepared narrowly dispersed ibuprofen−BSA− dextran nanogels by simply mixing BSA−dextran conjugate and ibuprofen (an organic acid with hydrophobic property), which is mainly driven by hydrophobic interaction.17 In addition, we found the loading capacity of the protein nanogels is greatly enhanced but depends on both pH and ionic strength similar to that of regular C3Ms.17 Received: July 28, 2012 Revised: September 7, 2012 Published: October 5, 2012 14553
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reported.31 Briefly, gelatin−dextran conjugate solutions were mixed with freshly prepared OPA reagent and incubated at room temperature for 3 min. Then the absorbance of the mixture was measured at 335 nm (UV-2405, Shimadzu) immediately. The working curve was obtained using L-leucine as a standard. The degree of conjugation is expressed as the changes in free amino group contents per one gram of protein in the solutions of gelatin alone or gelatin/dextran mixture or gelatin−dextran conjugates. Preparation of the C3Ms Composed of Gelatin−Dextran Conjugate and TPP. Solutions of the gelatin−dextran conjugate and TPP were used for the preparation of the C3Ms. First, the gelatin− dextran conjugate was mixed with TPP solutions at room temperature. The pH of the mixture was adjusted to the desired pH value with 0.1 M HCl solution or 0.1 M NaOH solution. Synthesis of the C3Ms between the gelatin−dextran conjugate and TPP at various weight ratios, concentrations, and pH values was performed. If not specified, the C3Ms were prepared at 1:1 weight ratio of TPP to gelatin, 1.0 mg mL−1 TPP, and pH 5.0. The total concentration was defined as the sum of the concentrations of TPP and gelatin. The cross-linked C3Ms were prepared by incubation with equivalent amounts of genipin (the final molar ratio of aldehyde groups in genipin to the amine groups in gelatin is 1:1) for 24 h at 4 °C. Dynamic Light Scattering (DLS) and ζ-Potential Measurements. The size distribution of the C3Ms was carried out on a Malvern Zetasizer Nano ZS equipped with a 4 mW He−Ne laser (633 nm). The measurements were performed at 25 °C and a fixed scattering angle of 173°. The Z-average size (apparent average hydrodynamic diameter, Dh) and polydispersity index (PDI) were obtained. Filtration of the solutions was avoided because it might remove the self-assembled large complexes. The concentration of the samples for DLS measurement was set at 1.0 mg mL−1 gelatin except those specified. Each sample was analyzed at least three times. ζ-potential measurements were also performed at 25 °C on a Malvern Zetasizer Nano ZS. ζ-potentials were obtained by the Dispersion Technology software according to Smoluchowski approximation in an automatic mode. Each sample was analyzed at least three times. TEM and SEM. TEM observation was conducted on a Microscope JEM-2100 at an accelerating voltage of 200 kV. Samples were prepared by depositing the C3Ms solution onto a carbon-coated copper grid, followed by removal of excess solution by blotting the grid with filter paper. The samples were dried for 72 h at room temperature in a desiccator containing dried silica gel. After that, the samples were negatively stained by phosphotungstic acid and dried for another 72 h before examined by TEM. SEM observation was conducted on a Nova NanoSEM 230 at an accelerating voltage of 10.0 kV. Samples were prepared by depositing the C3Ms solution onto the freshly cleaved silicon wafer surface, followed by a blow drying of nitrogen. The samples were dried for 72 h before being examined by SEM. Determination of the TPP Encapsulation Efficiency and Loading Capacity of the C3Ms. To determine the encapsulation efficiency and loading capacity of the C3Ms, the unloaded TPP was separated from the C3Ms by ultrafiltration (molecular weight cutoff of 3000 Da; Ultracel YM-3, Microcon, Millipore). The loaded TPP was calculated by subtracting the free TPP in the ultrafiltrate from the initial TPP in feed. TPP concentrations were determined by absorbance at 274 nm (UV-2450, Shimadzu) according to the working curve obtained using standard TPP solutions. All experiments were performed in triplicate and average data were reported. In Vitro Release. The release profile of TPP from the C3Ms was assessed using a dialysis method (cutoff molecular weight of 3500 Da). A solution of 10.0 mL of C3Ms or cross-linked C3Ms (1.0 mg mL−1) was transferred into a dialysis bag and dialyzed against 300 mL of release medium (0.05 M acetate buffer at pH 5.0 or 0.05 M phosphate buffer at pH 7.4). Then the release medium was continuously stirred for 24 h at room temperature. At predetermined sampling time, 3.0 mL of release medium was drawn out and replenished with 3.0 mL of
