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Poly(ethylene glycol) shell-sheddable TAT-modified core cross-linked nano-micelles: TAT-enhanced cellular uptake and lysosomal pH-triggered doxorubicin release
Colloids and Surfaces B: Biointerfaces 188 (2020) 110772
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Poly(ethylene glycol) shell-sheddable TAT-modified core cross-linked nanomicelles: TAT-enhanced cellular uptake and lysosomal pH-triggered doxorubicin release
T
Yuliu Zhanga, Yi Xiaob, Yushu Huanga, Yang Hea, Yanyun Xua, Wei Lua, Jiahui Yua,* a
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, PR China b Department of Radiology and Nuclear Medicine, Changzheng Hospital, Naval Medical University, Shanghai 200003, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: TAT-modified Enhanced endocytosis Core cross-linked nanomicelles Lysosomal pH-triggered doxorubicin release
This study aimed to develop sheddable polyethylene glycol (PEG) shells with TAT-modified core cross-linked nanomicelles as drug-delivery carriers of doxorubicin (DOX) to establish a programmed response against the tumor microenvironment, enhanced endocytosis, and lysosomal pH-triggered DOX release. First, poly(L-succinimide) (PSI) underwent a ring-opening reaction with ethylenediamine to generate poly(N-(2-aminoethyl)-Laspartamide) (P(ae-Asp)). Next, the thiolytic cleavable PEG, 3,4-dihydroxyphenylacetic acid, and TAT were grafted onto P(ae-Asp) to synthesize the amphiphilic graft copolymer of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. In aqueous solution, the amphiphilic polymer self-assembled into nanomicelles, encapsulating DOX into the hydrophobic core of micelles. TAT was shielded by the PEG corona during circulation to avoid non-specific transmembrane interaction with normal cells, while the tumor redox environment-responsive shedding of PEG could expose TAT to promote internalization of tumor cells. In order to improve the stability of nanomicelles and achieve pH-triggered drug release, a core cross-linking strategy based on the coordination of catechol and Fe3+ was adopted. In vitro studies demonstrated that core cross-linked nanomicelles maintained the nanostructure in 100 times dilution in pH 7.4 phosphate-buffered saline (PBS). Moreover, DOX release from DOX-loaded core cross-linked nanomicelles (DOX-TAT-CCLMs) was favored at simulated lysosomal conditions over simulated plasma conditions, indicating that these nanomicelles demonstrate characteristics of pH-triggered DOX release. The TAT modification considerably enhanced the mean fluorescence intensity of the nanomicelles endocytosed by MCF-7/ADR cells by 8 times, compared with DOX·HCl after 8 h of incubation. Notably, the IC50 value of nanomicelles (11.61 ± 0.95 μg/mL) was nearly 4 times lower than that of DOX·HCl against MCF-7/ADR cells, implying that the nanomicelles could overcome drug resistance observed in MCF-7/ADR cells. Furthermore, the DOX-TAT-CCLMs reported superior tumor growth suppression in a 4T1 tumor-bearing mouse model. Thus, the redox- and pH- stimuli stepwise-responsive novel nanomicelles fabricated from the mPEG-SS-g-P(ae-Asp)-MCADA-TAT graft copolymer exhibited multifunctionality and displayed great potential for drug delivery.
1. Introduction Polymeric nanomicelles and liposomes, have been rapidly developed to enhance the therapeutic efficacy of anticancer drugs and reduce the systemic toxicity associated with cancer therapy [1–4]. Notably, polymeric nanomicelles play an indispensable role in drug delivery systems due to the superior modification and biomedical potential [5]. Functional ligands are often modified to generate more intuitive nanomicelles. For example, modification of polyethylene glycol (PEG) on nanomicelles can lengthen the systemic circulation time, enhancing the
⁎
cumulation of anticancer drugs in cancer tissues [6,7]. Furthermore, the modification of nanomicelles with targeting ligands possessing high binding affinity with overexpressed receptors on cancer cells can improve the internalization efficiency of nanomicelles [8,9]. Another way to improve the internalization efficiency is to modify the cell-penetrating peptides (CPP) on nanomicelles. CPP can efficiently carry nanomicelles or macromolecules into cells without destroying the integrity of the cell membranes [10,11]. The main advantage of CPP is that it can penetrate the cytomembrane at low micromolar concentrations in vivo and in vitro without using any other receptors [12]. TAT,
Corresponding author. E-mail address: [email protected] (J. Yu).
https://doi.org/10.1016/j.colsurfb.2020.110772 Received 28 October 2019; Received in revised form 2 January 2020; Accepted 3 January 2020 Available online 20 January 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved.
