Research Article pubs.acs.org/journal/ascecg
pH-Triggered Charge-Reversal Silk Sericin-Based Nanoparticles for Enhanced Cellular Uptake and Doxorubicin Delivery Doudou Hu, Zongpu Xu, Zeyun Hu, Binhui Hu, Mingying Yang, and Liangjun Zhu* Institute of Applied Bioresource Research, College of Animal Science, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China S Supporting Information *
ABSTRACT: Silk-based nanoparticles have been exhibiting an increasing potential for use as drug delivery systems due to their great versatility. To extend applications of silk sericin in nanomedicine and improve the performance of silk-based nanoparticles in drug delivery, a facile two-step cross-linking is attempted, for the first time, to fabricate surface charge-reversal silk sericin-based nanoparticles (SSC@NPs) by introducing chitosan into silk sericin. The results suggest stable SSC@NPs are formed with a negative surface charge in a neutral environment. Under mildly acidic conditions, however, surface charge of SSC@NPs undergoes a negative-to-positive conversion. It proves that pH can regulate surface charge of SSC@NPs. It is the increased amino/carboxyl ratio in SSC@NPs that explains the underlying mechanism of the charge conversion property of SSC@NPs. Furthermore, the positively charged SSC@NPs triggered by tumor acidic microenvironment (pH 6.0) result in a 6.0fold higher cellular uptake than the negatively charged counterparts at pH 7.4. In addition, an anticancer drug doxorubicin (DOX) is readily loaded into SSC@NPs and released in a pH-dependent manner. This work provides a simple method to fabricate smart pH-responsive nanoparticles for anticancer drug delivery. KEYWORDS: Silk sericin, Chitosan, EDC, Cross-linking, pH-responsive, Nanocarriers, Drug delivery
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INTRODUCTION For cancer treatment, nanoparticle-based drug delivery systems (NDDSs) provide an opportunity to prolong blood circulation, reduce off-target toxicity, and accumulate at the targeted sites through enhanced permeability and retention (EPR) effect.1,2 In spite of these advantages, challenges such as inferior stability in vivo, poor tumor penetration, inefficient cellular uptake, and slow intracellular drug release remain to be addressed.3 On the basis of the first generation of nanoparticles, design and fabrication of nanoparticles with two or more functions could cross multiple physiological barriers to deliver their loads to target sites.4 For instance, multifunctional nanoparticles can control their properties and behavior to maximize the efficacy and minimize the side effects by responding to internal stimuli (such as pH,5 temperature,6 redox condition,7 and the activity of specific enzymes8) or external stimuli (such as magnetic field,9 ultrasound,10 and light11). Among these stimuli, the acidic extracellular environment of tumors (pH 6.0−7.0) and intracellular compartments (endosomes/lysosomes) are commonly exploited in the design of pH-responsive nanoparticles.12,13 One of the promising strategies is to develop charge-reversal nanoparticles which are able to undergo the negative-to-positive charge conversion in response to environmental pH changes.14 The nanoparticles remain negatively charged during blood circulation, which could reduce the nonspecial absorption by © 2016 American Chemical Society
serum proteins and prolong the circulating time. On the other hand, they become positively charged after accumulating in an acidic tumor extracellular microenvironment, facilitating the cellular uptake due to their high affinity for the negatively charged cell membranes. At present most of the building blocks of charge-reversal nanoparticles are dominated by synthetic polymers, like poly(βamino ester),15 polyethylenimine,16 poly(ε-caprolactone),17 and so on. However, the charge-reversal nanoparticles based on natural polymers are rarely reported up to now.18 Therefore, the formation of charge-reversal nanoparticles developed by natural polymers needs to be investigated to fill this gap. Silk proteins from Bombyx mori silkworms, silk fibroin and silk sericin, are natural biopolymers containing acidic and basic amino acids which are sensitive to pH variation. This feature makes them potential candidates for preparing charge-reversal nanoparticles. Additionally, because of their excellent biocompatibility, biodegradability, easy reconstitution, and mechanical properties, they are getting into the focus of biomedical research in the past decades.19 To date, a variety of approaches have been developed to fabricate silk-based nanoparticles. For example, desolvation, Received: October 4, 2016 Revised: December 16, 2016 Published: December 29, 2016 1638
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
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ACS Sustainable Chemistry & Engineering salting out, and self-assembly are the most used methods owing to the simplicity of operation and mild processing conditions.9,20−29 Besides, other preparation techniques include capillary microdot printing, microemulsion, electrospraying, supercritical fluid technique, and mechanical comminution.30−34 However, the unique feature, both side carboxyl and amino groups in silk proteins, has been neglected during preparing silk-based nanoparticles. Thus, these nanoparticles developed by traditional methods show relative inertia in response to pH stimuli, limiting the development of multifunctional silk-based nanoparticles. Nevertheless, to the best of our knowledge, the ability of silkbased nanoparticles to reverse surface charge via responding to physiological pH changes has remained unexplored. In the present work, we selected silk protein sericin, abundant in side carboxyl and amino groups, to develop sericin-based nanoparticles (SSC@NPs) by a two-step cross-linking approach, in which negatively charged sericin physically reacted with positively charged chitosan through charge-driven force in aqueous solution, followed by EDC chemical cross-linking for an improvement on the structural integrity of SSC@NPs. Meanwhile, this facile approach avoided the use of organic solvents, surfactants, or initiators in the preparation process. The charge-reversal property of SSC@NPs was characterized by zeta potential analysis and confirmed by cell uptake studies. Then, doxorubicin (DOX), a clinically used anticancer drug, was readily encapsulated into SSC@NPs. Therefore, SSC@NPs are proved to be potential nanocarriers in cancer therapy.
