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Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery
G Model JIEC 4581 No. of Pages 10
Journal of Industrial and Engineering Chemistry xxx (2019) xxx–xxx
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery Huifang Zhang, Mingliang Pei, Peng Liu* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 February 2019 Received in revised form 15 May 2019 Accepted 29 May 2019 Available online xxx
PK-SS-Hy-D drug-protein prodrug was obtained by conjugating doxorubicin (DOX) onto the PEGylated keratin (PK) with both bioreducible disulfide linkage and acid-cleavable hydrazone bond in a series connection mode. The controlled release profiles demonstrated the pH and reduction dual-responsive triggered release of DOX from the proposed drug-protein conjugate nanoparticles, with a low premature drug leakage of 5.5% in the simulated physiological medium. The in vitro experiments indicated that the proposed prodrug nanoparticles could delivery DOX into the cell nuclei, with an enhanced anti-tumor efficacy. It is expected as a potential candidate for future tumor chemotherapy with minimized toxic side effect. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Prodrug nanoparticles Drug-Protein conjugate Chicken feather keratin pH and reduction dual-responsive Series connection mode
Introduction In the last decades, polymer-based drug delivery systems (DDSs) have been intensely investigated for tumor chemotherapy to minimize the toxic side effects on normal tissues and multidrug resistance of the conventional chemotherapeutic drugs [1]. The polymer-based DDSs could be easily tailored into nanomedicines, as nanoparticles, nanogels or micelles, to enter and accumulate inside tumor tissues through passive targeting via the enhanced permeability and retention (EPR) effect [2], in which the conventional small chemotherapeutic drugs are loaded via noncovalent interactions or covalent conjugation as prodrugs [3]. The stimuli-responsive characteristics of the polymer carriers could also endow the stimuli-triggered drug release in the acidic tumor microenvironment with high reductant level, while the premature drug leakage could be minimized in the normal physiological medium. Thus, the toxic side effects of the chemotherapeutic drugs on normal tissues could be efficiently avoided and the bioavailability of the chemotherapeutic drugs could also be improved, especially for the prodrug nanoformulations in which the chemotherapeutics are covalently conjugated onto the polymer carriers via the acid-labile or reduction-cleavable linkages [4–7].
* Corresponding author. E-mail address: [email protected] (P. Liu).
Besides the acidic microenvironment (blood and healthy tissues of pH 7.4, while the tumor extracellular at pH 6.0–7.2, and pH 5.0– 6.0 in the endosomes and pH 4.0–5.0 in the lysosomes) and the higher reductant level (approximately 2–20 mM GSH in blood or extracellular matrices, while 0.5–10 mM inside cells) [8], endowing the pH and reduction responsive controlled drug release, tumors also overexpress certain enzymes, such as lysosomal proteinases [8,9], which could provide an enzyme-responsive triggered drug release, by degradation of the protein and polypeptide-based DDSs. To develop a pH/reduction dual-responsive nanomedicine for tumor chemotherapy, a multi-functional polymer should be selected at first, with various functional groups. Owing to their excellent biocompatibility and biodegradability, biopolymers have attracted more and more interest in the last decades, such as polysaccharides [10] and proteins [11]. As one kind of natural protein in wool, hair or feather, keratin has attracted research interest as biopolymer vehicle for anti-tumor DDSs recently [12], because of its massive cysteine residues (7–20 mol% of all amino acids) resulting disulfide crosslinkage, as well as plentiful amino and carboxyl functional groups, in which the disulfide crosslinking could be cleaved off into active thiol groups with reductant, and re-formed with oxidant, endowing keratin excellent redox-responsive property [13]. Such different functional groups are expected to respond to various stimuli intracellular tumor cells for the anti-tumor drug delivery. Furthermore, as a protein, keratin might be degraded by the proteinases in tumor cells, realizing an enzyme-responsive triggered drug release
https://doi.org/10.1016/j.jiec.2019.05.041 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Zhang, et al., Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.05.041
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[9,14]. Although the keratin proteins have been used as polymer vehicle in DDSs for various anti-tumor drugs [15–21], there is no report on the keratin-based drug-protein conjugate prodrug by now, to the best of our knowledge. In the present work, keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) were designed with acidlabile and reduction-cleavable linkages in series (Scheme 1), for the tumor intracellular pH/reduction dual-responsive triggered DOX release. Natural keratin contains more carboxyl groups than amino groups with a low isoelectric point (pI) of about 4.5 [17]. Such chemical structure would be against the acid-labile linkage in the proposed drug-protein conjugate prodrug. To solve the problem, the chicken feather keratin was PEGylated via the amidation of its carboxyl groups with amino-terminated methoxy poly(ethylene glycol) (mPEG-NH2) to raise its pI value. On the other hand, the PEGylation would improve the blood stability of the final prodrug nanoparticles [22]. Then, the chemotherapeutic drug, doxorubicin (DOX), was covalently conjugated onto the keratin from its thiol groups, with acid-labile and reduction-cleavable linkages in series. Such series connection mode would endow both the acid-triggered release of DOX by the hydrolysis of the acid-cleavable hydrazone linkage and the reduction-triggered release of its derivative (M-Hy-D) by the cleavage of the reduction-cleavable disulfide linkage. Furthermore, the covalently crosslinked prodrug nanoparticles could also be obtained by the inter-molecular oxidation coupling reaction of keratin between their free thiol groups, accompanying with the desired drug conjugation via the oxidation coupling reaction between the M-Hy-D and keratin. Thus, novel keratin-based drug-protein
conjugate prodrug nanoparticles were obtained with reductioncleavable disulfide crosslinkage and acid-labile and reductioncleavable drug conjugation. Experimental section Materials and reagents Chicken feather keratin was extracted from feather as described previously, with number average molecular weight (Mn) of 29 kDa [21]. Amino-terminated methoxy poly(ethylene glycol) (mPEGNH2, Mn of 2000, 98%) was purchased from Beijing Kaizheng Biotechnol Develop Co. Doxorubicin hydrochloride (DOXHCl, 99%) was purchased from Beijing Hvsf United Chemical Materials Co. Ltd.. All other chemicals in the experiments were of analytical grade and used without further purification. Double distilled water was used throughout. Synthesis of M-Hy-D The thiol-functionalized small molecular prodrug M-Hy-D was synthesized via the formation of acid-cleavable hydrazone bond between 3-mercaptopropanohydrazide (M-Hy) and DOX, as shown in Scheme 1. All reactions were conducted under N2 to protect the thiol groups from oxidation. 3-Mercaptopropanohydrazide (M-Hy) was synthesized via the amidation of hydrazine with methyl 3-mercaptopropionate. As reported previously [23], 2.0 mL of methyl 3-mercaptopropionate (99%) was dissolved into 15 mL methanol. Then 15 mL hydrazine
Scheme 1. Schematic illustration of the keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) with acid-labile and reduction-cleavable linkages in series.
Please cite this article in press as: H. Zhang, et al., Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.05.041
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hydrate aqueous solution (85%) was added, and the mixture was heated with stirring at 60 C for 4 h. After cooling to room temperature, methanol was removed by rotary evaporation and the unreacted ester was removed by extraction with ether. The solution was stored at 20 C to obtain the crystal of the product. The product was further purified to remove hydrazine by repeatedly dissolving in water followed by lyophilization. M-Hy was obtained with a yield of 47.1%. Its chemical structure was confirmed by 1H NMR (Fig. 1): solvent: CDC13; chemical shifts (d, in ppm): -SH, 1H, d = 1.25; -NH, 1H, d = 6.76; -NH2, 2H, d = 3.94; -S-CH2-, 2H, d = 2.84; C-CH2-C, 2H, d = 2.47. Then M-Hy-D was synthesized by conjugating DOX with M-Hy, according to the reported method [24]. 57.4 mg (0.1018 mmol) of DOXHCl was dissolved into 20 mL of methanol. After 0.60 mL of triethylamine (TEA, 99%) was added and the solution was stirred for 20 min, 10 mL of methanol solution containing 24.4 mg (0.2036 mmol) of M-Hy was added drop by drop. Then 30 mL of trifluoroacetic acid (TFA, 97%) was added and the mixture was stirred at room temperature for 48 h. The product was collected by precipitation in pH 7.4 phosphate-buffered saline (PBS), and dried. M-Hy-D was obtained with a yield of 49.7%. Its chemical structure was confirmed by 1H NMR (Fig. 1): solvent: DMSO-d6; chemical shifts (d, in ppm): a: CH3 in DOX, 3H, d = 1.15; b: OCH3 in DOX, 3H, d = 4.01; c,d,e: phenyl ring protons in DOX, 3H, d = 7.64, 7.82, and 7.90; f: -NH in M-Hy-D, 1H, d = 6.69, and g: -SH in M-Hy-D, 1H, d = 1.51.