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2.1. Synthesis of mPEG-SS-g-P(ae-Asp)
one such CPP, was isolated from the HIV-1 protein by Green et al. [13] and has been considered an effective CPP to transport different molecules into various cell lines [14,15]. However, the non-selectivity of TAT against all kinds of cells limits its application in anticancer drug delivery. Notably, several attempts have been made to solve this problem. An effective method to avoid non-specific transmembrane interaction with normal cells involves the use of PEG as a shield before the nanodrugs reach the tumor tissue. Upon reaching tumor tissues, the PEG shell of nanodrugs is rescinded in response to the redox environment of tumor tissues (GSH 10 mM), thereby exposing TAT to mediate the endocytosis of nanodrugs with high efficiency [16–18]. Nevertheless, the extracellular shedding of PEG is always accompanied by some challenges, such as the disintegration of drug-loaded micelles and premature drug release. Additionally, the access of nanomicelles to tumor tissues is a prerequisite for TAT to exert its function [19]. As previously known, following in vivo administration, drugloaded micelles usually undergo excessive dilution in the systemic circulation. The disintegration of drug-loaded micelles during hemodilution invalidates the functional ligands installed in nanomicelles, resulting in premature drug leakage and serious side effects. Therefore, the structural integrity of nanomicelles is crucial to drug delivery efficiency and treatment efficacy. In order to address these problems, core cross-linking has been utilized as an effective strategy to maintain the integrity of polymer micelles [20]. Several core cross-linking methods [21–23], including the use of reduction- or pH-sensitive chemical bonds to form cross-linked structures, have been reported. Among these strategies, the complexation between Fe3+ and catechol has attracted significant attention due to its dynamic response to pH [24–26]. This pH-dependent cross-linking strategy can store the drug in polymer micelles during systemic circulation, releasing the drug through controlled behavior at the lower pH value in lysosomes [27]. In this study, a new strategy to prepare PEG shell-sheddable TATmodified core cross-linked nanomicelles as the vector of doxorubicin (DOX) was proposed to enhance endocytosis and pH-triggered drug release. P(ae-Asp), which is rich in amino groups, was selected as the framework material. Here, P(ae-Asp) was modified with thiolytic cleavable PEG, 3,4-dihydroxyphenylacetic acid, and TAT. DOX was embedded in the hydrophobic core of the nanomicelles obtained from self-assembly of the amphiphilic polymer in aqueous solution. Since the low concentration of glutathione (GSH) in plasma (2−20 μM) is insufficient to separate PEG from nanomicelles, TAT can be shielded by the steric resistance of PEG during systemic circulation to avoid nonspecific interaction with normal cells. The GSH content in tumor tissues is four times higher compared with normal tissues [28]. Therefore, the PEG shells of DOX-TAT-CCLMs were removed in response to a high concentration of GSH in tumor tissues, and the exposed TAT efficiently mediated the uptake of DOX-TAT-CCLMs. After core cross-linking was achieved with the addition of Fe3+, the DOX-TAT-CCLMs were stable enough to maintain the nanostructure following PEG detachment by the redox tumor environment. Once the micelles were internalized into tumor cells, the coordination between catechol and Fe3+ was modified from bis-complexes to mono-complexes under acidic conditions in lysosomes, thus releasing DOX accurately (Fig. 1).