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DLC = (mass of DOX loaded in nanoparticles /mass of nanoparticles) × 100%
(1)
For in vitro DOX release assessment, freeze-dried DOX-SSC@NPs were dispersed into 1 mL of PBS (pH 7.4, 6.0, or 5.0) and encapsulated into a dialysis bag (MWCO of 3500 Da) before they were put into 5 mL of PBS under different pH values (pH 7.4, 6.0, or 5.0). Then, the releasing process was performed in a shaking table with a shaking speed of 150 rpm at 37 °C, and monitored by on a microplate reader at a time-course. Morphology of SSC@NPs and DOX-SSC@NPs. The morphological examinations were performed by transmission electron microscopy (TEM, JEM-1200, JEOL). The nanoparticle suspensions were dropped onto a carbon-coated copper grid and stained with 2% (w/v) uranyl acetate for observation. Size Distribution, Surface Zeta Potential, and Stability. The average size, size distribution, and zeta potential of SSC@NPs and DOX-SSC@NPs in PBS (pH 7.4) were monitored by dynamic light scattering (DLS) using a ZetaSizer Nano-ZS90 (Malvern Instruments, Malvern, U.K.). Each batch was analyzed in triplicate. To determine the stability of the nanoparticles in PBS (pH 7.4), the time-dependent hydrodynamic diameter and zeta potential of nanoparticles were also recorded by ZetaSizer Nano-ZS90. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra of the freeze-dried SSC@NPs and DOX-SSC@NPs were obtained with an FTIR spectrometer (8400S, Shimadzu, Japan) in the range 4000−400 cm−1. Before the measurement, KBr pellets were prepared by mixing the freeze-dried nanoparticles and KBr in the mass ratio of 1:100 (w/w). For each measurement, 40 scans were recorded with a resolution of 4 cm−1. Differential Scanning Calorimetry (DSC). The thermal behavior of the freeze-dried SSC@NPs and DOX-SSC@NPs were determined by a DSC822e differential scanning calorimeter (Mettler Toledo, Holland). The measurements were carried out in the range 50−400 °C under nitrogen at a scanning rate of 20 °C/min. Measurement of NH2 Content on SSC@NPs. The amino group content in the SSC@NPs was determined using the 2,4,6trinitrobenzenesulfonic acid (TNBS) method. Pure sericin was used as a control. A sample of 1 mL SSC@NPs or pure sericin solution with concentration of 0.5 mg/mL was incubated with 1 mL of 4% NaHCO3 and 1 mL of 0.1% TNBS at 37 °C for 2 h. The UV absorbance at 344 nm of all the samples was measured by the UV−vis spectrophotometer (UV-2450, Shimadzu, Japan) after the addition of 1 mL of HCl (1 N). The concentration of amine groups was calculated using the following equation:35
EXPERIMENTAL SECTION
Materials. Sericin powder (MWCO 30 000−40 000 Da) was purchased from Booocle Pharmaceutical Technology Co., Ltd., China. Chitosan (100−200 mPa s) and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC·HCl) were supplied by Aladdin Industrial Co., Ltd., China. Doxorubicin hydrochloride (DOX) was obtained from Shfeng Biological Technology Co. Ltd., China. Acetic acid, PBS, and other reagents of analytical grade were purchased from Sinopharm Chemical Reagents Co. Ltd., China. Deionized water was used throughout all experiments. Preparation of SSC@NPs. The silk sericin-based nanoparticles (SSC@NPs) were prepared by physically and chemically cross-linking of negatively charged sericin and positively charged chitosan. Briefly, a chitosan stock solution of 1 mg/mL was prepared by dissolving chitosan in 1% (w/v) acetate solution. Then, chitosan solution was diluted to 0.05 mg/mL using deionized water, and its pH was adjusted to 5.0. The resulting solution was added dropwise to sericin solution of 1 mg/mL at the weight ratio of 1:20 (chitosan/sericin) under magnetic stirring at room temperature, leading to the formation of SSC@NPs. Excess EDC was then introduced to the resulting nanoparticle suspension to improve the chemical stability of SSC@ NPs, followed by dialyzing the nanoparticles solution against excess PBS (pH 7.4, 6.0 or 5.0) with MWCO 3500 Da for 24 h. SSC@NPs were freeze-dried and stored at 25 °C for use. DOX Loading and in Vitro Release of DOX from DOX-SSC@ NPs under Different pH Values. To prepare DOX loaded SSC@ NPs (DOX-SSC@NPs), DOX aqueous solution (0.5 mg/mL) was added into the solution of nanoparticles with different pH values at a DOX/nanoparticles weight ratio of 1:10. After a 24 h adsorption period under magnetic stirring at room temperature, this orange mixture was introduced into a dialysis tube with MWCO of 3500 Da and dialyzed against PBS (pH 7.4, 6.0 or 5.0) for 12 h to remove unloaded drugs. The whole procedure was performed in the dark. The loading amount of doxorubicin was measured by UV absorbance at 490 nm, using a standard calibration curve experimentally obtained. The drug loading content (DLC) was defined as follows:
[NH 2] = AV /εlm
(2)
Here, the following abbreviations apply: [NH2] denotes the amine group content (mol/g sample); A is the absorbance at 344 nm; V is the solution volume (mL); ε is the molar absorption coefficient of trinitrophenyl lysine (1.46 × 104 L mol−1 cm−1); l is the path length (cm); and m is the weight of the sample (mg). In Vitro Cellular Uptake Studies. HeLa cells were used in cell culture and maintained in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in the presence of 5% CO2. The culture medium was replenished every 2 days. Preparation of FITC-Labeled Nanoparticles. To visualize the cell internalization, FITC-labeled SSC@NPs (FITC-SSC@NPs) were prepared. First, FITC (C21H11NO5S) of 1 mg/mL in 1 mL of deionized water was slowly added to 10 mL of a 0.5 mg/mL nanoparticles suspension; then the mixture was under magnetic stirring for 24 h in the dark at room temperature. Next, FITC-SSC@ NPs were obtained after dialyzing (MWCO 3500 Da) the solution against PBS (pH 7.4) to remove unconjugated FITC. Cellular Uptake Determined by Flow Cytometry. For quantitative analysis of the cellular uptake, HeLa cells were seeded at the density of 2 × 105 cells per well in 6-well plates and incubated for 24 h. To determine the cellular uptake efficiency of SSC@NPs at different pH values, the pH of the cell culture medium was adjusted 1639
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