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Preparation of PK-SS-Hy-D NPs PK graft copolymer was synthesized via the amidation of the carboxyl groups in keratin with mPEG-NH2, with a PEGylation degree of 58%, as reported previously [21]. The following oxidation coupling reaction was also conducted under N2 to protect the thiol groups from oxidation. 40 mg of PK was dissolved into 20 mL of pH 7.4 PBS. 20 mg of M-Hy-D was dissolved into 16 mL of DMF. The two solutions were combined with ultrasonication, following with addition of 2 mL of DMSO and further ultrasonication for 30 min. Then the mixture was heated at 45 C with stirring for 24 h. After the reaction, the suspension was dialyzed with a mixture of pH 7.4 PBS and DMF with volume ratio of 5:4, until a colorless dialysate. DMF in the suspension was removed by dialysis thoroughly with massive water. Finally, the PK-SS-Hy-D NPs were collected by lyophilization. To explore the effect of concentration on the final prodrug nanoparticles, the above oxidation coupling reaction between PK and M-Hy-D was also conducted with a half concentration with the similar procedure. Drug content and composition analysis of PK-SS-Hy-D NPs The drug content in the PK-SS-Hy-D NPs was determined after degradation. 1.0 mg of PK-SS-Hy-D NPs were dispersed into 5.0 mL of 0.20 M HCl + 50 mM GSH, followed with ultrasonication for 48 h. The obtained supernatant after centrifugation (12,000 rpm for
Fig. 1. 1H NMR spectra of DOX, M-Hy-D in DMSO-d6 and M-Hy in CDCl3.
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30 min) was used for the DOX concentration determination with UV–vis spectrometer. To identify the prodrug composition, 1.0 mg of PK-SS-Hy-D NPs were dispersed into 5.0 mL of DMSO with ultrasonication. Then the dispersion was placed into a dialysis bag (cut-off molecular weight of 3.5 kDa) and dialyzed with 20 mL of DMSO for 48 h, replacing the dialysate every 8 h. Finally, all dialysates were combined for the DOX concentration determination with UV–vis spectrometer.
The flow cytometry was used to monitor the HepG2 cell apoptosis with a flow cytometer (BD FACSCalibur), by measuring the cell associated fluorescence double stained with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI), after incubation with 10 mg/mL of PK, 5 mg/mL DOX of free DOX, 5 mg/mL DOX equiv/mL of the PK-SS-Hy-D NPs at 37 C for 24 h, respectively.
In vitro controlled release profiles
1 H NMR spectra were recorded with a Bruker 400 MHz NMR spectrometer at room temperature. Dynamic light scattering (DLS) technique was used to analyze the hydrodynamic diameter and distribution of the prodrug nanoparticles, with a Light Scattering System BI-200SM device (Brookhaven Instruments), using a 135 mW intense laser excitation at 532 nm at a detection angle of 90 using the aqueous dispersions of the samples directly at 25 C. The morphology of the prodrug nanoparticles was characterized with a JEM-1200 EX/S transmission electron microscope (TEM), sampling with their aqueous dispersion.
10 mL of different releasing media containing 10 mg of PK-SSHy-D NPs was loaded into a dialysis bag (cut-off molecular weight of 14 kDa). And the dialysis bag was placed into 110 mL of the corresponding releasing media at 37 C with constant stirring at a rate of 120 rpm. At certain time intervals, 5 mL of the dialysate was taken out and 5 mL of the corresponding fresh releasing medium was refilled to maintain the volume constant. The amount of the released DOX was measured by PerkinElmer Lambda UV–vis spectrometer at 480 nm with the calibration curves obtained with the corresponding releasing media.
Analysis and characterization
Results and discussion In vitro cellular experiments Synthesis and characterization of PK-SS-Hy-D NPs The cytotoxicity of the PK-SS-Hy-D NPs was evaluated via MTT assay. The HepG2 human liver cancer cells and the MCF7 human breast cancer cells were cultivated on 96-well plates (1.0 105 cells per well) in 100 mL DMEM containing 10% FBS, and incubated in atmospheric humidity (5% CO2, 95% air, 37 C) for 24 h. Then, the PK graft copolymer or the PK-SS-Hy-D NPs as well as the free DOX were added at different concentrations. After further cultivation for 24 h, 20.0 mL of MTT (5.0 mg/mL) was added into each well, followed with further incubation for 4 h and washing with PBS for three times. The absorbance of the solution was evaluated at 490 nm on a microplate reader. The cellular uptake of the PK-SS-Hy-D NPs was exhibited on a confocal fluorescence microscope (DMI 4000B, LEICA, Germany) using HepG2 cells after 24 h of incubation. The cell nuclei were stained with DAPI. The location of cellular fluorescence was validated with excitation wavelength of 480 nm (DOX) and 405 nm (DAPI), respectively.
The chicken feather keratin was extracted from chicken feather, and then PEGylated with mPEG-NH2 to partially eliminate its carboxyl groups. The PEGylated keratin (PK) was obtained with a PEGylation degree (mass ratio between the grafted PEG brushes and keratin backbone) of 58% and pI >10, with the mass feeding ratio of keratin and mPEG-NH2 of 1:2 [21]. Then the keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) were fabricated via the oxidation coupling reaction between the thiol groups in the PK graft copolymer and the DOX derivative (MHy-D) (Scheme 1). In the oxidation reactions, the one between the PK graft copolymer and M-Hy-D was desired, but the others between PK graft copolymers and between M-Hy-D might be occurred simultaneously as side reactions (Scheme 2). So the feeding ratio of the PK graft copolymer and M-Hy-D should be the determining factor to achieve well-defined PK-SS-Hy-D prodrug nanoparticles.
Scheme 2. The oxidation coupling reactions.
Please cite this article in press as: H. Zhang, et al., Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.05.041
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Considering the thiol content of about 1.14 mmol/g in feather keratin (about 1140 105 mmol/g of half-cystine available in feather keratin [25]), the available content of thiol groups in the PK graft copolymer was about 0.72 mmol/g, calculated from the PEGylation degree of 58%. So the feeding mass ratio of the PK graft copolymer and M-Hy-D was selected as 2:1, meaning a molar ratio of thiol groups in PK to M-Hy-D of 1:1.13, to facilitate the oxidation coupling reaction between PK and M-Hy-D, and restrain the side reactions between themselves of the two reactants (Scheme 2). In the oxidation coupling reaction between the thiol groups in PK and M-Hy-D, the resultant PK-SS-Hy-D polymer prodrug could self-assemble into micelles with the drug-conjugated keratin chains as core and the PEG brushes as shell, due to the conjugation of the hydrophobic drug derivative onto the hydrophilic PK graft copolymer. While the inter-molecular oxidation coupling reaction of the free thiol groups in keratin occurred unavoidably, leading to a covalent crosslinkage in the prodrug nanoparticles. The solubility of the two reactants and the desired product PK-SS-Hy-D prodrug would affect the self-assembly of the polymer prodrug, as well as the particle size and morphology of the final prodrug nanoparticles sebsuquently. After complete removal the unreacted M-Hy-D, the particle size and morphology of the final PK-SS-Hy-D NPs fabricated with different PK concentrations with a same mass ratio between PK graft copolymer and small molecular prodrug M-Hy-D of 2:1 are shown in Fig. 2. Both products did not show the typical micellar
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morphology, revealing the covalent crosslinking in the prodrug nanoparticles. At an initial PK concentration of 2.0 mg/mL, there were two regions in the hydrodynamic diameter distribution: smaller particles at 54–78 nm and bigger particles at 229–470 nm. The irregular shaped particles could be seen in the TEM image, with particle size <170 nm and a broad particle size distribution. While with a lower PK concentration of 1.0 mg/mL, only one relatively narrower hydrodynamic diameter distribution in 257– 452 nm was obtained from the DLS analysis. And the prodrug nanoparticles showed well-defined spherical shape in the size range of 175–250 nm. Besides the main products with the similar mean hydrodynamic diameter (328 nm and 341 nm) of the PK-SSHy-D NPs prepared with the initial PK concentration of 2.0 mg/mL and 1.0 mg/mL respectively, the smaller particles at 54–78 nm presented in the former one should be resulted from the crosslinked PK nanoparticles with very low DOX content. The PK-SS-Hy-D NPs prepared with different initial PK concentrations could be completely dissolved into an 0.20 M HCl aqueous solution containing 50 mM GSH with ultrasonication at 35 C in 72 h, while they could be partially dissolved into 0.20 M HCl aqueous solution without GSH. The results revealed the reduction-cleavable disulfide crosslinkage in the prodrug nanoparticles, via the inter-molecular oxidation coupling reaction of the free thiol groups in keratin, as shown in Scheme 2. The clear and transparent solution of the PK-SS-Hy-D NPs in the 0.20 M HCl + 50 mM GSH mixture was used to measure the drug content on
Fig. 2. Typical hydrodynamic diameter distribution and morphology of the PK-SS-Hy-D NPs fabricated with different concentrations of the PK graft copolymer.