P(ae-Asp) was obtained from the complete ring-opening of PSI with ethylenediamine, where PSI was synthesized as previously described [29]. mPEG-SS-COOH was synthesized from mPEG-NH2 and 3,3'-dithiodipropionic acid. Details have been provided in the supplementary information. mPEG-SS-COOH was activated to produce mPEG-SS-NHS to improve the reaction efficiency between mPEG-SS-COOH and P(aeAsp). Briefly, a mixture of mPEG-SS-COOH (1.0 g, 0.4 mmol), NHS (0.23 g, 2 mmol), and EDC·HCl (0.38 g, 2 mmol) in DCM (5 mL) was stirred for 12 h at 25℃. When the reaction was completed, the reaction solution was concentrated under vacuum to remove DCM. Subsequently, P(ae-Asp) (0.25 g, 1.6 mmol) and the obtained residue was dissolved in distilled water (5 mL) and stirred for 12 h. Then, the obtained mixture was dialyzed with distilled water for three days (molecular weight cutoff (MWCO) 5000 Da). Finally, mPEG-SS-g-P(ae-Asp) was obtained from the dialysate by filtration and freeze-drying. 2.2. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA 6-Maleimidocaproic acid (MCA) (0.11 g, 0.5 mmol) was dissolved in DMF (5 mL). Next, NHS (0.07 g, 0.6 mmol) and EDC·HCl (0.12 g, 0.6 mmol) were added to the above solution and stirred for 2 h under nitrogen atmosphere. To the resulting solution, mPEG-SS-g-P(ae-Asp) (0.5 g, 1 mmol) was added and then stirred for another 12 h at 25℃. Next, the resulting mixture was dialyzed with distilled water for three days (MWCO 5000 Da), and then filtered and freeze-dried to obtain mPEG-SS-g-P(ae-Asp)-MCA. 2.3. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA-DA mPEG-SS-g-P(ae-Asp)-MCA (0.47 g, 0.5 mmol) was dissolved in DMF (5 mL). Next, 3,4-dihydroxyphenylacetic acid (DOPA) (0.17 g, 1 mmol) and EDC·HCl (0.12 g, 1 mmol) was added to the above solution and stirred using a magnetic stirrer for 12 h under nitrogen atmosphere. The resulting mixture was dialyzed with distilled water for three days (MWCO 5000 Da), and then filtered and freeze-dried to obtain mPEGSS-g-P(ae-Asp)-MCA-DA. 2.4. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT A mixture of TAT (12 mg, 7 mmol) and mPEG-SS-g-P(ae-Asp)-MCADA (0.2 g, 1.3 mmol) in DMF (8 mL) was slowly stirred at 25℃ for 12 h in a sealed flask. The resultant solution was purified by dialysis with distilled water for three days (MWCO 5000 Da), and freeze-dried to obtain mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. 2.5. Preparation of micelles For the preparation of micelles, mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT (20 mg) was thoroughly dissolved in DMSO (0.6 mL). Then, the solution was slowly added to stirring Milli-Q water (5 mL). After stirring for another 1 h, the solution was purified by dialysis with deionized water (MWCO 3500 Da). Finally, the uncross-linked micellar (TAT-UCLMs) solution was obtained. The core cross-linked micelles (TAT-CCLMs) were prepared by adding the appropriate 40 mM FeCl3 solution into the TAT-UCLMs solution (molar ratio of catechol:Fe3+ = 2:1). Next, the pH of the mixture was adjusted to 7.4 using the phosphate buffer (2 M) to induce Fe3+-catechol bis-complex formation [30–32]. After 2 h of reaction, the mixture was dialyzed to remove the extra ferric ions. Ultimately, the TAT-UCLMs solution and the TAT-CCLMs solution were reduced to the same concentration (2 mg/mL). Referring to the preparation method of TAT-CCLMs, the core crosslinked micelles without penetrating peptide (CCLMs) were prepared as the control group using mPEG-SS-g-P(ae-Asp)-MCA-DA.