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ACS Sustainable Chemistry & Engineering with 0.1 M HCl to a desired pH, and FITC-SSC@NPs were prepared prior to use. HeLa cells were incubated in fresh medium (pH 7.4, 6.5, and 6.0) containing 50 μg/mL FITC-SSC@NPs for 1 h. Cells incubated in medium without treatment were used as controls. After incubation, the cells were rinsed with PBS (pH 7.4) twice and collected by trypsin treatment. The harvested cells were suspended in PBS and centrifuged at 1000 rpm for 5 min. The supernatants were discarded, and the cell pellets were resuspended with PBS to obtain the cell suspension, which was analyzed by flow cytometry (BD FACSCalibur, USA). To determine the cellular uptake of free DOX and DOX-SSC@ NPs, cell culture medium was replaced by fresh medium with free DOX or DOX-SSC@NPs at a equivalent DOX concentration of 1 μg/ mL and incubated HeLa cells for 0.5 and 6 h, respectively. The acquisition of corresponding cell suspension was performed as described above. Intracellular Localization Imaging by Confocal Laser Scanning Microscopy (CLSM). HeLa cells were seeded onto 12 mm coverslips in 24-well plates with 2 × 104 cells per well in 1 mL DMEM culture medium with 10% FBS and incubated in a humidified 5% CO2 atmosphere for 24 h. For intracellular colocalization of FITCSSC@NPs, FITC-SSC@NPs were added to medium to reach a concentration of 50 μg/mL. After 2 h, 50 nM LysoTrack Red (Beyotime, China) was added to the medium for another 1 h to stain late endosomes/lysosomes. Then cells were washed twice with PBS (pH 7.4) and fixed with fresh 4% paraformaldehyde for 15 min at room temperature. The cells were counterstained with 4′,6-diamidino2-phenylindole (DAPI) for the cell nucleus before being imaged on a confocal microscope (LSM 780 Meta, Carl Zeiss Inc., Thornwood, NY). For cellular uptake of FITC-SSC@NPs, FITC-SSC@NPs were mixed to into a culture medium (pH 7.4, 6.5, or 6.0, respectively) with ultrasonic dispersion for 60 s and incubated with HeLa cells for 1 h. For further investigation of the cellular distribution of free DOX and DOX-SSC@NPs, HeLa cells were treated with fresh medium containing free DOX and DOX-SSC@NPs at an equivalent DOX concentration of 1 μg/mL for 0.5 and 6 h, respectively. After incubation, cell immobilization and staining were processed as described above. In Vitro Cytotoxicity by CCK8 Assay. HeLa cells were seeded in a 96-well plate at a density of 5 × 103 cells per well. After 24 h, the culture medium was replaced with 100 μL of fresh medium (pH 7.4, 6.5, or 6.0, respectively) containing free DOX or DOX-SSC@NPs (DOX concentrations were from 0.1 to 10 μg/mL). The cell viability was measured by CCK8 assay. At the end of incubation, the cell culture medium was aspirated and supplemented with fresh medium followed by incubation with 10 μL of CCK8 for 3 h at 37 °C. The absorbance of solutions was monitored at 450 nm on a microplate reader (BioTek Instruments, Inc.). The results were expressed as the percentage of viable cells over untreated control cells. Statistical Analysis. Statistical analysis was performed with SPSS statistics software. All data were reported as mean ± standard deviation unless specified otherwise. Statistically significant differences (*P < 0.05 or **P < 0.01) were determined by one-way ANOVA with Student’s t-test.
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Scheme 1. (A) Schematic Representation of Formation of SSC@NPs and DOX-SSC@NPs, (B) Decrease in pH Inducing Surface Charge Reversal of SSC@NPs, Facilitating Cellular Uptake of SSC@NPs, and (C) Intracellular Drug Release and Distribution of DOX-SSC@NPs
NPs was observed by TEM (Figure 1C). It was found that the particle size measured by TEM was smaller than that by DLS, which could be attributed to the loss of hydrated layer of SSC@ NPs during TEM measurement. Chemotherapeutic agent doxorubicin (DOX) was added to SSC@NPs suspension followed by incubation at room temperature. DOX loaded SSC@NPs (DOX-SSC@NPs) presented as an orange translucent suspension (Figure 1D). No remarkable changes in particle size, size distribution, and morphology were observed between SSC@NPs and DOXSSC@NPs (Table 1 and Figure 1E,F). Notably, the zeta potential of DOX-SSC@NPs decreased to −6.25 ± 1.2 mV after DOX was loaded (Table 1), which was explained by the shielding effect of positively charged DOX binding to the surface of SSC@NPs. In pilot studies we varied the sericin/chitosan ratio during the preparation of nanoparticles. It was observed that only a specific range of sericin/chitosan ratios could lead to the suspension of SSC@NPs. Here, we selected a 20:1 weight ratio of sericin/chitosan, which resulted in the formation of SSC@ NPs with desired size and surface charge. From a thermodynamic point of view, the interaction of proteins and polyelectrolytes is entropically driven.36 Hence, proteins will strongly bind to the oppositely charged linear polyelectrolytes, leading to the formation of complexed nanoparticles via the electrostatic interactions.37,38 In our work, sericin, a hydrophilic protein, carries negatively charged carboxyl groups, whereas chitosan is a cationic polyelectrolyte bearing a number of positively charged amino groups. Therefore, electrostatic interactions of negatively charged sericin and positively charged chitosan were the primary driving force for SSC@NPs formation. Previous studies found that the surface charge of silk-based nanoparticles were in a wide range of approximately −50 to
RESULTS AND DISCUSSION
Fabrication of SSC@NPs and DOX-SSC@NPs. As illustrated in Scheme 1A, novel silk sericin-based nanoparticles (SSC@NPs) were successfully prepared by a two-step crosslinking approach. As shown in Figure 1A, SSC@NPs in PBS (pH 7.4) exhibited a light-milky colored suspension which was clear and transparent. The particle size distribution and zeta potential of SSC@NPs were determined by DLS. The average particle size of SSC@NPs was 213 ± 5.8 nm with a narrow size distribution (Table 1 and Figure 1B). The zeta potential of SSC@NPs was −10.1 ± 2.4 mV (Table 1). TEM technique provides direct and detailed information on the morphology of SSC@NPs. A well-defined spherical solid structure of SSC@ 1640
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Figure 1. Digital images, the size distribution, and TEM images of SSC@NPs (A−C) and DOX-SSC@NPs (D−F). Scale bar, 200 nm.