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PerkinElmer Lambda UV–vis spectrometry at 480 nm with the standard curve in the corresponding medium (y = 16.586x + 0.108 with R2 = 0.9875), as 27.8% and 29.4% for the PK-SS-Hy-D NPs prepared with the initial PK concentration of 2.0 mg/mL and 1.0 mg/mL, respectively. The drug contents were slightly higher than the theoretical value of 26.8% assuming that all thiol groups in the PK graft copolymer had been completely conjugated with DOX, demonstrating that some dimer of DOX (D-Hy-SS-Hy-D) formed by the inter-molecular oxidation coupling reaction of thiol groups in M-Hy-D molecules had been encapsulated into the drug-protein conjugate nanopartles. The PK-SS-Hy-D NPs prepared with the higher initial PK concentration (2.0 mg/mL) possessed a lower drug content, in comparison with the one prepared with the lower initial PK concentration (1.0 mg/mL). It should be due to the formation of the smaller particles at 54–78 nm in the former one, the disulfidecrosslinked PK nanoparticles with very low DOX content. The thiol groups in the PK graft copolymer tend to react with each other under a higher concentration, forming the crosslinked PK nanoparticles. Due to the crosslinked structure, the small molecular prodrug M-Hy-D could hardly diffuse into the nanoparticles. While under the lower concentration, the thiol groups in the PK graft copolymer tend to react with the thiol group in M-Hy-D, forming the desired PK-SS-Hy-D prodrug. With the drug conjugation degree increased, the drug conjugated keratin became hydrophobic, and the PK-SS-Hy-D polymer prodrug self-assembled into micelles with the drug-conjugated keratin chains as core and the PEG brushes as shell. Because the unavoidable inter-molecular oxidation coupling reaction of thiol groups in keratin occurred along with the drug conjugation, both the micellization and crosslinking occurred simultaneously, so the products were nanoparticles, but not micelles or core-crosslinked micelles which were crosslinked after micellization. To further reveal the formation of the pH/reduction dualresponsive small prodrug D-Hy-SS-Hy-D, the PK-SS-Hy-D NPs were dialyzed against DMSO (cut-off molecular weight of 3.5 kDa) for 48 h. The DOX concentration in the dialysate was determined with UV–vis spectrometer. It was found that the small prodrug DHy-SS-Hy-D had been produced by the side oxidation coupling reaction of the M-Hy-D for both PK-SS-Hy-D NPs prepared with higher or lower concentration, with a content of 22.4% and 17.9% respectively. The drug content in the PK-SS-Hy-D polymer prodrug, either crosslinked or not, could be calculated to be 11.5% and 17.4% after deducting the small prodrug D-Hy-SS-Hy-D. The results indicated that lower initial concentration was benificial to the conjugation of M-Hy-D onto the PK graft copolymer. Based on the above discussion, the PK-SS-Hy-D NPs prepared with the lower initial PK concentration were selected for the further investigation, due to their higher DOX-conjugation, as well as narrower diameter distribution and higher drug content. On the other hand, the presence of the small prodrug D-Hy-SS-Hy-D enhanced the drug content significantly, from 17.4% to 29.4%, so the PK-SS-Hy-D NPs were used directly for the further investigation without separation of the small prodrug D-Hy-SS-Hy-D. In vitro drug release profiles The pH/reduction dual-responsive triggered drug release performance was then evaluated at 37 C in different releasing media with different acidities and reductant levels: pH 7.4 PBS, pH 7.4 PBS + 10 mM GSH mimicking the normal physiological medium, pH 7.4 + 10 mM GSH, pH 5.0 acetate buffered solution (ABS), pH 5.0 ABS + 10 mM GSH, and pH 5.0 ABS + 10 mM GSH mimicking the tumor intracellular microenvironment, respectively (Fig. 3). No obvious burst was found for the drug release profiles in the six
Fig. 3. Cumulative drug release profiles from the PK-SS-Hy-D NPs in different releasing media.
releasing media. At pH 7.4, a very low drug release of 3.3% was found in 107 h. While at the simulated normal physiological medium, pH 7.4 PBS + 10 mM GSH, the cumulative DOX release ratio increased slowly to 5.5% within 107 h. The higher cumulative DOX release ratio than that without GSH showed the reduction-triggered release. With such low reductant level, the disulfide-crosslinkage could not be decrosslinked and the prodrug nanoparticles remained the crosslinked structure, and only the reductioncleavable disulfide conjugation on the surface of the prodrug nanoparticles was partially cleaved off to release the DOX derivative (M-Hy-D), which water-solubility is poorer than DOX. It has been reported that the premature drug leakage of the free DOX reached >70% in the simulated physiological medium within 7 h [21]. In the present work, both the two factors, the stable crosslinked structure of the proposed prodrug nanoparticles in such medium and the poor water-soluble M-Hy-D possibly released from the prodrug nanoparticles, resulted in a very low premature drug leakage in the simulated normal physiological medium, which is expected to efficiently reduce the toxic side effect of the chemotherapeutic drugs on the normal cells and tissues. In the weak basic medium with a high GSH level (pH 7.4 + 10 mM GSH), the drug could be released much faster than in the pH 7.4 PBS without GSH or low GSH level (10 mM). The results showed that both the reduction-cleavable disulfide-crosslinkage and the disulfide conjugation could be cleaved off with a high reductant level, as a result, the propdrug nanoparticles were decrosslinked and the DOX derivative (M-Hy-D) were released with a cumulative release ratio of 18.57% within 107 h. As in the pH 5.0 ABS, the acid-cleavable hydrazone bond between the protein copolymer and drug was cut off to release DOX in a sustained release mode, showing an endosomal pHtriggered release characteristics [26]. After the proposed PK-SSHy-D NPs are uptaken by tumor cells and trapped in endosome. The weakly acidic environment (pH 5.0–5.5) would trigger the cleavage of the hydrazone conjugate to release DOX in endosome. However, the cumulative DOX release ratio was only 21.7% in 107 h, because the disulfide-crosslinkage in the prodrug nanoparticles was maintained in such releasing medium. In the acidic medium with a low reductant level (pH 5.0 + 10 mM GSH), the cumulative drug release ratio increased to 33.69% in 107 h. With such low reductant level, only the disulfide crosslinkage and conjugation on
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the surface of the prodrug nanoparticles could be cleaved off, besides the release of M-Hy-D due to the cleavage of the drugprotein conjugation, the DOX release was also accelerated because of the surface decrosslinking of the prodrug nanoparticles. As for the simulated tumor intracellular microenvironment, pH 5.0 ABS + 10 mM GSH, the cumulative DOX release ratio increased up to 42.8% in 107 h, also in a sustained release mode. The much faster drug release was resulted from the decrosslinking of the prodrug nanoparticles with the high reductant level (10 mM GSH), which induced the disintegration of the prodrug nanoparticles. As a result, the acid-cleavable hydrazone conjugation is easily to be attacked by H+ and the released DOX molecules are facilitated to diffuse out of the DDSs. It is expected that the prodrug nanoparticles could disintegrate into soluble polymer in the reductive cytosol, after endosomal escape [27]. The hydrodynamic diameter distribution of the prodrug nanoparticles were traced with DLS technique, in order to explore their disintegration behavior in the simulated tumor intracellular microenvironment. The change in the hydrodynamic diameter distribution was affected with the two main factors: drug releasing and decrosslinking [21]. With the pH/reduction dual-responsive drug releasing upon the acid and reduction stimuli, their hydrodynamic diameter should decline. However, the hydrophilicity of the prodrug nanoparticles enhanced after the drug release, so the swelling degree increased, leading a rise in hydrodynamic diameter. As for the reduction-responsive decrosslinking, it would cause higher swelling, as well as disintegration. As shown in Fig. 4, the hydrodynamic diameter distribution became broarder in a range of 258–676 nm due to higher swelling, with a few small species of 31–46 nm in 24 h, indicating the disintegration of the prodrug nanoparticles. The higher swelling favored the diffusion of H+ and GSH into the nanoparticles, accelerating the drug release. So the hydrodynamic diameter distribution shifted to the smaller direction, 18–26 nm and 196– 436 nm after 48 h. Prolonging the releasing time to 72 h, a smaller fragment could be seen with diameter of 0.9–1.8 nm,
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demonstrating the water-soluble PK graft copolymer. Further increasing to 96 h, the proportion of the smaller fragment increased obviously, indicating the disintegration of the prodrug nanoparticles. Such post-endocytic disintegration of the prodrug nanoparticles is expected to improve nuclear translocation and efficacy of the chemotherapeutics. To further undertand the releasing mechanism from the proposed prodrug nanoparticles, the drug release profiles were fitted with the Higuchi and Korsmeyer–Peppas models. The fitted plots are shown in Fig. S1 and the main parameters are summarized in Table 1. The higher correlation coefficent (R2) was obtained from the Higuchi model than from the KorsmeyerPeppas model for all six releasing media, suggesting that the DOX release mechanism from the proposed prodrug nanoparticles was the extraction of the drug by simple diffusion or leaching of the drug by the releasing media [28]. As for the Korsmeyer–Peppas model, although with lower fitted coefficents, all the diffusion exponents (n) were obtained in the range of 0.45 < n < 0.89, indicating a non-Fickian or anomalous diffusion [29], due to the swelling effect of the prodrug nanoparticles above-discussed, mainly caused by the main factors: drug releasing and decrosslinking.
Table 1 Fitted data with the Higuchi and Korsmeyer–Peppas models. Releasing media
Higuchi 2
pH pH pH pH pH pH
7.4 7.4 + 10 mM GSH 7.4 + 10 mM GSH 5.0 5.0 + 10 mM GSH 5.0 + 10 mM GSH
Korsmeyer–Peppas
R
k
R2
n
0.9465 0.9389 0.9736 0.9950 0.9844 0.9947
0.0447 0.0640 0.2611 0.2909 0.4965 0.6079
0.9171 0.9295 0.9573 0.9672 0.9836 0.9753
0.5800 0.4556 0.8731 0.6264 0.7189 0.7854
Fig. 4. Typical hydrodynamic diameter distribution of the PK-SS-Hy-D NPs during the DOX release in the simulated tumor intracellular microenvironment.
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Fig. 5. Viability of HepG2 cells after incubated with PK, PK-SS-Hy-D and free DOX at various concentrations for 24 h (a), with PK-SS-Hy-D NPs at various concentrations for 24 h and 48 h (b), and MCF7 cells after incubated with PK, PK-SS-Hy-D and free DOX at various concentrations for 24 h (c).