2. Experimental L-aspartic acid, diisopropyl azodiformate (DIAD), N-hydroxysuccinimide (NHS), GSH, 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDC·HCl), and 3,3'-dithiodipropionic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methoxy polyethylene glycol (mPEG, molecular weight: 2000 Da) was obtained from Vita Chemical Reagent Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) were purified prior to use. TAT (CYGRKKRRQRRR) (95 %) was purchased from ChinaPeptides Co., Ltd. (Suzhou, China). 2
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Fig. 1. Illustration of DOX-TAT-CCLMs self-assembled by mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT and lysosomal pH-triggered drug release.
after the removal of the PEG shell. Therefore, we added GSH to simulate the reductive environment of the tumor tissue. To achieve this, the TATCCLMs solution was mixed with GSH (10 mM) in PBS buffer and vibrated on a shaker at 37℃. The nanoparticle size changes of micelles were measured by DLS at different time points.
2.6. Characterization of micelles We characterized the micelle morphology using transmission electron microscopy (TEM) after the samples were dried on the copper mesh. Dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Limited, UK) was employed to measure the average size and the critical aggregation concentration (CAC) of the micelles [33].
2.9. Release profiles of DOX from micelles in vitro
2.7. Fabrication of DOX-loaded nanomicelles
The release characteristics of DOX-loaded micelles in vitro were evaluated in phosphate buffers (pH 5.0 or pH 7.4). Firstly, each dialysis tube (MWCO 1000 Da) was filled with 1 mL of DOX-TAT-CCLMs solution. Next, they were immersed in a 20 mL release medium and shaken at 37°C. At a preset time point, 200 μL medium was removed for the measurement of fluorescence intensity, and 200 μL of fresh phosphate buffer was added to the release medium [34]. Finally, a microplate reader (SpectraMax M5, Molecular Devices) was employed to determine the concentration of DOX in the samples at 595 nm.
mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT (20 mg) was completely dissolved in DMSO (0.6 mL). To this solution, DOX·HCl and triethylamine (40 μL) were added and stirred for 4 h. Then, the above solution was slowly added to the stirred ultra-pure water (6 mL). After stirring for another 1 h, the solution was dialyzed with deionized water to remove DMSO and extra DOX·HCl (MWCO 3500 Da). Next, the DOX-loaded uncross-linked micelle (DOX-TAT-UCLMs) solution was obtained. The DOX-loaded core cross-linked micelle (DOX-TAT-CCLMs) solution was prepared by adding the appropriate 40 mM FeCl3 solution to the DOXTAT-UCLMs solution (molar ratio of catechol: Fe3+ = 2:1). Then, the pH of the mixture was adjusted to 7.4 using the phosphate buffer (2 M) to induce Fe3+-catechol bis-complex formation. After 2 h of reaction, the mixture was dialyzed to remove extra Fe3+. Ultimately, the DOXTAT-CCLMs solution was obtained. Using the preparation method of DOX-TAT-CCLMs, the DOX-loaded micelles group without penetrating peptide (DOX-CCLMs) was prepared as the control. UV–vis spectroscopy was used to determine the drug-loading content (DLC) and drug-loading efficiency (DLE). First, the DOX-TATCCLMs and DOX-CCLMs solutions were lyophilized to obtain a solid red powder. Then, the solid red powder was dissolved in DMSO and the absorbance at 480 nm was determined using UV–vis spectroscopy. A fitting calibration curve was obtained by dissolving different formulations of DOX in DMSO. DLC and DLE were calculated using formulas (1) and (2).