DOX-SSC@NPs (Figure S1Ad), which could be assigned to the CO vibration of ketone group from DOX.45 The results implied that DOX was loaded into SSC@NPs. Figure S1B shows the DSC curves of chitosan and pure sericin, SSC@NPs and DOX-SSC@NPs. Chitosan presented an exothermic peak at 326.57 °C (Figure S1Ba), which was due to the decomposition of the polysaccharide chains. On the other hand, sericin displayed two distinct endothermic peaks: a strong one at 214.26 °C and a weak one at 340.27 °C (Figure S1Bb), corresponding to the thermal decomposition of the amorphous region and crystalline region of sericin, respectively.46 It can be seen from Figure S1Bc that the first endothermic peak at around 100 °C was obviously due to the loss of free water. More importantly, it was of interest to note that only one endothermic peak of SSC@NPs was detected, which revealed that sericin and chitosan were highly compatible and no phase separation between them occurred. In comparison with pure sericin, the decomposition temperature of SSC@NPs increased from 214.26 to 218.71 °C (Figure S1Bc), indicating that SSC@NPs were more thermally stable than pure sericin. After loading DOX into SSC@NPs (Figure S1Bd), thermal stability of DOX-SSC@NPs was further increased (from 218.71 to 221.36 °C), which could be related to the electrostatic interactions between DOX and SSC@NPs. Stability and pH-Responsive Surface Charge-Reversal Behavior over a Physiologically Relevant pH Range. The colloidal stability of nanoparticles is important for applications in vivo. The stability of SSC@NPs under PBS (pH 7.4) was examined by monitoring the variation of particle size and zeta potential with the incubation time. Over a time period of 24 h, no obvious changes were noted in the particle size and zeta potential of SSC@NPs (P > 0.05; Figure 2A,B). In fact, SSC@ NPs suspension could be stored at room temperature for more than a week without obvious aggregation. In earlier work, however, the exposure of silk fibroin nanoparticles to PBS induced time-dependent aggregation in just a few minutes.47,48
Table 1. Characterization of SSC@NPs and DOX-SSC@NPs sample
particle size (nm)
polydispersity index
zeta potential (mV)
SSC@NPs DOX-SSC@NPs
213 ± 5.8 231 ± 7.3
0.049 0.067
−10.1 ± 2.4 −6.25 ± 1.2
−15 mV.9,29 However, the surface charge of SSC@NPs in our study was about −10 mV. It was because the amount of negative charge on sericin slightly exceeded positive charge on chitosan. This slightly negative charge of nanoparticles was suggested to reduce the undesirable clearance by the reticuloendothelial system (RES) in vivo.39 Characterization of SSC@NPs and DOX-SSC@NPs. To understand the chemical and structural information on SSC@ NPs and DOX-SSC@NPs, they were characterized by FTIR spectra and DSC, with pure sericin and chitosan as controls. For the FTIR spectra of pure sericin (Figure S1Ab), characteristic amide absorption peaks were observed at 1654 cm−1 (amide I, CO stretching), 1540 cm−1 (amide II, NH bending), and 1247 cm−1 (amide III, CN stretching and N H bending), which were indicative of the presence of a random coil structure.40 The other peak at 1397 cm−1 was attributed to CH and OH bending and COH stretching vibrations of hydroxyl amino acid side chains, such as serine.41 The peak at 1155 cm−1, the characteristic of saccharide structure (CO C stretching) of pure chitosan (Figure S1Aa), was detected as a shoulder peak in the spectra of SSC@NPs (Figure S1Ac), suggesting chitosan was integrated into SSC@NPs. Furthermore, the peak at 1397 cm−1 was barely identifiable while new bands appeared at 2424, 987, 862, and 529 cm−1, indicating that carboxyl groups in sericin might react with available amino or hydroxyl groups in both sericin and chitosan in the presence of EDC.42 At the same time, the increased intensity of amide I and decreased intensity of amide II indicated the formation of a new amide linkage in
[email protected],44 In contrast to SSC@NPs, a shoulder peak at 1730 cm−1 was observed in the spectra of 1641
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
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Figure 2. Stability and pH-responsive properties of SSC@NPs. Particle size (A) and zeta potential (B) of SSC@NPs in PBS (pH 7.4) within 24 h. Zeta potential (C) and particle size (D) of SSC@NPs in PBS at different pH values. Representative TEM images of SSC@NPs at different pH values (E−G).
SSC@NPs increased (Table S1). Therefore, the amino/ carboxyl ratio in SSC@NPs increased, resulting in the augment in pI of SSC@NPs. It should be pointed out that it was the increased amino/carboxyl ratio that endowed SSC@NPs with charge-reversal capacity of responding to the tumor acidic microenvironment (pH 6.0−7.0). This also indicated that the pI of SSC@NPs could be manipulated by regulating the ratio of amino and carboxyl groups. Similarly, another study also illustrated that the pI of ovalbumin increased on addition of chitosan.50 Other nanoparticles synthesized by zwitterionic copolymers of poly(L-glutamic acid) and poly(L-lysine) also achieved the charge conversion according to this mechanism.51 In comparison, traditional silk-based nanoparticles are not able to switch their surface charge to positive in this particular pH range,48,52 because the pI values of these silk proteins are too low (around 453) to reach the transition pH at which the surface charge of the nanoparticles reverses. For instance, Huang et al.29 developed tumor-targeted sericin-based nanoparticles by synthesizing sericin with DOX and folate, which
Thus, the superior stability of SSC@NPs was probably attributed to the nature of the hydrophilicity of sericin. The surface charge-reversal behavior of SSC@NPs was investigated on the basis of the changes of zeta potential after incubation at pH 7.4, 6.3, and 5.1. Obviously, the zeta potential of SSC@NPs underwent a charge transition from negative (−11.2 mV) to positive (15.7 mV) as the pH decreased (Figure 2C). Interestingly, the surface charge of SSC@NPs at pH 6.3 was only 1.1 mV, implying that the isoelectric point (pI) of SSC@NPs was a little higher than 6.3. However, the pI of pure sericin was determined to be 4.2 (Figure S2), which was consistent with previous literature results.49 On the basis of the results, we thus speculated that the increase of pI of SSC@NPs was associated with the ratio of amino and carboxyl group in SSC@NPs. It is known that EDC is commonly used as a cross-linker for the formation of amide between available carboxyl and amino groups. After EDC crosslinking, available carboxyl in sericin and amino groups in chitosan were both consumed, whereas overall amino groups in 1642
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Figure 3. Drug loading (A) and in vitro DOX release (B) of SSC@NPs at different pH values.