Fig. 6. Representative CLSM images recorded for HepG2 cells incubated with the PK-SS-Hy-D NPs for 24 h in comparison with free DOX, respectively (scale bar = 50 mm).
In vitro cellular experiments The cellular toxicity of the PK graft copolymer and the PK-SSHy-D prodrug nanoparticles were in vitro evaluated with different concentrations. After co-incubation of the HepG2 human liver cancer cells for 24 h (Fig. 5a), the cell viability decreased with increasing the concentration of the blank copolymer and the prodrug nanoparticles, as well as free DOX. However, the cell viability of >92% was achieved for the blank copolymer, even with a high concentration of 20 mg/mL, demonstrating the excellent
cytocompatibility of the PEGylated keratin protein. The cell viability decreased to 87.3%, 79.6%, 72.9% and 66.1% with increasing the concentration of PK-SS-Hy-D to 2.5, 5.0, 10.0, and 20.0 mg/mL respecively, revealing the obvious cytotoxicity of the proposed prodrug nanoparticles. Compared with the free DOX, the cytotoxicity of the prodrug nanoparticles seemed lower, because of the higher viability under the same concentration. It should be due to the drug content and the sustained release performance of the prodrug nanoparticles. Taking account of the drug content of 29.4% and the cumulative
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excellent cytocompatibility of the PK graft copolymer. However, the rate of apoptotic and necrotic cells induced by the PK-SS-Hy-D NPs was 99.79%, similar to that by DOX of 99.95%, consistent with the cell viability data. Conclusions
Fig. 7. Apoptosis in HepG2 cells detected by flow cytometry with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining. Lower left quadrant: normal viable cells; lower right quadrant: early stage apoptotic cells; upper right quadrant: late stage apoptotic cells; upper left quadrant: necrotic cells.
release ratio of 18.1% at the simulated tumor intracellular microenvironment, the actual released DOX concentration was only 1 mg/mL after 24 h of incubation at a concentration of 20.0 mg/ mL. The cell viability at such case was 66.1%, near to the viability of 64.2% with free DOX at 2.5 mg/mL. The results showed that the proposed prodrug nanoparticles possessed an enhanced antitumor efficacy than the free DOX [30]. Further prolonging the incubation time of HepG2 cells with different concentrations of the PK-SS-Hy-D nanoparticles to 48 h, the viabilities at each concentration were obviously lower than those data after 24 h (Fig. 5b). The results demonstrated a stronger cellular toxicity of the prodrug nanoparticles with longer incubation time, due to the long-term and slow sustained drug release. As for the MCF7 human breast cancer cells after co-incubation for 24 h (Fig. 5c), high cell viability of >90% was achieved even with 20.0 mg/mL of the bare PK graft copolymer. However, the cell viability decreased to 68.40%, 60.52%, 53.17% and 41.14% with increasing the concentration of PK-SS-Hy-D to 2.5, 5.0, 10.0, and 20.0 mg/mL, respectively. All the data were similar or slightly lower than the data with the same free DOX concentration. The results were another direct evidence for the enhanced anti-tumor efficacy of the proposed drug-protein conjugate nanoparticles. Then the cellular uptake of the PK-SS-Hy-D NPs was also explored in HepG2 cells with the confocal fluorescence technique after 24 h of incubation. The blue fluorescence showed the position of the cell nuclei stained with DAPI (Fig. 6). Different from the very weak red fluorescence in the blank sample, the HepG2 cells after incubation for 24 h with both free DOX and PK-SS-Hy-D NPs showed strong red fluorescence of DOX. The red fluorescence of DOX completely overlapped with the blue fluorescence of the cell nuclei stained with DAPI after 24 h of incubation with the PK-SSHy-D NPs, similar as the free DOX, demonstrating the prodrug nanoparticles had been uptaken by the cells and then the released DOX was mainly accumulated in the cell nuclei [31]. Therefore, an enhanced anti-tumor efficacy was achieved within 24 h, although a lower cumulative release ratio of 18.1% within 24 h. Finally, cell apoptosis was measured with the flow cytometry technique to evaluate the in vitro antitumor effects of the proposed drug-protein conjugate nanoparticles. As shown in Fig. 7, the quantitative analysis results of the apoptotic HepG2 cells incubation with the bare PK graft copolymer were similar as the control sample with the survival of cells >93%, demonstrating the
In summary, chicken feather keratin-based prodrug nanoparticles, PK-SS-Hy-D NPs, were designed for pH and reduction dual-responsive tumor intracellular triggered DOX delivery, via a facile oxidation coupling reaction based on the thiol groups in the keratin protein. During the conjugating DOX onto the PEGylated keratin backbone with both bioreducible disulfide linkage and acid-cleavable hydrazone bond in series, the disulfide crosslinkage was also formed simultaneously between the thiol groups in the drug-protein conjugate PK-SS-Hy-D. The in vitro controlled release profiles demonstrated the pH/reduction dual-responsive triggered release of DOX from the optimized drug-protein conjugate nanopartles with a drug content of 29.4%, with a cumulative release up to 43% within 107 h in a sustained release manner in the stimulated tumor intracellular microenvironment, while a low drug premature leakage of 5.5% occurred in the normal physiological medium. The CLSM analysis showed that the prodrug nanoparticles could be uptaken by HepG2 cells, and then intracellular delivery DOX into nuclei. And the MTT assays indicated an enhanced anti-tumor efficacy of the prodrug nanoparticle than the free DOX, and the cytotoxicity increased with prolonging the incubation time, due to the long-term and slow sustained drug release. Such tumor intracellular DOX delivery makes it a potential candidate for future tumor chemotherapy with minimized toxic side effect on the normal tissues. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jiec.2019.05.041. References [1] R. Davoodi, L.Y. Lee, Q.X. Xu, V. Sunil, Y.J. Sun, S. Soh, C.-H. Wang, Adv. Drug Deliv. Rev. 132 (2018) 104. [2] T. Ojha, V. Pathak, Y. Shi, W.E. Hennink, C.T.W. Moonen, G. Storm, F. Kiessling, T. Lammers, Adv. Drug Deliv. Rev. 19 (2017) 44. [3] K. Ulbrich, K. Hola, V. Subr, A. Bakandritsos, J. Kucek, R. Zboril, Chem. Rev. 116 (2016) 5338. [4] L. Bildstein, D.P. Couvreur, Adv. Drug Deliv. Rev. 63 (2011) 3. [5] J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Jarvinen, J. Savolainen, Nat. Rev. Drug Discov. 7 (2008) 255. [6] P.F. Gou, W.W. Liu, W.W. Mao, J.B. Tang, Y.Q. Shen, M.H. Sui, J. Mater. Chem. B 1 (2013) 284. [7] H.J. Song, J. Zhang, W.W. Wang, P.S. Huang, Y.M. Zhang, J.F. Liu, C. Li, D.L. Kong, Colloid Surf. B-Biointerfaces 136 (2015) 365. [8] R. Mo, Z. Gu, Mater. Today 19 (2016) 274. [9] M.M. Mohamed, B.F. Sloane, Nat. Rev. Cancer 6 (2006) 764. [10] F. Seidi, R. Jenjob, T. Phakkeeree, D. Crespy, J. Control Release 284 (2018) 188. [11] K. DeFrates, T. Markiewicz, P. Gallo, A. Rack, A. Weyhmiller, B. Jarmusik, X. Hu, Int. J. Mol. Sci. 19 (2018) 1717. [12] A. Shavandi, T.H. Silva, A.A. Bekhit, A.E. Bekhit, Biomater. Sci. 5 (2017) 1699. [13] D. Fass, C. Thorpe, Chem. Rev. 118 (2018) 293. [14] N. Zhang, F.F. Zhao, Q.L. Zou, Y.X. Li, G.H. Ma, X.H. Yan, Small 12 (2016) 5936. [15] Q. Li, L. Zhu, R. Liu, D. Huang, X. Jin, N. Che, Z. Li, X. Qu, H. Kang, Y. Huang, J. Mater. Chem. 22 (2012) 19964. [16] Q. Li, S. Yang, L. Zhu, H. Kang, X. Qu, R. Liu, Y. Huang, Polym. Chem. 6 (2015) 2869. [17] M. Curcio, B. Blanco-Fernandez, L. Diaz-Gomez, A. Concheiro, C. AlvarezLorenzo, Bioconjugate Chem. 26 (2015) 1900. [18] X.L. Zhi, Y.F. Wang, P.F. Li, J. Yuan, J. Shen, RSC Adv. 5 (2015) 82334. [19] Y. Li, X. Zhi, J. Lin, X. You, J. Yuan, Mater. Sci. Eng. C 73 (2017) 189. [20] A. Aluigia, M. Ballestria, A. Guerrinia, G. Sotgiua, C. Ferronia, F. Corticellib, M.B. Gariboldic, E. Montic, G. Varchia, Mater. Sci. Eng. C 90 (2018) 476. [21] H.F. Zhang, P. Liu, Int. J. Bio. Macromol. 123 (2019) 1150. [22] J. Hwang, D. Lee, Y. Seo, J. Son, Y. Jo, K. Lee, C. Park, J. Choi, J. Ind. Eng. Chem. 66 (2018) 20. [23] J.A. Maassen, T.P.G.M. Thielen, W. Moller, Eur. J. Biochem. 134 (1983) 327.