DLC% =
weight of loaded drug × 100% weight of drug loaded micelle
(1)
DLE% =
weight of loaded drug × 100% weight of drug in feed
(2)
2.10. Cellular uptake evaluation and DOX efflux assay MCF-7 cells and MCF-7/ADR cells were grown in 24-well plates (1 × 105 cells/well) for 24 h, respectively. Afterward, the medium was drained and replaced with fresh medium (with or without 10 mM GSH) containing DOX-loaded micelles or DOX·HCl (DOX concentration: 1 μg/ mL). After 4 h of incubation, the medium was drained, and the plates were washed with PBS for 3 times. Finally, an inverted fluorescence microscope (Olympus, TH4-200 with Olympus UHGLGPS) was employed to capture the fluorescence pictures. The cellular uptake efficiency of DOX-TAT-CCLMs was measured with a fluorescence detector (SpectraMax M5, Molecular Devices) [35,36]. The specific process is as follows: firstly, MCF-7/ADR cells and MCF-7 cells were grown in 6-well plates (2 × 105 cells/well), respectively. After 24 h of incubation, replace the old medium with fresh medium (with 10 mM GSH) containing DOX-loaded micelles or DOX·HCl (DOX concentration: 5 μg/mL). After incubation for another 2, 4, or 8 h, for quantitative analysis of cellular uptake efficiency, cell lysis buffer (0.01 M HCl, 10 % sodium dodecyl sulfonate and 5 % isobutyl alcohol) was added to break the cell structure after removing DOXloaded micelles and DOX·HCl solutions. The fluorescence emission intensity at each time point was determined at 595 nm with a microplate reader (SpectraMax M5, Molecular Devices) to calculate cellular uptake efficiency. In the case of DOX efflux assays, MCF-7/ADR cells were grown in 24-well plates. Twenty-four hours later, fresh medium (with 10 mM
2.8. Reduction-sensitized micelles in vitro According to the design, TAT in nanomicelles can only function 3
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aspartic acid. Subsequently, the five-membered rings of PSI were completely opened with ethylenediamine, which was confirmed by the disappearance of the peak at 5.25 ppm (Fig. S3). As shown in Fig. S4A, the appearance of the peak at 3.23 ppm indicated the successful synthesis of polymer mPEG-SS-g-P(ae-Asp). According to the following formula (4), the grafting degree (DG) of mPEG was 20 %.
GSH) containing DOX-TAT-CCLMs or DOX HCl (DOX concentration: 10 μg/mL) replaced the old medium. After incubation for 4 h, the DOXTAT-CCLMs and DOX·HCl solutions were drained out and then washed with PBS. Subsequently, fresh medium was added to each well and incubated for another 1 and 4 h. To quantify the DOX mass pumped from the cells at each time point, a fluorescence detector (SpectraMax M5, Molecular Devices) was used to determine the fluorescence emission intensity of the medium at 595 nm. The DOX mass accumulated inside the cells was determined following incubation with the corresponding dose of DOX.
DG = S1/(3S2)
In the formula, S2 is the integral area of 4.34–4.75 ppm assigned to methylene protons in the aspartic acid unit, and S1 is the integral area of terminal methyl protons at 3.22–3.25 ppm in PEG. Fig. S4B shows the 1H-NMR spectrum of mPEG-SS-g-P(ae-Asp)MCA-DA. The appearance of peaks at 7.0 ppm and 6.43–6.68 ppm was attributed to the maleimide groups of MCA and the protons of catechols, respectively. The DG of DOPA and MCA were 7 % and 14 %, respectively, determined using the following formula:
2.11. In vitro cytotoxicity assays To perform the in vitro cytotoxicity assays, MCF-7/ADR cells and MCF-7 cells were grown in 96-well plates (5000 cells/well), respectively. Twenty-four hours later, the culture medium was removed and 180 μL of fresh medium (with 10 mM GSH) was added. Subsequently, the culture medium containing different concentrations of DOX-loaded micelles (20 μL) was filled into the wells and incubated for 72 h. Then, 10 μL of MTT solution (5 mg/mL) was added to each well after the removal of 80 μL of culture media. After 4 h of incubation, a pyrolysis solution (50 mL/well) was added to dissolve the crystals. An automatic BIO-TEK microplate reader (Powerwave XS, USA) was employed to measure the absorbance of each well. The cells incubated with 200 μL medium were employed as a control (ODcontrol). The culture media without cells was employed as a blank (ODblank). Cell viability was calculated according to formula (3).