Figure 4. Cell viability of HeLa cells in distinct pH environments after 24 h incubation (A). CLSM images of FITC-SSC@NPs at pH 7.4, 6.5, and 6.0. Scale bar, 50 μm (B). Cellular uptake of FITC-SSC@NPs at pH 7.4, 6.5, and 6.0 after 1 h incubation investigated by flow cytometry. Cells without any treatment were used as a negative control to detect autofluorescence (C). The FITC mean fluorescence intensity of the HeLa cells incubated with FITC-SSC@NPs at pH 7.4, 6.5, and 6.0 for 1 h. Cells without any treatment as a control. Indicated values were mean ± SD (n = 3). *P < 0.05, **P < 0.01 (D).
repulsion at pH 6.3 caused the formation of agglomeration of SSC@NPs. These findings are in agreement with previous reports that the neutralization of surface charge during the charge transition process may result in the formation of agglomeration.12 Otherwise, some studies have reported that the size of these charge-reversal nanoparticles increased even to micrometers with a reduced pH.54−56 Drug Loading and in Vitro Release. Drug loading of pharmaceutical agents in nanoparticles is a key parameter to evaluate the therapeutic efficacy of nanocarriers by in vitro and in vivo models. Drug loading content of DOX at pH 7.4, 6.0, and 5.0 were 31.4, 23.7, and 20.1 μg/mg SSC@NPs, respectively (Figure 3A). It was obvious that the difference in
was aimed at controlling the drug release by cleaving a type of pH-responsive bonds (hydrazone bonds) in sericin-DOX conjugates in acid conditions. Although drug released faster at pH 5.0 than at pH 7.4, the sericin-based nanoparticles failed to reverse their surface charge in this pH range. Figure 2D shows the changes in the particle size of SSC@ NPs at different pH values. The particle sizes at pH 7.4 and 5.1 were similar, while a sharp increase in particle size was detected at pH 6.3. The changes in the particle size of SSC@NPs were further verified by TEM (Figure 2E−G). It can be seen that SSC@NPs dispersed well with nearly unchanged particle size at pH 7.4 and 5.1, whereas they aggregated at pH 6.3. Taken together, this illustrated that the lack of sufficient electrostatic 1643
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
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In order to evaluate the intracellular uptake efficiency, CLSM was used to identify the intracellular drug release behaviors of free DOX and DOX-SSC@NPs after incubating HeLa cells for 0.5 and 6 h, respectively. As clearly shown in Figure 5A, free
drug loading content was mainly due to the distinct surface charge of the SSC@NPs, which was discussed above. DOX loading content of SSC@NPs at pH 7.4 was comparable to that of the silk fibroin nanoparticles prepared by desolvation,20 and higher than silk-poly(amino acid)s particles (4 μg/mg).57 The DOX release under various pH environments was pHdependent. As can be seen in Figure 3B, the amount of DOX released from DOX-SSC@NPs at pH 6.0 (∼38%) was slightly higher than DOX released at pH 7.4 (∼32%) over 24 h. Drug release at pH 5.0 was, however, much faster with about 67% of total drug being released during the time period. The increased drug release may be associated with the change of electrostatic interaction between the SSC@NPs and DOX. Meanwhile, the enhanced solubility of DOX at a lower pH also accelerated its diffusion from the nanoparticles. Enhanced Cellular Uptake of SSC@NPs in the Slightly Acidic Environment. To investigate the pH-responsive uptake of SSC@NPs, we first determined cell viability of HeLa cells at different pH values. No significant difference in cell viability was obtained in the range of pH 7.4−6.0 (P > 0.05) (Figure 4A). The results indicated that the slightly acidic environment did not affect viability of HeLa cells, which laid a foundation for further experiments. Then, we fluorescently labeled SSC@NPs with FITC (FITCSSC@NPs) to visualize the internalization of the nanoparticles in HeLa cells by confocal laser scanning microscopy (CLSM). As shown in the CLSM images (Figure 4B), the green fluorescence signal became more intense when pH decreased from 7.4 to 6.0, suggesting the intracellular accumulation of FITC-SSC@NPs increased at a lower pH. Quantitative analysis obtained from flow cytometry (Figure 4C) indicated that slight acidity (pH 6.5 and 6.0) did enhance the cellular uptake of FITC-SSC@NPs and that a more remarkable increase was observed at pH 6.0. As illustrated in Figure 4D, the mean FITC fluorescence intensity from HeLa cells incubated with FITCSSC@NPs at pH 6.5 and 6.0 were approximately 1.4-fold (P < 0.05) and 6.0-fold (P < 0.01) higher than that of cells incubated at pH 7.4, respectively. The more efficient cellular uptake of SSC@NPs was presumably attributed to the fact that surface charge switched from negative to positive as a result of a lower pH (Scheme 1B). At pH 7.4, the negative charge on the surface of SSC@NPs resulted in limited cellular internalization due to repulsive electrostatic interaction with negatively charged phospholipid head groups, glycans, as well as proteins on cell membranes. However, at pH 6.5, the approximate pI of SSC@ NPs, the neutralization of surface charge weakened the repulsive electrostatic interaction, leading to improved cellular uptake of SSC@NPs. As pH further decreased to 6.0, the surface charge of SSC@NPs turned to be positive, which showed a higher affinity for the cell membranes, and thus, SSC@NPs were readily internalized by HeLa cells. The findings corroborated the property of surface charge-reversal of SSC@ NPs at the cellular level. Intracellular Drug Release and Distribution of DOXSSC@NPs. To achieve pH-dependent drug release in lysosomes, SSC@NPs were first required to be internalized into lysosomes. As illustrated in Figure S3, the green fluorescence of FITC-SSC@NPs was observed in cells after 2 h incubation and colocalized with red fluorescence of endosomes/lysosomes (visualized as yellow fluorescence). Consistent with earlier work,20,47 the results proved that SSC@NPs were taken up by the cells and transported to endosomes/lysosomes.
Figure 5. CLSM images of the distribution of free DOX (A, B) and DOX-SSC@NPs (C, D) by HeLa cells after incubation for 0.5 h (A, C) and 6 h (B, D), respectively. Scale bar, 50 μm.