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Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery Huifang Zhang, Mingliang Pei, Peng Liu* State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 February 2019 Received in revised form 15 May 2019 Accepted 29 May 2019 Available online xxx
PK-SS-Hy-D drug-protein prodrug was obtained by conjugating doxorubicin (DOX) onto the PEGylated keratin (PK) with both bioreducible disulfide linkage and acid-cleavable hydrazone bond in a series connection mode. The controlled release profiles demonstrated the pH and reduction dual-responsive triggered release of DOX from the proposed drug-protein conjugate nanoparticles, with a low premature drug leakage of 5.5% in the simulated physiological medium. The in vitro experiments indicated that the proposed prodrug nanoparticles could delivery DOX into the cell nuclei, with an enhanced anti-tumor efficacy. It is expected as a potential candidate for future tumor chemotherapy with minimized toxic side effect. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Prodrug nanoparticles Drug-Protein conjugate Chicken feather keratin pH and reduction dual-responsive Series connection mode
Introduction In the last decades, polymer-based drug delivery systems (DDSs) have been intensely investigated for tumor chemotherapy to minimize the toxic side effects on normal tissues and multidrug resistance of the conventional chemotherapeutic drugs [1]. The polymer-based DDSs could be easily tailored into nanomedicines, as nanoparticles, nanogels or micelles, to enter and accumulate inside tumor tissues through passive targeting via the enhanced permeability and retention (EPR) effect [2], in which the conventional small chemotherapeutic drugs are loaded via noncovalent interactions or covalent conjugation as prodrugs [3]. The stimuli-responsive characteristics of the polymer carriers could also endow the stimuli-triggered drug release in the acidic tumor microenvironment with high reductant level, while the premature drug leakage could be minimized in the normal physiological medium. Thus, the toxic side effects of the chemotherapeutic drugs on normal tissues could be efficiently avoided and the bioavailability of the chemotherapeutic drugs could also be improved, especially for the prodrug nanoformulations in which the chemotherapeutics are covalently conjugated onto the polymer carriers via the acid-labile or reduction-cleavable linkages [4–7].
* Corresponding author. E-mail address: [email protected] (P. Liu).
Besides the acidic microenvironment (blood and healthy tissues of pH 7.4, while the tumor extracellular at pH 6.0–7.2, and pH 5.0– 6.0 in the endosomes and pH 4.0–5.0 in the lysosomes) and the higher reductant level (approximately 2–20 mM GSH in blood or extracellular matrices, while 0.5–10 mM inside cells) [8], endowing the pH and reduction responsive controlled drug release, tumors also overexpress certain enzymes, such as lysosomal proteinases [8,9], which could provide an enzyme-responsive triggered drug release, by degradation of the protein and polypeptide-based DDSs. To develop a pH/reduction dual-responsive nanomedicine for tumor chemotherapy, a multi-functional polymer should be selected at first, with various functional groups. Owing to their excellent biocompatibility and biodegradability, biopolymers have attracted more and more interest in the last decades, such as polysaccharides [10] and proteins [11]. As one kind of natural protein in wool, hair or feather, keratin has attracted research interest as biopolymer vehicle for anti-tumor DDSs recently [12], because of its massive cysteine residues (7–20 mol% of all amino acids) resulting disulfide crosslinkage, as well as plentiful amino and carboxyl functional groups, in which the disulfide crosslinking could be cleaved off into active thiol groups with reductant, and re-formed with oxidant, endowing keratin excellent redox-responsive property [13]. Such different functional groups are expected to respond to various stimuli intracellular tumor cells for the anti-tumor drug delivery. Furthermore, as a protein, keratin might be degraded by the proteinases in tumor cells, realizing an enzyme-responsive triggered drug release
https://doi.org/10.1016/j.jiec.2019.05.041 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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[9,14]. Although the keratin proteins have been used as polymer vehicle in DDSs for various anti-tumor drugs [15–21], there is no report on the keratin-based drug-protein conjugate prodrug by now, to the best of our knowledge. In the present work, keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) were designed with acidlabile and reduction-cleavable linkages in series (Scheme 1), for the tumor intracellular pH/reduction dual-responsive triggered DOX release. Natural keratin contains more carboxyl groups than amino groups with a low isoelectric point (pI) of about 4.5 [17]. Such chemical structure would be against the acid-labile linkage in the proposed drug-protein conjugate prodrug. To solve the problem, the chicken feather keratin was PEGylated via the amidation of its carboxyl groups with amino-terminated methoxy poly(ethylene glycol) (mPEG-NH2) to raise its pI value. On the other hand, the PEGylation would improve the blood stability of the final prodrug nanoparticles [22]. Then, the chemotherapeutic drug, doxorubicin (DOX), was covalently conjugated onto the keratin from its thiol groups, with acid-labile and reduction-cleavable linkages in series. Such series connection mode would endow both the acid-triggered release of DOX by the hydrolysis of the acid-cleavable hydrazone linkage and the reduction-triggered release of its derivative (M-Hy-D) by the cleavage of the reduction-cleavable disulfide linkage. Furthermore, the covalently crosslinked prodrug nanoparticles could also be obtained by the inter-molecular oxidation coupling reaction of keratin between their free thiol groups, accompanying with the desired drug conjugation via the oxidation coupling reaction between the M-Hy-D and keratin. Thus, novel keratin-based drug-protein
conjugate prodrug nanoparticles were obtained with reductioncleavable disulfide crosslinkage and acid-labile and reductioncleavable drug conjugation. Experimental section Materials and reagents Chicken feather keratin was extracted from feather as described previously, with number average molecular weight (Mn) of 29 kDa [21]. Amino-terminated methoxy poly(ethylene glycol) (mPEGNH2, Mn of 2000, 98%) was purchased from Beijing Kaizheng Biotechnol Develop Co. Doxorubicin hydrochloride (DOXHCl, 99%) was purchased from Beijing Hvsf United Chemical Materials Co. Ltd.. All other chemicals in the experiments were of analytical grade and used without further purification. Double distilled water was used throughout. Synthesis of M-Hy-D The thiol-functionalized small molecular prodrug M-Hy-D was synthesized via the formation of acid-cleavable hydrazone bond between 3-mercaptopropanohydrazide (M-Hy) and DOX, as shown in Scheme 1. All reactions were conducted under N2 to protect the thiol groups from oxidation. 3-Mercaptopropanohydrazide (M-Hy) was synthesized via the amidation of hydrazine with methyl 3-mercaptopropionate. As reported previously [23], 2.0 mL of methyl 3-mercaptopropionate (99%) was dissolved into 15 mL methanol. Then 15 mL hydrazine
Scheme 1. Schematic illustration of the keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) with acid-labile and reduction-cleavable linkages in series.
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hydrate aqueous solution (85%) was added, and the mixture was heated with stirring at 60 C for 4 h. After cooling to room temperature, methanol was removed by rotary evaporation and the unreacted ester was removed by extraction with ether. The solution was stored at 20 C to obtain the crystal of the product. The product was further purified to remove hydrazine by repeatedly dissolving in water followed by lyophilization. M-Hy was obtained with a yield of 47.1%. Its chemical structure was confirmed by 1H NMR (Fig. 1): solvent: CDC13; chemical shifts (d, in ppm): -SH, 1H, d = 1.25; -NH, 1H, d = 6.76; -NH2, 2H, d = 3.94; -S-CH2-, 2H, d = 2.84; C-CH2-C, 2H, d = 2.47. Then M-Hy-D was synthesized by conjugating DOX with M-Hy, according to the reported method [24]. 57.4 mg (0.1018 mmol) of DOXHCl was dissolved into 20 mL of methanol. After 0.60 mL of triethylamine (TEA, 99%) was added and the solution was stirred for 20 min, 10 mL of methanol solution containing 24.4 mg (0.2036 mmol) of M-Hy was added drop by drop. Then 30 mL of trifluoroacetic acid (TFA, 97%) was added and the mixture was stirred at room temperature for 48 h. The product was collected by precipitation in pH 7.4 phosphate-buffered saline (PBS), and dried. M-Hy-D was obtained with a yield of 49.7%. Its chemical structure was confirmed by 1H NMR (Fig. 1): solvent: DMSO-d6; chemical shifts (d, in ppm): a: CH3 in DOX, 3H, d = 1.15; b: OCH3 in DOX, 3H, d = 4.01; c,d,e: phenyl ring protons in DOX, 3H, d = 7.64, 7.82, and 7.90; f: -NH in M-Hy-D, 1H, d = 6.69, and g: -SH in M-Hy-D, 1H, d = 1.51.
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Preparation of PK-SS-Hy-D NPs PK graft copolymer was synthesized via the amidation of the carboxyl groups in keratin with mPEG-NH2, with a PEGylation degree of 58%, as reported previously [21]. The following oxidation coupling reaction was also conducted under N2 to protect the thiol groups from oxidation. 40 mg of PK was dissolved into 20 mL of pH 7.4 PBS. 20 mg of M-Hy-D was dissolved into 16 mL of DMF. The two solutions were combined with ultrasonication, following with addition of 2 mL of DMSO and further ultrasonication for 30 min. Then the mixture was heated at 45 C with stirring for 24 h. After the reaction, the suspension was dialyzed with a mixture of pH 7.4 PBS and DMF with volume ratio of 5:4, until a colorless dialysate. DMF in the suspension was removed by dialysis thoroughly with massive water. Finally, the PK-SS-Hy-D NPs were collected by lyophilization. To explore the effect of concentration on the final prodrug nanoparticles, the above oxidation coupling reaction between PK and M-Hy-D was also conducted with a half concentration with the similar procedure. Drug content and composition analysis of PK-SS-Hy-D NPs The drug content in the PK-SS-Hy-D NPs was determined after degradation. 1.0 mg of PK-SS-Hy-D NPs were dispersed into 5.0 mL of 0.20 M HCl + 50 mM GSH, followed with ultrasonication for 48 h. The obtained supernatant after centrifugation (12,000 rpm for
Fig. 1. 1H NMR spectra of DOX, M-Hy-D in DMSO-d6 and M-Hy in CDCl3.