In this work, we adopted a green process for nanoencapsulation of TPP to avoid the use of toxic chemicals. In order to obtain narrowly dispersed micelles for TPP delivery, new double hydrophilic biopolymers, gelatin−dextran conjugate was synthesized. To the best of our knowledge, this is the first report of gelatin−dextran conjugate. Instead of coupling PEG for most double hydrophilic copolymers, herein, gelatin conjugated with dextran via the Maillard reaction. The Maillard reaction is a naturally occurring reaction requiring no additional chemicals that conjugates polysaccharide and protein by linking the reducing end carbonyl groups in the former to the amino groups in the latter.28,29 Noting that dextran, a watersoluble polysaccharide and biomimetic alternative to PEG as antifouling coating components but showing little affinity for TPP, was selected as shell-forming segments.18,30 Afterward, we simply mixed the gelatin−dextran conjugate with TPP to produce the C3Ms. This new kind of C3Ms is based on the complex coacervation of gelatin−dextran conjugate and TPP where the insoluble complex coacervation core of gelatin and TPP is protected from macroaggregation by the conjugated dextran shell. Several merits of these C3Ms are expected including green process, narrow size distribution, high loading capacity, and sustained release. In this study, the physicochemical properties, morphology, self-assembly process, and mechanism of the C3Ms were characterized. In addition, we evaluated the antitumor activity of the C3Ms using cell cytotoxicity assay.
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MATERIALS AND METHODS
Materials. Gelatin (type B, 300 bloom), 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonic acid) (ABTS), and genipin were purchased from Sigma-Aldrich. Dextran with a molecular weight of 62 kDa was purchased from Pharmacia AB (Uppsala, Sweden). Tea polyphenol (TPP, total polyphenolic content >98.0%, total catechin content is 78.2%, EGCG content is 45.6%) was supplied by Beijing Baishun Chemical Technology Co. Ltd. o-Phthaldialdehyde (OPA) of chemical grade was obtained from Sino-China Pharm Co. Ltd. Calf serum was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. All solutions were prepared using deionized water purified by a Milli-Q Water Purification System (Millipore, Bedford, MA, USA) to a resistance of 18.2 MΩ cm. Synthesis of Gelatin−Dextran Conjugates. Gelatin−dextran conjugates were synthesized via the Maillard reaction.28 Briefly, gelatin and dextran with a certain weight ratio were dissolved together in water at 37 °C, then the pH of the mixture was adjusted to 8.0 using 0.1 M NaOH, and the mixture was lyophilized. The lyophilized powder was heated at 60 °C under 79% relative humidity in a desiccator containing saturated KBr solution for 24 h. The resultant Maillard product (gelatin−dextran conjugate) was kept at −20 °C before use. If not specified, the gelatin−dextran conjugate was prepared at 1:1 weight ratio of dextran to gelatin. The product was used without separation of unreacted gelatin and dextran. The BSA− dextran conjugate was also prepared through the same procedure. SDS-PAGE. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a gel electrophoresis apparatus (JM250, JM-X Scientific Co., Dalian, China) to confirm the formation of gelatin−dextran conjugate. Two separate gels were formed with a 10% acrylamide separating gel and a 5% acrylamide staking gel containing 0.1% SDS and were run in electrophoresis at the same time. After electrophoresis, one gel was stained for protein by Coomassie Blue R-250 and the other one was stained for carbohydrate by periodate-fuchsin solution. The protein stain was destained with 10% acetic acid (v/v) containing 10% methanol (v/v). OPA assay. The degree of conjugation of the gelatin−dextran conjugate was analyzed through an OPA assay based on the loss of free amino groups of gelatin after the Maillard reaction as previously 14554
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fresh buffer. All of the samples were protected from light. The amount of released TPP was measured by the UV method as described above. Cell Cytotoxicity. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cell cytotoxicity of TPP against MCF-7 cell lines. The cells were seeded in 96-well plates at a density of 4 × 104 cells per mL. The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin and kept in humidified environment at 37 °C containing 5% CO2. After the cells anchored to the wells, they were treated with fresh culture solution with free TPP and C3Ms and incubated for 24 h, respectively. After incubation, 20 μL of MTT solution (final concentration 0.5 mg mL−1) was added to each well and incubated for 4 h at 37 °C in 5% CO2. Then the plates were centrifuged at 2000 rpm and the medium was aspirated. The MTTformazan crystals formed by metabolically viable cells were dissolved in 200 μL DMSO. Finally, the absorbance of each well was monitored by a microplate reader (Bio-Tek ELx800) at the wavelength of 570 and 630 nm. The cell viability is expressed as follows: Cell viability (%) = AT/A0 × 100; where AT is the absorbance of treated cells and A0 the control absorbance. Data of cell viability are given as mean ± standard deviation (S.D). In addition, data of cell viability for each concentration are statistically compared between free TPP and the C3Ms using Student’s t-test in Microsoft Excel 2003. The level of significance used is p < 0.05.