DOX (red fluorescence) rapidly localized into the cell cytoplasm and nucleus (DAPI, blue staining) as early as 30 min postincubation. After incubation for 6 h, free DOX was found to produce an intense staining only into the nucleus (Figure 5B). In contrast to free DOX, a 0.5 h incubation of FITC-DOX-SSC@NPs with the cells led to an overlap between red and green fluorescence (yellow fluorescence in merged images) in the cell cytoplasm (Figure 5C), suggesting that DOX-SSC@NPs were successfully transported into cytoplasm without leakage of the entrapped DOX. Six hours later, however, DOX was detached from FITC-SSC@NPs and translocated into the cell nucleus. FITC-SSC@NPs (green fluorescence) still dispersed in cytoplasm, as presented in Figure 5D. This finding was in line with an earlier observation, which also showed that free DOX entered cells faster than DOX loaded silk-elastin-like nanoparticles.28 Therefore, several conclusions could be drawn according to the results. First, entrapped DOX onto SSC@NPs was taken up into lysosomes, where DOX detached from SSC@NPs due to the lowered pH environment. Second, the time needed for DOX release from SSC@NPs was about 0.5−6 h. Third, SSC@NPs remained in the lysosomes without entering into nucleus (Scheme 1C). Quantitative analysis of intracellular DOX content was further investigated by flow cytometry. As shown in Figure 6, it indicated that intracellular DOX content released form DOXSSC@NPs was slightly higher than that of free DOX for both 0.5 and 6 h. In Vitro Cytotoxicity. The cytotoxicity of SSC@NPs was evaluated against HeLa cells by CCK8 assay (Figure 7A). SSC@NPs showed nearly no cytotoxicity to HeLa cells even up to a concentration of 1000 μg/mL, demonstrating the high potential of SSC@NPs as a chemotherapy drug carrier. Cells treated with DOX-SSC@NPs displayed a dose-dependent 1644
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
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CONCLUSIONS
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ASSOCIATED CONTENT
In this study, we successfully developed SSC@NPs via physical and chemical cross-linking between silk sericin and chitosan. The preparation process was simple, and neither organic solvents nor harsh conditions were involved. SSC@NPs displayed pH-responsive surface charge-reversal from negative charge in a neutral environment to positive charge in tumor intracellular acidic microenvironment (pH 6.0), due to the increased amino/carboxyl ratio in SSC@NPs. More importantly, the charge-reversal property of SSC@NPs favored HeLa cellular uptake. Furthermore, DOX was loaded onto SSC@NPs and released in a pH-dependent manner. DOX-SSC@NPs can be taken up by HeLa cells and accumulate in endosomes/ lysosomes where DOX was released to the nucleus of cancer cells. In summary, the surface charge-reversal SSC@NPs are able to serve as a promising functional nanocarrier for pHresponsive drug delivery systems.
S Supporting Information *
Figure 6. Flow cytometry analysis of HeLa cells treated with free DOX and DOX-SSC@NPs at an equivalent DOX concentration of 1 μg/mL for 0.5 h (A) and 6 h (B), respectively. Cells without any treatment were used as a negative control to detect autofluorescence.
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02392. Structural characterization of SSC@NPs and DOXSSC@NPs, zeta potential as a function of the pH values for aqueous silk sericin solution, CLSM images of the uptake of FITC-SSC@NPs by HeLa cells, and absorbance and NH2 content of pure sericin and SSC@NPs (PDF)
inhibition (Figure 7B). Compared with free DOX at pH 7.4, DOX-SSC@NPs at pH 7.4 had similar cytotoxicity at low DOX concentrations. Notably, DOX-SSC@NPs were even more effective than free DOX at 1 mg/mL, which was confirmed by intracellular DOX content measured by flow cytometry (Figure 6). Free DOX at high doses, however, was superior to DOXSSC@NPs in cell inhibition (P < 0.05). This may be attributed to DOX rapid diffusion into the cell and nucleus at high concentrations.58 Meanwhile, at a lower pH, DOX-SSC@NPs had an increased anticancer effect compared with at pH 7.4. This indicated that enhanced cellular uptake and rapid drug release at a mildly acidic condition promoted the cytotoxicity. In vivo studies using this novel pH-responsive nanoparticles are in progress, and the results will be reported soon.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +86-0571-88982185. Fax: +86-0571-88982185. ORCID
Liangjun Zhu: 0000-0001-9606-5287 Notes
The authors declare no competing financial interest.
Figure 7. Cytotoxicity of SSC@NPs with different concentrations on HeLa cells after 48 h incubation (A). Cell viability of HeLa cells after 24 h incubation of free DOX and DOX-SSC@NPs with different DOX dosage at pH 7.4, 6.5, and 6.0, measured by CCK8 assay (B). 1645