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30 min) was used for the DOX concentration determination with UV–vis spectrometer. To identify the prodrug composition, 1.0 mg of PK-SS-Hy-D NPs were dispersed into 5.0 mL of DMSO with ultrasonication. Then the dispersion was placed into a dialysis bag (cut-off molecular weight of 3.5 kDa) and dialyzed with 20 mL of DMSO for 48 h, replacing the dialysate every 8 h. Finally, all dialysates were combined for the DOX concentration determination with UV–vis spectrometer.
The flow cytometry was used to monitor the HepG2 cell apoptosis with a flow cytometer (BD FACSCalibur), by measuring the cell associated fluorescence double stained with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI), after incubation with 10 mg/mL of PK, 5 mg/mL DOX of free DOX, 5 mg/mL DOX equiv/mL of the PK-SS-Hy-D NPs at 37 C for 24 h, respectively.
In vitro controlled release profiles
1 H NMR spectra were recorded with a Bruker 400 MHz NMR spectrometer at room temperature. Dynamic light scattering (DLS) technique was used to analyze the hydrodynamic diameter and distribution of the prodrug nanoparticles, with a Light Scattering System BI-200SM device (Brookhaven Instruments), using a 135 mW intense laser excitation at 532 nm at a detection angle of 90 using the aqueous dispersions of the samples directly at 25 C. The morphology of the prodrug nanoparticles was characterized with a JEM-1200 EX/S transmission electron microscope (TEM), sampling with their aqueous dispersion.
10 mL of different releasing media containing 10 mg of PK-SSHy-D NPs was loaded into a dialysis bag (cut-off molecular weight of 14 kDa). And the dialysis bag was placed into 110 mL of the corresponding releasing media at 37 C with constant stirring at a rate of 120 rpm. At certain time intervals, 5 mL of the dialysate was taken out and 5 mL of the corresponding fresh releasing medium was refilled to maintain the volume constant. The amount of the released DOX was measured by PerkinElmer Lambda UV–vis spectrometer at 480 nm with the calibration curves obtained with the corresponding releasing media.
Analysis and characterization
Results and discussion In vitro cellular experiments Synthesis and characterization of PK-SS-Hy-D NPs The cytotoxicity of the PK-SS-Hy-D NPs was evaluated via MTT assay. The HepG2 human liver cancer cells and the MCF7 human breast cancer cells were cultivated on 96-well plates (1.0 105 cells per well) in 100 mL DMEM containing 10% FBS, and incubated in atmospheric humidity (5% CO2, 95% air, 37 C) for 24 h. Then, the PK graft copolymer or the PK-SS-Hy-D NPs as well as the free DOX were added at different concentrations. After further cultivation for 24 h, 20.0 mL of MTT (5.0 mg/mL) was added into each well, followed with further incubation for 4 h and washing with PBS for three times. The absorbance of the solution was evaluated at 490 nm on a microplate reader. The cellular uptake of the PK-SS-Hy-D NPs was exhibited on a confocal fluorescence microscope (DMI 4000B, LEICA, Germany) using HepG2 cells after 24 h of incubation. The cell nuclei were stained with DAPI. The location of cellular fluorescence was validated with excitation wavelength of 480 nm (DOX) and 405 nm (DAPI), respectively.
The chicken feather keratin was extracted from chicken feather, and then PEGylated with mPEG-NH2 to partially eliminate its carboxyl groups. The PEGylated keratin (PK) was obtained with a PEGylation degree (mass ratio between the grafted PEG brushes and keratin backbone) of 58% and pI >10, with the mass feeding ratio of keratin and mPEG-NH2 of 1:2 [21]. Then the keratin-based drug-protein conjugate prodrug nanoparticles (PK-SS-Hy-D NPs) were fabricated via the oxidation coupling reaction between the thiol groups in the PK graft copolymer and the DOX derivative (MHy-D) (Scheme 1). In the oxidation reactions, the one between the PK graft copolymer and M-Hy-D was desired, but the others between PK graft copolymers and between M-Hy-D might be occurred simultaneously as side reactions (Scheme 2). So the feeding ratio of the PK graft copolymer and M-Hy-D should be the determining factor to achieve well-defined PK-SS-Hy-D prodrug nanoparticles.
Scheme 2. The oxidation coupling reactions.
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Considering the thiol content of about 1.14 mmol/g in feather keratin (about 1140 105 mmol/g of half-cystine available in feather keratin [25]), the available content of thiol groups in the PK graft copolymer was about 0.72 mmol/g, calculated from the PEGylation degree of 58%. So the feeding mass ratio of the PK graft copolymer and M-Hy-D was selected as 2:1, meaning a molar ratio of thiol groups in PK to M-Hy-D of 1:1.13, to facilitate the oxidation coupling reaction between PK and M-Hy-D, and restrain the side reactions between themselves of the two reactants (Scheme 2). In the oxidation coupling reaction between the thiol groups in PK and M-Hy-D, the resultant PK-SS-Hy-D polymer prodrug could self-assemble into micelles with the drug-conjugated keratin chains as core and the PEG brushes as shell, due to the conjugation of the hydrophobic drug derivative onto the hydrophilic PK graft copolymer. While the inter-molecular oxidation coupling reaction of the free thiol groups in keratin occurred unavoidably, leading to a covalent crosslinkage in the prodrug nanoparticles. The solubility of the two reactants and the desired product PK-SS-Hy-D prodrug would affect the self-assembly of the polymer prodrug, as well as the particle size and morphology of the final prodrug nanoparticles sebsuquently. After complete removal the unreacted M-Hy-D, the particle size and morphology of the final PK-SS-Hy-D NPs fabricated with different PK concentrations with a same mass ratio between PK graft copolymer and small molecular prodrug M-Hy-D of 2:1 are shown in Fig. 2. Both products did not show the typical micellar
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morphology, revealing the covalent crosslinking in the prodrug nanoparticles. At an initial PK concentration of 2.0 mg/mL, there were two regions in the hydrodynamic diameter distribution: smaller particles at 54–78 nm and bigger particles at 229–470 nm. The irregular shaped particles could be seen in the TEM image, with particle size <170 nm and a broad particle size distribution. While with a lower PK concentration of 1.0 mg/mL, only one relatively narrower hydrodynamic diameter distribution in 257– 452 nm was obtained from the DLS analysis. And the prodrug nanoparticles showed well-defined spherical shape in the size range of 175–250 nm. Besides the main products with the similar mean hydrodynamic diameter (328 nm and 341 nm) of the PK-SSHy-D NPs prepared with the initial PK concentration of 2.0 mg/mL and 1.0 mg/mL respectively, the smaller particles at 54–78 nm presented in the former one should be resulted from the crosslinked PK nanoparticles with very low DOX content. The PK-SS-Hy-D NPs prepared with different initial PK concentrations could be completely dissolved into an 0.20 M HCl aqueous solution containing 50 mM GSH with ultrasonication at 35 C in 72 h, while they could be partially dissolved into 0.20 M HCl aqueous solution without GSH. The results revealed the reduction-cleavable disulfide crosslinkage in the prodrug nanoparticles, via the inter-molecular oxidation coupling reaction of the free thiol groups in keratin, as shown in Scheme 2. The clear and transparent solution of the PK-SS-Hy-D NPs in the 0.20 M HCl + 50 mM GSH mixture was used to measure the drug content on
Fig. 2. Typical hydrodynamic diameter distribution and morphology of the PK-SS-Hy-D NPs fabricated with different concentrations of the PK graft copolymer.