distribution of the Maillard products, which can be ascribed to the heterogeneous reaction between the different amino groups in the protein and the reducing end carbonyl groups in the polysaccharide32,33 and the broad molecular weight distribution in the parent gelatin molecules (shown in Figure 1B). In addition, thicker high molecular weight bands of the gelatin−dextran conjugate on the tops of the separating gels with increasing the molar ratio of dextran to gelatin are observed for both the polysaccharide and protein staining gel. However, the color changes in the gels are not so obvious upon addition of dextran, which may be due to the gradual but nonlinear increase of the conjugated dextran. We assessed the degree of conjugation using the OPA assay and discovered that the degree of conjugation is about 12% in the gelatin−dextran conjugate at the weight ratio of 1:1. The amount of average free amino groups per 1 g of gelatin is about 0.38 mmol, whereas about 0.33 mmol free amino groups is retained in 1 g of gelatin after the Maillard reaction. Self-Assembly of the C3Ms. Effect of Maillard Conjugates. The interaction of TPP with gelatin is so strong that it easily leads to hazes or precipitates at acidic pH.34,35 In this work, however, interaction of TPP with gelatin was well controlled by the introduction of the gelatin−dextran conjugate. On addition of TPP to a solution of the gelatin− dextran conjugate (1.0 mg mL−1 gelatin) at pH 5.0, no precipitate is observed but homogeneous dispersion occurs (Scheme 1, inset). This phenomenon is in sharp contrast to that of the solutions of gelatin or gelatin/dextran physical mixture in the presence of TPP, which cause obvious precipitates. The results suggested spontaneous formation of micelles through the complexation of TPP with the gelatin− dextran conjugate. Dextran is highly water-soluble and nonionic but has little interaction either with TPP or with gelatin.30 For this reason, the dextran conjugating with gelatin can serve as a stabilizing agent and protect the insoluble core of TPP/gelatin complex against macroaggregation (Scheme 1), whereas free dextran in the physical mixture cannot. DLS measurements revealed that the C3Ms are about 86 nm with narrow distribution (PDI close to 0.1). Though the conjugate was directly used for the self-assembly without further separation, the effect of the conjugate in terms of conjugation degree on the self-assembly was manipulated if we added the equivalent unreacted gelatin/dextran mixture after removing a fraction of the gelatin−dextran conjugate. We observed larger Dh and PDI values on increasing the fraction of the unreacted gelatin/ dextran mixture. As mentioned above, only the fraction of dextran covalently conjugating gelatin provides a steric hindrance effect for the micellar assembly, the decrease in the fraction of conjugated dextran holds a decrease in total specific surface area resulting in the increase in Dh. Effect of pH. Figure 2 shows that minimum Dh and maximum scattering intensity (at constant attenuator settings) are observed at pH 5.0, which is the isoelectric point (pI) of gelatin (pI ≈ 5). In addition, we found that the C3Ms maintain good suspension in a wide range of pH 3−8, though the Dh and PDI are gradually increased. At the critical pH (around pH 5.0), the obvious change in Dh and intensity means that a structural transition of the C3Ms. The increase of the intensity is induced by an increase in molecular mass of the C3Ms, and the decrease of the Dh could be due to the shrinkage of the complex. It indicates that TPP exhibits the strongest binding with gelatin at pH 5.0. This result is consistent with previous observations which demonstrated polyphenol has the strongest interaction
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RESULTS AND DISCUSSION Synthesis of Gelatin−Dextran Conjugates. The Maillard reaction is a natural and nontoxic reaction which occurs during the food processing and conjugates polysaccharide and protein by linking the reducing end carbonyl groups to the amino groups. Herein, water-soluble gelatin−dextran conjugates were prepared via the Maillard reaction by controlling the reaction in its early stages: Amadori rearrangements. Formation of Maillard conjugates was confirmed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) technique. As shown in Figure 1A, a new broad smearing band is clearly observed at the boundary of the separating and stacking gels in
Figure 1. SDS-PAGE patterns of the gelatin−dextran conjugate: (A) carbohydrate stain; (B) protein stain. Lanes 1−5: BSA−dextran conjugate, gelatin, dextran, gelatin/dextran mixture, molecular weight marker (kDa); lanes 6−10 for gelatin−dextran conjugates at different weight ratios of 0.25:1, 0.5:1, 1:1, 2:1, and 4:1, respectively. The amount of the protein sample in each lane was 30 μg except that in lane 5 was 20 μg; the amount of dextran in lane 3 was 30 μg.