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(19) Vepari, C.; Kaplan, D. L. Silk as a biomaterial. Prog. Polym. Sci. 2007, 32 (8−9), 991−1007. (20) Seib, F. P.; Jones, G. T.; Rnjak-Kovacina, J.; Lin, Y.; Kaplan, D. L. pH-dependent anticancer drug release from silk nanoparticles. Adv. Healthcare Mater. 2013, 2 (12), 1606−1611. (21) Kundu, J.; Chung, Y.-I.; Kim, Y. H.; Tae, G.; Kundu, S. C. Silk fibroin nanoparticles for cellular uptake and control release. Int. J. Pharm. 2010, 388 (1−2), 242−250. (22) Das, S. K.; Dey, T.; Kundu, S. C. Fabrication of sericin nanoparticles for controlled gene delivery. RSC Adv. 2014, 4 (5), 2137−2142. (23) Chen, M.; Shao, Z.; Chen, X. Paclitaxel-loaded silk fibroin nanospheres. J. Biomed. Mater. Res., Part A 2012, 100A (1), 203−210. (24) Lammel, A. S.; Hu, X.; Park, S.-H.; Kaplan, D. L.; Scheibel, T. R. Controlling silk fibroin particle features for drug delivery. Biomaterials 2010, 31 (16), 4583−4591. (25) Slotta, U. K.; Rammensee, S.; Gorb, S.; Scheibel, T. An engineered spider silk protein forms microspheres. Angew. Chem., Int. Ed. 2008, 47 (24), 4592−4594. (26) Lammel, A.; Schwab, M.; Hofer, M.; Winter, G.; Scheibel, T. Recombinant spider silk particles as drug delivery vehicles. Biomaterials 2011, 32 (8), 2233−2240. (27) Mandal, B. B.; Kundu, S. C. Self-assembled silk sericin/ poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery. Nanotechnology 2009, 20 (35), 355101. (28) Xia, X.-X.; Wang, M.; Lin, Y.; Xu, Q.; Kaplan, D. L. Hydrophobic drug-triggered self-assembly of nanoparticles from silkelastin-like protein polymers for drug delivery. Biomacromolecules 2014, 15 (3), 908−914. (29) Huang, L.; Tao, K.; Liu, J.; Qi, C.; Xu, L.; Chang, P.; Gao, J.; Shuai, X.; Wang, G.; Wang, Z.; Wang, L. Design and fabrication of multifunctional sericin nanoparticles for tumor targeting and phresponsive subcellular delivery of cancer chemotherapy drugs. ACS Appl. Mater. Interfaces 2016, 8 (10), 6577−6585. (30) Zhao, Z.; Chen, A. Z.; Li, Y.; Hu, J. Y.; Liu, X.; Li, J. S.; Zhang, Y.; Li, G.; Zheng, Z. J. Fabrication of silk fibroin nanoparticles for controlled drug delivery. J. Nanopart. Res. 2012, 14 (4), 736. (31) Qu, J.; Liu, Y.; Yu, Y.; Li, J.; Luo, J.; Li, M. Silk fibroin nanoparticles prepared by electrospray as controlled release carriers of cisplatin. Mater. Sci. Eng., C 2014, 44, 166−174. (32) Myung, S. J.; Kim, H.-S.; Kim, Y.; Chen, P.; Jin, H.-J. Fluorescent silk fibroin nanoparticles prepared using a reverse microemulsion. Macromol. Res. 2008, 16 (7), 604−608. (33) Gupta, V.; Aseh, A.; Rios, C. N.; Aggarwal, B. B.; Mathur, A. B. Fabrication and characterization of silk fibroin-derived curcumin nanoparticles for cancer therapy. Int. J. Nanomed. 2009, 4 (1), 115− 122. (34) Roy, K.; Patel, Y. S.; Kanwar, R. K.; Rajkhowa, R.; Wang, X.; Kanwar, J. R. Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells. Int. J. Nanomed. 2016, 11, 25−41. (35) Everaerts, F.; Torrianni, M.; van Luyn, M.; van Wachem, P.; Feijen, J.; Hendriks, M. Reduced calcification of bioprostheses, crosslinked via an improved carbodiimide based method. Biomaterials 2004, 25 (24), 5523−5530. (36) Becker, A. L.; Henzler, K.; Welsch, N.; Ballauff, M.; Borisov, O. Proteins and polyelectrolytes: A charged relationship. Curr. Opin. Colloid Interface Sci. 2012, 17 (2), 90−96. (37) Chen, L.; Subirade, M. Chitosan/β-lactoglobulin core−shell nanoparticles as nutraceutical carriers. Biomaterials 2005, 26 (30), 6041−6053. (38) Qi, J.; Yao, P.; He, F.; Yu, C.; Huang, C. Nanoparticles with dextran/chitosan shell and BSA/chitosan core-Doxorubicin loading and delivery. Int. J. Pharm. 2010, 393 (1−2), 177−185. (39) Xiao, K.; Li, Y.; Luo, J.; Lee, J. S.; Xiao, W.; Gonik, A. M.; Agarwal, R. G.; Lam, K. S. The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles. Biomaterials 2011, 32 (13), 3435−3446.
ACKNOWLEDGMENTS This study was supported by the earmarked fund (CARS-22ZJ0402) for China Agriculture Research System (CARS) and the National Natural Science Foundation of China (21172194).
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REFERENCES
(1) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science 2012, 338 (6109), 903−910. (2) Tammam, S. N.; Azzazy, H. M.; Lamprecht, A. Biodegradable particulate carrier formulation and tuning for targeted drug delivery. J. Biomed. Nanotechnol. 2015, 11 (4), 555−577. (3) Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Bioresponsive polymeric nanotherapeutics for targeted cancer chemotherapy. Nano Today 2015, 10 (5), 656−670. (4) Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Discovery 2014, 13 (11), 813−827. (5) Zhang, J. M.; Chen, R. I. E.; Fang, X. F.; Chen, F. Q.; Wang, Y. T.; Chen, M. W. Nucleolin targeting AS1411 aptamer modified pHsensitive micelles for enhanced delivery and antitumor efficacy of paclitaxel. Nano Res. 2015, 8 (1), 201−218. (6) Chen, K.-J.; Liang, H.-F.; Chen, H.-L.; Wang, Y.; Cheng, P.-Y.; Liu, H.-L.; Xia, Y.; Sung, H.-W. A thermoresponsive bubble-generating liposomal system for triggering localized extracellular drug delivery. ACS Nano 2013, 7 (1), 438−446. (7) Hu, Y.-w.; Du, Y.-z.; Liu, N.; Liu, X.; Meng, T.-t.; Cheng, B.-l.; He, J.-b.; You, J.; Yuan, H.; Hu, F.-q. Selective redox-responsive drug release in tumor cells mediated by chitosan based glycolipid-like nanocarrier. J. Controlled Release 2015, 206, 91−100. (8) Xiong, M.-H.; Bao, Y.; Yang, X.-Z.; Wang, Y.-C.; Sun, B.; Wang, J. Lipase-sensitive polymeric triple-layered nanogel for ″on-demand″ drug delivery. J. Am. Chem. Soc. 2012, 134 (9), 4355−4362. (9) Tian, Y.; Jiang, X.; Chen, X.; Shao, Z.; Yang, W. Doxorubicinloaded magnetic silk fibroin nanoparticles for targeted therapy of multidrug-resistant cancer. Adv. Mater. 2014, 26 (43), 7393−7398. (10) Schroeder, A.; Honen, R.; Turjeman, K.; Gabizon, A.; Kost, J.; Barenholz, Y. Ultrasound triggered release of cisplatin from liposomes in murine tumors. J. Controlled Release 2009, 137 (1), 63−68. (11) Yu, J.; Ju, Y.; Zhao, L.; Chu, X.; Yang, W.; Tian, Y.; Sheng, F.; Lin, J.; Liu, F.; Dong, Y.; Hou, Y. Multistimuli-regulated photochemothermal cancer therapy remotely controlled via Fe5C2 nanoparticles. ACS Nano 2016, 10 (1), 159−169. (12) Mizuhara, T.; Saha, K.; Moyano, D. F.; Kim, C. S.; Yan, B.; Kim, Y.