Cell viability% =
ODsample - ODblank × 100% ODcontrol - ODblank
(4)
DG = S3/(2S2)
(5)
DG = S4/(2S2)
(6)
where S3 is the integral area of the maleimide groups of MCA, and S4 is the integral area of the protons of catechols. Finally, TAT was grafted onto mPEG-SS-g-P(ae-Asp)-MCA-DA via the Michael addition reaction. Fig. S4C shows the 1H-NMR spectrum of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. The appearance of TAT characteristic peaks indicated that TAT was successfully grafted onto the copolymers. The 1H-NMR spectrum of TAT is shown in Fig. S2B. 3.2. Fabrication and characterization of nanomicelles
(3)
DLS and TEM were utilized for further analysis of the particle size and observation of the morphology of TAT-UCLMs and TAT-CCLMs. Fig. S5A and S5C demonstrate that the TAT-UCLMs were exhibited as nano-spheres with an average size of 188.7 nm (PDI: 0.166). As shown in Fig. S5B, the TAT-CCLMs morphology was observed as spherical nanomicelles with enhanced contrast in the center of the TAT-CCLMs, which could be attributed to the high atomic amount of iron. As shown in Fig. S5D, the average size of TAT-CCLMs was 180.1 nm (PDI: 0.156). The particle size measured by DLS was larger than that measured by TEM, which may be attributed to the shrinkage of the hydrophilic PEG shell during TEM sample preparation. It is well known that CAC plays an important role in micellization. Polymer micelles as drug carriers should possess lower CAC values to resist extensive blood dilution in the systemic circulation. Here, DLS was employed to determine the CAC values. The CAC value of mPEGSS-g-P(ae-Asp)-MCA-DA-TAT micelles was determined as 1.41 × 10−2 mg/mL (Fig. S6). The lower CAC value indicated that the nanomicelles have the potential to maintain the integrity of nanostructures during blood circulation. In addition, sample dilution experiments were also performed. As shown in Fig. S7, the size distribution of TAT-UCLMs and TAT-CCLMs diluted 100 times with PBS. Fig. S7A demonstrates that the diluted TAT-UCLMs sample was unsuitable for DLS measurement. In contrast, the average size of TAT-CCLMs was 238.7 nm (Fig. S7B), and its size distribution was relatively narrow (PDI: 0.355). This suggested that TAT-CCLMs have the potential to withstand dilution to avoid disassembly of nanomicelles and premature drug release during in vivo circulation.
2.12. In vivo biodistribution For biodistribution analysis, the tumor model was established by subcutaneously injecting about 1.0 × 106 4T1 cells into the right upper armpit of female BALB/C mice (4–6 weeks old, weighing approximately 20 g). When tumor volumes reached about 400–500 mm3, the tumorbearing mice were randomly divided into three groups (n = 3) and intravenously administered DOX-TAT-CCLMs, DOX-CCLMs, and free DOX at a dose of 5 mg DOX/kg body weight per mouse. The mice were executed post 24 h of administration. Tumors and main organs, including heart, liver, spleen, lung, and kidney were excised carefully and washed with saline, weighed after drying, and homogenized in 1 mL mixture of water and DMSO (V/V: 1/1). The supernatant fractions were collected, and the concentration of DOX in the samples was determined using a microplate reader (SpectraMax M5, Molecular Devices) at 595 nm. 2.13. In vivo antitumor efficacy To further evaluate the anticancer efficacy of the DOX-TAT-CCLMs in vivo, 4T1 cells were subcutaneously injected into the right upper armpit of female BALB/C mice (1 × 106 cells per mouse). When tumors reached to a volume of approximately 30 mm3 (determined as day 0), mice were randomly divided into five groups (n = 4). Then 4T1 tumor bearing mice were injected through the tail vein with saline, TATCCLMs, DOX-CCLMs, DOX-TAT-CCLMs, or free DOX at 3-day intervals (0, 3, 6, and 9 days, and DOX dosage: 3 mg/kg).