the lane of the gelatin−dextran conjugate for the carbohydrate staining gel. On the other hand, no bands appear either in the lane of gelatin or in the lane of the gelatin/dextran mixture. Dextran can not migrate into the gel because of lacking of charges. Hence, the carbohydrate staining experiments strongly verified that dextran is covalently linked to gelatin which drags dextran into the gels via their covalent linkage. The broad band in the lane of the conjugates suggests a heterogeneous 14555
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Scheme 1. Preparation of C3Ms Composed of Gelatin−Dextran Conjugate and TPPa
a
Inset: Photograph of the gelatin/TPP physical mixture (A); gelatin/dextran/TPP physical mixture (B); and the C3Ms composed of the gelatin− dextran conjugate and TPP (C). The samples were prepared at pH 5.0 and 1:1 weight ratio.
Figure 2. Dh, PDI, and intensity of the C3Ms at different pHs.
with protein near the pI of protein where the electrostatic repulsion of the protein itself is minimized. 34 Similar phenomena were found in the complex coacervation of whey proteins and gum arabic which formed an electrostatic complex in a specific pH range.36 Effect of Mixing Ratio. In addition to pH, the mixing ratio is also crucial to the self-assembly of complex coacervation, which usually determines if the complex formation is stoichiometric or not.37 At a fixed concentration of the gelatin−dextran conjugate (1.0 mg mL−1 gelatin) at pH 5.0, Dh was plotted as a function of TPP concentration (Figure 3A). Although the DLS instrument has limitations to obtaining accurate Dh results when the size distribution of suspension is broad, it does help us to determine an optimal condition for a good dispersion with narrow distribution. In the absence of TPP, the gelatin− dextran conjugate has weak scattering intensity (data not shown) and broad size distribution (high PDI value shown in Figure 3A), indicating limited aggregates. On addition of TPP, Figure 3A demonstrates a two-stage process including an initial sharp decrease of Dh and PDI followed by a slightly gradual
Figure 3. (A) Dh of C3Ms vs TPP concentration at pH 5.0. Inset: PDI of C3Ms vs. TPP concentration. (B) Size distributions of C3Ms obtained from different TPP concentrations (a−f: 0, 0.05, 0.20, 1.0, 10.0, and 15.0 mg mL−1).
increase of Dh and PDI. The transition between these two stages is at a TPP concentration around 1.0 mg mL−1. In the first stage, cumulant analysis showed no specific Dh and PDI 14556
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(resulting in large error bars), suggesting a heterogeneous population of aggregates, in agreement with the complexation of a glycosylated proline-rich protein (PRP) called II-1 and EGCG.38 However, upon further addition of TPP in the latter stage, cumulant analysis gave a specific Dh and PDI. To further understand this uncommon aggregation behavior of micelles, the size distribution of the C3Ms was also plotted against a broad TPP concentration range of 0.05−15.0 mg mL−1 (Figure 3B). In the absence or at low concentration of TPP ( 7, which are attributed to the surface adsorption of TPP containing negative phenolic groups. Interaction between TPP and the Gelatin−Dextran Conjugate. A lot of methods, such as isothermal titration microcalorimetry and capillary electrophoresis, have been used to illustrate the interactions between proteins and polyphenols.41,42 Here, three destabilizing agents NaCl, SDS, and urea, which affect the binding mode in different ways, were used to gain insight into the complex coacervation mechanism of the C3Ms. DLS was also used to monitor the association/ dissociation behavior in the solutions. At first, the effect of ionic strength on the dissociation of C3Ms was investigated. The Dh of the C3Ms has little change upon addition of 0.6 M NaCl (Table 1), indicating that the C3Ms are not stabilized by electrostatic interaction. In contrast, urea (disrupting hydrogen bonding) and SDS (destroying both hydrophobic interaction and electrostatic interaction) have a more profound effect on the colloidal stability of the C3Ms. Significant changes in the scattering intensity, Dh, and PDI are observed in the presence of urea or SDS (Table 1), suggesting the C3Ms are disintegrated in these cases. Thus, we proposed the principle of complex coacervation in this C3Ms is based on hydrophobic interaction and hydrogen bonding instead of electrostatic interaction which is always lying in regular C3Ms. Those C3Ms normally dissociate when the ionic strength of the solution reaches the critical value.26 Because the encapsulation and release properties are largely influenced by the stability in the micellar dynamics, this relationship was further discussed in the following text. Morphology. TEM image reveals that the C3Ms are spherical in shape with an obvious core−shell structure (Figure 6A). The core is in light color and the shell is in dark color. According to the precipitation of gelatin with TPP and steric stabilization properties of dextran, we supposed that the core is composed of the coacervate of gelatin with TPP and the shell is composed of the conjugated dextran segments. Under the negative staining by phosphotungstic acid, the loose and hydrophilic dextran shell is stained dark, whereas the coacervation core is relatively compact and cannot be fully stained leading to light-colored spheres. In Figure 6A, most of the particles have a diameter smaller than 120 nm, and the average diameter value is calculated to be about 80 nm. In Figure 6B, SEM image identifies that the C3Ms are narrowly