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PerkinElmer Lambda UV–vis spectrometry at 480 nm with the standard curve in the corresponding medium (y = 16.586x + 0.108 with R2 = 0.9875), as 27.8% and 29.4% for the PK-SS-Hy-D NPs prepared with the initial PK concentration of 2.0 mg/mL and 1.0 mg/mL, respectively. The drug contents were slightly higher than the theoretical value of 26.8% assuming that all thiol groups in the PK graft copolymer had been completely conjugated with DOX, demonstrating that some dimer of DOX (D-Hy-SS-Hy-D) formed by the inter-molecular oxidation coupling reaction of thiol groups in M-Hy-D molecules had been encapsulated into the drug-protein conjugate nanopartles. The PK-SS-Hy-D NPs prepared with the higher initial PK concentration (2.0 mg/mL) possessed a lower drug content, in comparison with the one prepared with the lower initial PK concentration (1.0 mg/mL). It should be due to the formation of the smaller particles at 54–78 nm in the former one, the disulfidecrosslinked PK nanoparticles with very low DOX content. The thiol groups in the PK graft copolymer tend to react with each other under a higher concentration, forming the crosslinked PK nanoparticles. Due to the crosslinked structure, the small molecular prodrug M-Hy-D could hardly diffuse into the nanoparticles. While under the lower concentration, the thiol groups in the PK graft copolymer tend to react with the thiol group in M-Hy-D, forming the desired PK-SS-Hy-D prodrug. With the drug conjugation degree increased, the drug conjugated keratin became hydrophobic, and the PK-SS-Hy-D polymer prodrug self-assembled into micelles with the drug-conjugated keratin chains as core and the PEG brushes as shell. Because the unavoidable inter-molecular oxidation coupling reaction of thiol groups in keratin occurred along with the drug conjugation, both the micellization and crosslinking occurred simultaneously, so the products were nanoparticles, but not micelles or core-crosslinked micelles which were crosslinked after micellization. To further reveal the formation of the pH/reduction dualresponsive small prodrug D-Hy-SS-Hy-D, the PK-SS-Hy-D NPs were dialyzed against DMSO (cut-off molecular weight of 3.5 kDa) for 48 h. The DOX concentration in the dialysate was determined with UV–vis spectrometer. It was found that the small prodrug DHy-SS-Hy-D had been produced by the side oxidation coupling reaction of the M-Hy-D for both PK-SS-Hy-D NPs prepared with higher or lower concentration, with a content of 22.4% and 17.9% respectively. The drug content in the PK-SS-Hy-D polymer prodrug, either crosslinked or not, could be calculated to be 11.5% and 17.4% after deducting the small prodrug D-Hy-SS-Hy-D. The results indicated that lower initial concentration was benificial to the conjugation of M-Hy-D onto the PK graft copolymer. Based on the above discussion, the PK-SS-Hy-D NPs prepared with the lower initial PK concentration were selected for the further investigation, due to their higher DOX-conjugation, as well as narrower diameter distribution and higher drug content. On the other hand, the presence of the small prodrug D-Hy-SS-Hy-D enhanced the drug content significantly, from 17.4% to 29.4%, so the PK-SS-Hy-D NPs were used directly for the further investigation without separation of the small prodrug D-Hy-SS-Hy-D. In vitro drug release profiles The pH/reduction dual-responsive triggered drug release performance was then evaluated at 37 C in different releasing media with different acidities and reductant levels: pH 7.4 PBS, pH 7.4 PBS + 10 mM GSH mimicking the normal physiological medium, pH 7.4 + 10 mM GSH, pH 5.0 acetate buffered solution (ABS), pH 5.0 ABS + 10 mM GSH, and pH 5.0 ABS + 10 mM GSH mimicking the tumor intracellular microenvironment, respectively (Fig. 3). No obvious burst was found for the drug release profiles in the six
Fig. 3. Cumulative drug release profiles from the PK-SS-Hy-D NPs in different releasing media.
releasing media. At pH 7.4, a very low drug release of 3.3% was found in 107 h. While at the simulated normal physiological medium, pH 7.4 PBS + 10 mM GSH, the cumulative DOX release ratio increased slowly to 5.5% within 107 h. The higher cumulative DOX release ratio than that without GSH showed the reduction-triggered release. With such low reductant level, the disulfide-crosslinkage could not be decrosslinked and the prodrug nanoparticles remained the crosslinked structure, and only the reductioncleavable disulfide conjugation on the surface of the prodrug nanoparticles was partially cleaved off to release the DOX derivative (M-Hy-D), which water-solubility is poorer than DOX. It has been reported that the premature drug leakage of the free DOX reached >70% in the simulated physiological medium within 7 h [21]. In the present work, both the two factors, the stable crosslinked structure of the proposed prodrug nanoparticles in such medium and the poor water-soluble M-Hy-D possibly released from the prodrug nanoparticles, resulted in a very low premature drug leakage in the simulated normal physiological medium, which is expected to efficiently reduce the toxic side effect of the chemotherapeutic drugs on the normal cells and tissues. In the weak basic medium with a high GSH level (pH 7.4 + 10 mM GSH), the drug could be released much faster than in the pH 7.4 PBS without GSH or low GSH level (10 mM). The results showed that both the reduction-cleavable disulfide-crosslinkage and the disulfide conjugation could be cleaved off with a high reductant level, as a result, the propdrug nanoparticles were decrosslinked and the DOX derivative (M-Hy-D) were released with a cumulative release ratio of 18.57% within 107 h. As in the pH 5.0 ABS, the acid-cleavable hydrazone bond between the protein copolymer and drug was cut off to release DOX in a sustained release mode, showing an endosomal pHtriggered release characteristics [26]. After the proposed PK-SSHy-D NPs are uptaken by tumor cells and trapped in endosome. The weakly acidic environment (pH 5.0–5.5) would trigger the cleavage of the hydrazone conjugate to release DOX in endosome. However, the cumulative DOX release ratio was only 21.7% in 107 h, because the disulfide-crosslinkage in the prodrug nanoparticles was maintained in such releasing medium. In the acidic medium with a low reductant level (pH 5.0 + 10 mM GSH), the cumulative drug release ratio increased to 33.69% in 107 h. With such low reductant level, only the disulfide crosslinkage and conjugation on
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the surface of the prodrug nanoparticles could be cleaved off, besides the release of M-Hy-D due to the cleavage of the drugprotein conjugation, the DOX release was also accelerated because of the surface decrosslinking of the prodrug nanoparticles. As for the simulated tumor intracellular microenvironment, pH 5.0 ABS + 10 mM GSH, the cumulative DOX release ratio increased up to 42.8% in 107 h, also in a sustained release mode. The much faster drug release was resulted from the decrosslinking of the prodrug nanoparticles with the high reductant level (10 mM GSH), which induced the disintegration of the prodrug nanoparticles. As a result, the acid-cleavable hydrazone conjugation is easily to be attacked by H+ and the released DOX molecules are facilitated to diffuse out of the DDSs. It is expected that the prodrug nanoparticles could disintegrate into soluble polymer in the reductive cytosol, after endosomal escape [27]. The hydrodynamic diameter distribution of the prodrug nanoparticles were traced with DLS technique, in order to explore their disintegration behavior in the simulated tumor intracellular microenvironment. The change in the hydrodynamic diameter distribution was affected with the two main factors: drug releasing and decrosslinking [21]. With the pH/reduction dual-responsive drug releasing upon the acid and reduction stimuli, their hydrodynamic diameter should decline. However, the hydrophilicity of the prodrug nanoparticles enhanced after the drug release, so the swelling degree increased, leading a rise in hydrodynamic diameter. As for the reduction-responsive decrosslinking, it would cause higher swelling, as well as disintegration. As shown in Fig. 4, the hydrodynamic diameter distribution became broarder in a range of 258–676 nm due to higher swelling, with a few small species of 31–46 nm in 24 h, indicating the disintegration of the prodrug nanoparticles. The higher swelling favored the diffusion of H+ and GSH into the nanoparticles, accelerating the drug release. So the hydrodynamic diameter distribution shifted to the smaller direction, 18–26 nm and 196– 436 nm after 48 h. Prolonging the releasing time to 72 h, a smaller fragment could be seen with diameter of 0.9–1.8 nm,
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demonstrating the water-soluble PK graft copolymer. Further increasing to 96 h, the proportion of the smaller fragment increased obviously, indicating the disintegration of the prodrug nanoparticles. Such post-endocytic disintegration of the prodrug nanoparticles is expected to improve nuclear translocation and efficacy of the chemotherapeutics. To further undertand the releasing mechanism from the proposed prodrug nanoparticles, the drug release profiles were fitted with the Higuchi and Korsmeyer–Peppas models. The fitted plots are shown in Fig. S1 and the main parameters are summarized in Table 1. The higher correlation coefficent (R2) was obtained from the Higuchi model than from the KorsmeyerPeppas model for all six releasing media, suggesting that the DOX release mechanism from the proposed prodrug nanoparticles was the extraction of the drug by simple diffusion or leaching of the drug by the releasing media [28]. As for the Korsmeyer–Peppas model, although with lower fitted coefficents, all the diffusion exponents (n) were obtained in the range of 0.45 < n < 0.89, indicating a non-Fickian or anomalous diffusion [29], due to the swelling effect of the prodrug nanoparticles above-discussed, mainly caused by the main factors: drug releasing and decrosslinking.
Table 1 Fitted data with the Higuchi and Korsmeyer–Peppas models. Releasing media
Higuchi 2
pH pH pH pH pH pH
7.4 7.4 + 10 mM GSH 7.4 + 10 mM GSH 5.0 5.0 + 10 mM GSH 5.0 + 10 mM GSH
Korsmeyer–Peppas
R
k
R2
n
0.9465 0.9389 0.9736 0.9950 0.9844 0.9947
0.0447 0.0640 0.2611 0.2909 0.4965 0.6079
0.9171 0.9295 0.9573 0.9672 0.9836 0.9753
0.5800 0.4556 0.8731 0.6264 0.7189 0.7854
Fig. 4. Typical hydrodynamic diameter distribution of the PK-SS-Hy-D NPs during the DOX release in the simulated tumor intracellular microenvironment.
Please cite this article in press as: H. Zhang, et al., Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.05.041
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Fig. 5. Viability of HepG2 cells after incubated with PK, PK-SS-Hy-D and free DOX at various concentrations for 24 h (a), with PK-SS-Hy-D NPs at various concentrations for 24 h and 48 h (b), and MCF7 cells after incubated with PK, PK-SS-Hy-D and free DOX at various concentrations for 24 h (c).
Fig. 6. Representative CLSM images recorded for HepG2 cells incubated with the PK-SS-Hy-D NPs for 24 h in comparison with free DOX, respectively (scale bar = 50 mm).