3.3. pH-Sensitive coordination between catechol and Fe3+ 3. Results and discussion The pH-sensitive coordination of catechol and Fe3+ was determined by UV–vis spectroscopy under different pH values. As shown in Fig. 3A, the absorption peak at 470 nm was attributed to the formation of triscomplexes between catechol and ferric ions at pH 10.0. The UV absorption peak shifted to 540 nm and 590 nm when the pH values were modified to 7.4 and 5.0, indicating that catechol and Fe3+ formed bis-
3.1. Synthesis and characterization of mPEG-SS-g-P(ae-Asp)-MCA-DATAT Fig. 2 represents the detailed synthetic route of mPEG-SS-g-P(aeAsp)-MCA-DA-TAT. Firstly, PSI was synthesized by condensation of L4
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Fig. 2. The detailed synthetic route of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT.
fracture. Additionally, another size distribution of 100−200 nm was assigned to the core cross-linked TAT-CCLMs after the addition of GSH in nanomicelles. The above results implied that the PEG shell of nanomicelles can be removed in the redox environment of tumor tissue, while TAT-CCLMs could still exist stably due to the cross-linking of catechol with Fe3+.
complexes and mono-complexes, respectively. In addition, the pH-responsive variations of coordination between catechol and Fe3+ were further confirmed by obvious color changes of the complex (Fig. 3B). The color of the TAT-CCLMs solution was orange at pH 10.0, changed to violet at pH 7.4, and pale yellow at pH 5.0. 3.4. Reduction-sensitivity of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT micelles
3.5. Drug encapsulation and drug release in vitro The disulfide bonds in mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT were susceptible to the tumor redox environment. This is helpful for the tumor tissue-specific detachment of the PEG shell and the exposure of TAT. DLS was employed to measure the size change in nanomicelles in response to GSH (10 mM) in PBS buffer (pH 7.4). As shown in Fig. 3C, nanoparticles with a diameter of about 20 nm appeared after the addition of GSH to the nanomicelles for 20 min. After 200 min, the small particles increased slightly. The appearance of small particles can probably be attributed to the peeling of PEG shells due to disulfide bond
DOX was loaded in TAT-CCLMs via the dialysis method. According to the design, the synthesized amphiphilic polymer self-assembled in water. Owing to the hydrophobic interaction between the DA segment and hydrophobic DOX, DOX was enfolded in the hydrophobic core of nanomicelles. DLC and DLE of DOX were measured as 10.18 ± 0.24 % and 61.16 ± 1.65 %, respectively. The TEM image shows the spherical shape of DOX-TAT-CCLMs (Fig. S8A). As shown in Fig. S8B, the particle size of DOX-TAT-CCLMs was 251.7 nm, with a narrow PDI of 0.297. It is
Fig. 3. (A) Determination of pH-responsive coordination between catechol with Fe3+ by UV–vis. (B) Color changes under diverse pH values. (C) Size change of TATCCLMs in response to GSH (10 mM). 5
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Fig. 4. (A) Drug release curve of DOX-TAT-CCLMs at different pH values. (B) Drug release curve of DOX-TAT-UCLMs and DOX-TAT-CCLMs at pH 7.4.
S10, in MCF-7/ADR and MCF-7 cells, no differences were observed between the cellular internalization of DOX-CCLMs and DOX-TATCCLMs without the addition of GSH. This result validated that the cellpenetration effect of TAT could be effectively shielded by the PEG shell with a grafting ratio of 20 %, which is in line with results reported by Kuai et al. [16]. Conversely, as demonstrated in Fig. 6, the fluorescent intensity of DOX in MCF-7/ADR cells treated with DOX-TAT-CCLMs and 10 mM GSH was much higher than that without GSH. This phenomenon could be due to the removal of the PEG shell in DOX-TATCCLMs in response to GSH, contributing to the exposure of TAT and subsequent facilitated cellular internalization of nanomicelles. Furthermore, it is worth noting that the addition of GSH or not had no influence on the cellular internalization of DOX·HCl. Similar cellular uptake results of DOX·HCl and DOX-TAT-CCLMs were observed in MCF7 cells (Fig. S11). The cellular internalization of DOX·HCl, DOX-CCLMs, and DOX-TAT-CCLMs was further