Figure 6. TEM (A) and SEM (B) images of the C3Ms composed of the gelatin−dextran conjugate and TPP.
distributed and have a spherical shape with an average diameter of about 75 nm. The particle size from TEM or SEM is somewhat smaller than the Dh from DLS. Probably, DLS reveals the hydrodynamic diameter (Dh) for the C3Ms swollen in solution, but TEM and SEM display the size of the micellar particles spread and collapsed on the surfaces of solid matrixes. Loading Capacity and Encapsulation Efficiency. Previous studies demonstrated that specific and nonspecific interactions can significantly improve micellar loading capacity.17,43,44 Interestingly, although the preferred complexation of the C3Ms is presented at the pH 5.0 as mentioned above, we found that C3Ms exhibit almost the same TPP encapsulation efficiency (about 70%) in the pH range of 3−7 (Figure 7A). The loading behavior of the C3Ms in this work is quite different from that of other C3Ms which showed a pHdependent loading and release behavior.17,25 Notably, Lvov and co-workers reported a similar trend of pH-independent EGCG encapsulation into gelatin nanoparticles.10 It is known that the pKa of TPP is in the range of 7.7−8.7,15 and the phenolic groups of TPP are mostly protonated at acidic pH (e.g., pH 5.0). Hence, the results further supported that the driving forces for TPP encapsulation into C3Ms are mainly based on hydrophobic interaction and hydrogen bonding instead of electrostatic interaction. At a pH above 7.0 TPP is more deprotonated and the encapsulation efficiency decreases gradually, which is in line with the weakened hydrogen bonding and enhanced electrostatic repulsion at basic pH. In particular, the encapsulation efficiency of the C3Ms keeps unchangeable (Figure 7B) when the concentration of TPP is above 0.5 mg mL−1, which means the loading capacity has a quasilinear relationship with TPP concentration. That is to say, the loading capacity can be controlled via adjusting the concentration of TPP in the feed. The loading capacity of the C3Ms for TPP at 5.0 mg mL−1 is calculated to be about 360 wt % (weight/weight of protein), much higher than that of other TPP-encapsulated nanovehicles.6,45 The results implied that the C3Ms composed of the gelatin−dextran conjugate and TPP have a high loading capacity of TPP for sufficient delivery. In Vitro Drug Release. As the pH of extracellular fluid in most tumors is relatively lower than that in normal tissues,46 the release profiles of C3Ms were determined at pH 5.0 in 0.05 M of acetic acid buffer and at pH 7.4 in 0.05 M phosphate buffer. As illustrated in Figure 8A,B, the C3Ms show a slower release process compared with free TPP. At pH 5.0, 81% of free 14558
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TPP diffuses across the dialysis membrane after 4 h, whereas about 61% TPP releases from C3Ms. The C3Ms continuously release TPP until completely released at 14 h (ca. 97%). At pH 7.4, the release of TPP from the C3Ms is a little faster than that at pH 5.0. This result is consistent with the above pHdependent assembly result, in which larger size particles are formed at higher pH (Figure 2), due to the weak interaction between gelatin and TPP as well as the high solubility of TPP at the high pH. The sustained release behavior of C3Ms is presumably due to the existence of nonspecific interactions between the carrier and TPP which overcomes a burst release of TPP. In addition, we found that the cross-linked C3Ms further slow the release rate of TPP, though the results sound inconspicuously. We observed a complete release within 24 h for both cross-linked and noncross-linked C3Ms. Meanwhile, a recent work reported a complete release of EGCG within 2.5 h in both water and PBS from the poly(D,L-lactic-co-glycolic acid)−block-poly(ethylene glycol) nanoparticles, whereas EGCG itself dissolved immediately.8 We speculated that, due to the highly hydrophilic nature and small molecular size of TPP, TPP can diffuse out the C3Ms after a certain time in spite of the sustained release. Hence, in contrast to free TPP, the C3Ms can deliver much more TPP into the tumors due to their enhanced permeability and retention (EPR) effect and release TPP in a sustained and effective way in the tumor microenvironments. Cell cytotoxicity of the C3Ms. TPP has been documented to possess antitumor activities against human breast carcinoma MCF-7 cells.2−5 In order to investigate whether TPP inside the C3Ms could maintain the antitumor properties toward MCF-7 cells, we compared the cell viability treated with free TPP or C3Ms using MTT assay. As shown in Figure 9, both free TPP
Figure 7. Encapsulation efficiency of the C3Ms vs pH (A) and vs TPP concentration (B).