In vitro cellular experiments The cellular toxicity of the PK graft copolymer and the PK-SSHy-D prodrug nanoparticles were in vitro evaluated with different concentrations. After co-incubation of the HepG2 human liver cancer cells for 24 h (Fig. 5a), the cell viability decreased with increasing the concentration of the blank copolymer and the prodrug nanoparticles, as well as free DOX. However, the cell viability of >92% was achieved for the blank copolymer, even with a high concentration of 20 mg/mL, demonstrating the excellent
cytocompatibility of the PEGylated keratin protein. The cell viability decreased to 87.3%, 79.6%, 72.9% and 66.1% with increasing the concentration of PK-SS-Hy-D to 2.5, 5.0, 10.0, and 20.0 mg/mL respecively, revealing the obvious cytotoxicity of the proposed prodrug nanoparticles. Compared with the free DOX, the cytotoxicity of the prodrug nanoparticles seemed lower, because of the higher viability under the same concentration. It should be due to the drug content and the sustained release performance of the prodrug nanoparticles. Taking account of the drug content of 29.4% and the cumulative
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excellent cytocompatibility of the PK graft copolymer. However, the rate of apoptotic and necrotic cells induced by the PK-SS-Hy-D NPs was 99.79%, similar to that by DOX of 99.95%, consistent with the cell viability data. Conclusions
Fig. 7. Apoptosis in HepG2 cells detected by flow cytometry with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining. Lower left quadrant: normal viable cells; lower right quadrant: early stage apoptotic cells; upper right quadrant: late stage apoptotic cells; upper left quadrant: necrotic cells.
release ratio of 18.1% at the simulated tumor intracellular microenvironment, the actual released DOX concentration was only 1 mg/mL after 24 h of incubation at a concentration of 20.0 mg/ mL. The cell viability at such case was 66.1%, near to the viability of 64.2% with free DOX at 2.5 mg/mL. The results showed that the proposed prodrug nanoparticles possessed an enhanced antitumor efficacy than the free DOX [30]. Further prolonging the incubation time of HepG2 cells with different concentrations of the PK-SS-Hy-D nanoparticles to 48 h, the viabilities at each concentration were obviously lower than those data after 24 h (Fig. 5b). The results demonstrated a stronger cellular toxicity of the prodrug nanoparticles with longer incubation time, due to the long-term and slow sustained drug release. As for the MCF7 human breast cancer cells after co-incubation for 24 h (Fig. 5c), high cell viability of >90% was achieved even with 20.0 mg/mL of the bare PK graft copolymer. However, the cell viability decreased to 68.40%, 60.52%, 53.17% and 41.14% with increasing the concentration of PK-SS-Hy-D to 2.5, 5.0, 10.0, and 20.0 mg/mL, respectively. All the data were similar or slightly lower than the data with the same free DOX concentration. The results were another direct evidence for the enhanced anti-tumor efficacy of the proposed drug-protein conjugate nanoparticles. Then the cellular uptake of the PK-SS-Hy-D NPs was also explored in HepG2 cells with the confocal fluorescence technique after 24 h of incubation. The blue fluorescence showed the position of the cell nuclei stained with DAPI (Fig. 6). Different from the very weak red fluorescence in the blank sample, the HepG2 cells after incubation for 24 h with both free DOX and PK-SS-Hy-D NPs showed strong red fluorescence of DOX. The red fluorescence of DOX completely overlapped with the blue fluorescence of the cell nuclei stained with DAPI after 24 h of incubation with the PK-SSHy-D NPs, similar as the free DOX, demonstrating the prodrug nanoparticles had been uptaken by the cells and then the released DOX was mainly accumulated in the cell nuclei [31]. Therefore, an enhanced anti-tumor efficacy was achieved within 24 h, although a lower cumulative release ratio of 18.1% within 24 h. Finally, cell apoptosis was measured with the flow cytometry technique to evaluate the in vitro antitumor effects of the proposed drug-protein conjugate nanoparticles. As shown in Fig. 7, the quantitative analysis results of the apoptotic HepG2 cells incubation with the bare PK graft copolymer were similar as the control sample with the survival of cells >93%, demonstrating the
In summary, chicken feather keratin-based prodrug nanoparticles, PK-SS-Hy-D NPs, were designed for pH and reduction dual-responsive tumor intracellular triggered DOX delivery, via a facile oxidation coupling reaction based on the thiol groups in the keratin protein. During the conjugating DOX onto the PEGylated keratin backbone with both bioreducible disulfide linkage and acid-cleavable hydrazone bond in series, the disulfide crosslinkage was also formed simultaneously between the thiol groups in the drug-protein conjugate PK-SS-Hy-D. The in vitro controlled release profiles demonstrated the pH/reduction dual-responsive triggered release of DOX from the optimized drug-protein conjugate nanopartles with a drug content of 29.4%, with a cumulative release up to 43% within 107 h in a sustained release manner in the stimulated tumor intracellular microenvironment, while a low drug premature leakage of 5.5% occurred in the normal physiological medium. The CLSM analysis showed that the prodrug nanoparticles could be uptaken by HepG2 cells, and then intracellular delivery DOX into nuclei. And the MTT assays indicated an enhanced anti-tumor efficacy of the prodrug nanoparticle than the free DOX, and the cytotoxicity increased with prolonging the incubation time, due to the long-term and slow sustained drug release. Such tumor intracellular DOX delivery makes it a potential candidate for future tumor chemotherapy with minimized toxic side effect on the normal tissues. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jiec.2019.05.041. References [1] R. Davoodi, L.Y. Lee, Q.X. Xu, V. Sunil, Y.J. Sun, S. Soh, C.-H. Wang, Adv. Drug Deliv. Rev. 132 (2018) 104. [2] T. Ojha, V. Pathak, Y. Shi, W.E. Hennink, C.T.W. Moonen, G. Storm, F. Kiessling, T. Lammers, Adv. Drug Deliv. Rev. 19 (2017) 44. [3] K. Ulbrich, K. Hola, V. Subr, A. Bakandritsos, J. Kucek, R. Zboril, Chem. Rev. 116 (2016) 5338. [4] L. Bildstein, D.P. Couvreur, Adv. Drug Deliv. Rev. 63 (2011) 3. [5] J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Jarvinen, J. Savolainen, Nat. Rev. Drug Discov. 7 (2008) 255. [6] P.F. Gou, W.W. Liu, W.W. Mao, J.B. Tang, Y.Q. Shen, M.H. Sui, J. Mater. Chem. B 1 (2013) 284. [7] H.J. Song, J. Zhang, W.W. Wang, P.S. Huang, Y.M. Zhang, J.F. Liu, C. Li, D.L. Kong, Colloid Surf. B-Biointerfaces 136 (2015) 365. [8] R. Mo, Z. Gu, Mater. Today 19 (2016) 274. [9] M.M. Mohamed, B.F. Sloane, Nat. Rev. Cancer 6 (2006) 764. [10] F. Seidi, R. Jenjob, T. Phakkeeree, D. Crespy, J. Control Release 284 (2018) 188. [11] K. DeFrates, T. Markiewicz, P. Gallo, A. Rack, A. Weyhmiller, B. Jarmusik, X. Hu, Int. J. Mol. Sci. 19 (2018) 1717. [12] A. Shavandi, T.H. Silva, A.A. Bekhit, A.E. Bekhit, Biomater. Sci. 5 (2017) 1699. [13] D. Fass, C. Thorpe, Chem. Rev. 118 (2018) 293. [14] N. Zhang, F.F. Zhao, Q.L. Zou, Y.X. Li, G.H. Ma, X.H. Yan, Small 12 (2016) 5936. [15] Q. Li, L. Zhu, R. Liu, D. Huang, X. Jin, N. Che, Z. Li, X. Qu, H. Kang, Y. Huang, J. Mater. Chem. 22 (2012) 19964. [16] Q. Li, S. Yang, L. Zhu, H. Kang, X. Qu, R. Liu, Y. Huang, Polym. Chem. 6 (2015) 2869. [17] M. Curcio, B. Blanco-Fernandez, L. Diaz-Gomez, A. Concheiro, C. AlvarezLorenzo, Bioconjugate Chem. 26 (2015) 1900. [18] X.L. Zhi, Y.F. Wang, P.F. Li, J. Yuan, J. Shen, RSC Adv. 5 (2015) 82334. [19] Y. Li, X. Zhi, J. Lin, X. You, J. Yuan, Mater. Sci. Eng. C 73 (2017) 189. [20] A. Aluigia, M. Ballestria, A. Guerrinia, G. Sotgiua, C. Ferronia, F. Corticellib, M.B. Gariboldic, E. Montic, G. Varchia, Mater. Sci. Eng. C 90 (2018) 476. [21] H.F. Zhang, P. Liu, Int. J. Bio. Macromol. 123 (2019) 1150. [22] J. Hwang, D. Lee, Y. Seo, J. Son, Y. Jo, K. Lee, C. Park, J. Choi, J. Ind. Eng. Chem. 66 (2018) 20. [23] J.A. Maassen, T.P.G.M. Thielen, W. Moller, Eur. J. Biochem. 134 (1983) 327.
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Please cite this article in press as: H. Zhang, et al., Keratin-based drug-protein conjugate with acid-labile and reduction-cleavable linkages in series for tumor intracellular DOX delivery, J. Ind. Eng. Chem. (2019), https://doi.org/10.1016/j.jiec.2019.05.041