-K.; Rotello, V. M. Acylsulfonamide-functionalized zwitterionic gold nanoparticles for enhanced cellular uptake at tumor pH. Angew. Chem., Int. Ed. 2015, 54 (22), 6567−6570. (13) Danhier, F.; Feron, O.; Preat, V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 2010, 148 (2), 135− 146. (14) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 2013, 12 (11), 991−1003. (15) Chen, F.; Zhang, J.; Wang, L.; Wang, Y.; Chen, M. Tumor pH(e)-triggered charge-reversal and redox-responsive nanoparticles for docetaxel delivery in hepatocellular carcinoma treatment. Nanoscale 2015, 7 (38), 15763−15779. (16) Guan, X.; Li, Y.; Jiao, Z.; Chen, J.; Guo, Z.; Tian, H.; Chen, X. A pH-sensitive charge-conversion system for doxorubicin delivery. Acta Biomater. 2013, 9 (8), 7672−7678. (17) Yuan, Y.-Y.; Mao, C.-Q.; Du, X.-J.; Du, J.-Z.; Wang, F.; Wang, J. Surface charge switchable nanoparticles based on zwitterionic polymer for enhanced drug delivery to tumor. Adv. Mater. 2012, 24 (40), 5476−5480. (18) Hu, Y. C.; Gong, X.; Zhang, J. M.; Chen, F. Q.; Fu, C. M.; Li, P.; Zou, L.; Zhao, G. Activated charge-reversal polymeric nano-system: the promising strategy in drug delivery for cancer therapy. Polymers 2016, 8 (4), 99. 1646
DOI: 10.1021/acssuschemeng.6b02392 ACS Sustainable Chem. Eng. 2017, 5, 1638−1647
Research Article
ACS Sustainable Chemistry & Engineering (40) Zhu, L. J.; Arai, M.; Hirabayashi, K. Gelation of silk sericin and physical properties of the gel. J. Seric. Sci. Jpn. 1995, 64 (5), 415−419. (41) Teramoto, H.; Miyazawa, M. Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy. Biomacromolecules 2005, 6 (4), 2049−2057. (42) Choi, Y. S.; Hong, S. R.; Lee, Y. M.; Song, K. W.; Park, M. H.; Nam, Y. S. Studies on gelatin-containing artificial skin: II. Preparation and characterization of cross-linked gelatin-hyaluronate sponge. J. Biomed. Mater. Res. 1999, 48 (5), 631−639. (43) Kang, M. L.; Ko, J.-Y.; Kim, J. E.; Im, G.-I. Intra-articular delivery of kartogenin-conjugated chitosan nano/microparticles for cartilage regeneration. Biomaterials 2014, 35 (37), 9984−9994. (44) Ma, D. H.-K.; Lai, J.-Y.; Cheng, H.-Y.; Tsai, C.-C.; Yeh, L.-K. Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells. Biomaterials 2010, 31 (25), 6647−6658. (45) Arora, H. C.; Jensen, M. P.; Yuan, Y.; Wu, A.; Vogt, S.; Paunesku, T.; Woloschak, G. E. Nanocarriers enhance doxorubicin uptake in drug-resistant ovarian cancer cells. Cancer Res. 2012, 72 (3), 769−778. (46) Zhang, H.; Deng, L.; Yang, M.; Min, S.; Yang, L.; Zhu, L. Enhancing effect of glycerol on the tensile properties of bombyx mori cocoon sericin films. Int. J. Mol. Sci. 2011, 12 (5), 3170−3181. (47) Wang, S.; Xu, T.; Yang, Y.; Shao, Z. Colloidal Stability of Silk fibroin nanoparticles coated with cationic polymer for effective drug delivery. ACS Appl. Mater. Interfaces 2015, 7 (38), 21254−21262. (48) Wongpinyochit, T.; Uhlmann, P.; Urquhart, A. J.; Seib, F. P. PEGylated silk nanoparticles for anticancer drug delivery. Biomacromolecules 2015, 16 (11), 3712−3722. (49) Yang, M.; Zhou, G.; Shuai, Y.; Wang, J.; Zhu, L.; Mao, C. Ca2+induced self-assembly of Bombyx mori silk sericin into a nanofibrous network-like protein matrix for directing controlled nucleation of hydroxylapatite nano-needles. J. Mater. Chem. B 2015, 3 (12), 2455− 2462. (50) Yu, S. Y.; Hu, J. H.; Pan, X. Y.; Yao, P.; Jiang, M. Stable and pHsensitive nanogels prepared by self-assembly of chitosan and ovalbumin. Langmuir 2006, 22 (6), 2754−2759. (51) Huang, Y.; Tang, Z. H.; Zhang, X. F.; Yu, H. Y.; Sun, H.; Pang, X.; Chen, X. S. pH-triggered charge-reversal polypeptide nanoparticles for cisplatin delivery: preparation and in vitro evaluation. Biomacromolecules 2013, 14 (6), 2023−2032. (52) Hofer, M.; Winter, G.; Myschik, J. Recombinant spider silk particles for controlled delivery of protein drugs. Biomaterials 2012, 33 (5), 1554−1562. (53) Kim, U. J.; Park, J. Y.; Li, C. M.; Jin, H. J.; Valluzzi, R.; Kaplan, D. L. Structure and properties of silk hydrogels. Biomacromolecules 2004, 5 (3), 786−792. (54) Deng, X.; Wang, Y.; Zhang, F.; Yin, Z.; Hu, Q.; Xiao, X.; Zhou, Z.; Wu, Y.; Sheng, W.; Zeng, Y. Acidic pH-induced charge-reversal nanoparticles for accelerated endosomal escape and enhanced microRNA modulation in cancer cells. Chem. Commun. 2016, 52 (15), 3243−3246. (55) Kim, D.; Lee, E. S.; Oh, K. T.; Gao, Z. G.; Bae, Y. H. Doxorubicin-loaded polymeric micelle overcomes multidrug resistance of cancer by double-targeting folate receptor and early endosomal pH. Small 2008, 4 (11), 2043−2050. (56) Li, S.; Hu, K.; Cao, W.; Sun, Y.; Sheng, W.; Li, F.; Wu, Y.; Liang, X.-J. pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery. Nanoscale 2014, 6 (22), 13701−13709. (57) Serban, M. A.; Kaplan, D. L. pH-sensitive ionomeric particles obtained via chemical conjugation of silk with poly(amino acid)s. Biomacromolecules 2010, 11 (12), 3406−3412. (58) Li, D.; Bu, Y.; Zhang, L.; Wang, X.; Yang, Y.; Zhuang, Y.; Yang, F.; Shen, H.; Wu, D. Facile construction of ph- and redox-responsive micelles from a biodegradable poly(beta-hydroxyl amine) for drug delivery. Biomacromolecules 2016, 17 (1), 291−300.
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