Figure 9. Cell cytotoxicity of free TPP and the C3Ms against MCF-7 cells at various TPP concentrations after 24 h incubation. Asterisk means a significant difference (p < 0.05).
and C3Ms display concentration-dependent cytotoxicity properties. The cell viability gradually decreases as the TPP concentration increases for both free TPP and the C3Ms. The C3Ms shows a comparable cytotoxicity to free TPP in most cases (p > 0.05). However, at a TPP concentration of 50 μg mL−1, the C3Ms have stronger cytotoxicity than free TPP (p < 0.05). Only 35% cells survive for the C3Ms, whereas more than 50% of MCF-7 cells survive for free TPP. Moreover, we found that the difference of the cytotoxicity between the cross-linked and noncross-linked C3Ms is insignificant (data not shown). The conjugate has no obvious cytotoxicity under the same conditions (the cell viability is about 99.7 ± 7.4%). Generally,
Figure 8. In vitro release of TPP from the C3Ms in 0.05 M acetate buffer at pH 5.0 (A) and in 0.05 M phosphate buffer at pH 7.4 (B).
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two factors would contribute to the difference in cytotoxicity between the C3Ms and free TPP: (1) TPP release from the C3Ms and (2) the uptake of C3Ms by MCF-7 cells. However, the exact mechanism of cytotoxicity remains unknown. Nevertheless, the EPR effect of nanosized drug delivery systems is a key factor in tumor therapy. After systemic administration of free drug, the drug diffuses rapidly in plasma without differential biodistribution, resulting in low bioavailability. The C3Ms are nanosized with narrow distribution, thus they could selectively accumulate around the leaky regions of tumors. Further, the C3Ms possess high loading capacity of TPP, which ensures enough TPP interaction with the MCF-7 cells. The C3Ms in this work will be very helpful for the treatment of breast cancer because the threshold concentration of TPP to kill breast cancer cells is relatively high.
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CONCLUSION A new kind of biopolymeric C3Ms was prepared via the selfassembly of the gelatin−dextran Maillard conjugate with TPP. The procedure is simple with no involvement of toxic chemicals. The driving forces for the formation of C3Ms are hydrophobic interaction and hydrogen bonding between the gelatin−dextran conjugate and TPP. The C3Ms are of nanosize with narrow distribution, and have core−shell structures. In addition, the C3Ms exhibit high and controllable loading behavior, and sustained release of TPP in vitro. The MTT assay results reveal that the C3Ms have comparable or even stronger cytotoxicity against MCF-7 breast cancer cells than free TPP. Therefore, the nanoencapsulation of hydrophilic TPP by this new C3Ms holds promise for increasing the bioavailability of TPP and merits further investigation in cancer chemoprevention.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-731-88879616. Tel.: +86-731-88836964. E-mail:
[email protected] (J.L.);
[email protected] (Y.-N.L.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 20904066, 21076232, and 21104096), China Postdoctoral Science Foundation (No. 2011M501281), and the Fundamental Research Funds for the Central Universities (No. 2011QNZT052). The authors express grateful thanks to Professor Zhongshi Wu of Second Xiangya Hospital of Central South University for help with MTT studies.
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