quantified by the fluorescent intensity measurement of DOX. As shown in Fig. 7A, compared with DOX·HCl and DOX-CCLMs, DOX-TAT-CCLMs were abundantly internalized into MCF-7/ADR cells. Specifically, after incubation for 8 h, the mean fluorescence intensity of DOX-TAT-CCLMs in MCF-7/ADR cells was 8 times higher than that of DOX·HCl. These results verified that the enhancement of TAT can remarkably increase the tumor cell intracellular accumulation of DOX. Similar to MCF-7/ADR cells, the DOXTAT-CCLMs also showed the highest mean fluorescence intensity of DOX in MCF-7 cells compared with DOX·HCl and DOX-CCLMs (Fig. S12A). In order to demonstrate that the low fluorescent intensity of
obvious that the particle diameter of DOX-TAT-CCLMs was larger than TAT-CCLMs, resulting from the encapsulation of DOX in micelles. The stability of DOX-TAT-CCLMs was determined by DLS measurement after dilution in fetal bovine serum (FBS; HyClone, Logan, UT) at 37℃. As shown in Fig. S9, the size of DOX-TAT-CCLMs was not significantly altered within 4 days. The results showed that DOX-TAT-CCLMs was relatively stable in FBS for 4 days. The in vitro drug release analysis of DOX-TAT-CCLMs was performed by the dialysis method. It has been anticipated that the core crosslinked DOX-TAT-CCLMs could minimize premature leakage of DOX in blood circulation while accomplishing pH-triggered DOX release in cancer cells. As shown in Fig. 4A, the core cross-linked DOX-TATCCLMs exhibited a pH-dependent drug release profile. Since the coordination ratio between catechol and ferric ions reduced from 2 (at pH 7.4) to 1 (at pH 5.0), DOX release was more favorable at pH 5.0 than at pH 7.4. Furthermore, the drug release behavior of DOX-TAT-UCLMs at pH 7.4 was evaluated. Fig. 4B demonstrates the rapid release of DOX from the DOX-TAT-UCLMs compared with DOX-TAT-CCLMs. All these results demonstrated that the DOX-TAT-CCLMs could effectively constrain DOX in the cross-linked core of nanomicelles during systemic circulation, while effectively releasing DOX in the acidic lysosomal environment. 3.6. Cellular uptake and DOX efflux assay DOX emits red fluorescence when stimulated, which can be utilized to measure the cellular uptake efficiency. As exhibited in Figs. 5 and
Fig. 5. Fluorescence images of the MCF-7/ADR cells incubated with DOX-CCLMs and DOX-TAT-CCLMs without GSH treatment (incubation for 4 h, bar represents 50 μm). 6
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Fig. 6. Fluorescence images of the MCF-7/ADR cells incubated with DOX-TAT-CCLMs and DOX·HCl with or without GSH treatment (incubation for 4 h, bar represents 50 μm).
Fig. 7. (A) Mean fluorescence intensity of MCF-7/ADR cells incubated with DOX·HCl, DOX-TAT-CCLMs, and DOX-CCLMs at 2, 4, and 8 h. (B) DOX efflux percentage of MCF-7/ADR cells incubated with DOX·HCl and DOX-TATCCLMs. (C) Survival rate of MCF-7/ADR cells cultured in different concentrations of blank micelles for 72 h. (D) Inhibitory activity on MCF-7/ADR cells in different groups (DOX·HCl, DOX-TAT-CCLMs, DOX-CCLMs). Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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Fig. 8. (A) Quantitative evaluation of in vivo biodistribution of different formulations in tumors and main organs normalized with organ or tumor weights (post 24 h of administration). (B) 4T1 tumor growth curves after intravenous administration of five types of formulations. (C) Photos of tumor tissues of five groups on day 15 after intravenous injection. (D) The quantitative summary of tumor weights after treatment with five different formulations on day 15. (E) The body weight variations of 4T1 tumor-bearing mice during treatment. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
DOX·HCl in MCF-7/ADR cells was caused by drug efflux, DOX efflux analysis was performed with 10 μg/mL DOX. As shown in Fig. 7B, the DOX efflux against MCF-7/ADR cells was performed in a time-dependent manner and the DOX efflux ratio increased to 25 % within 4 h. However, the DOX-TAT-CCLMs showed a rather low DOX efflux ratio of 4.5 %. These results indicated that the DOX·HCl failed to efficiently enter into multidrug-resistant MCF-7/ADR cells owing to the high expression of drug efflux pumps such as P-glycoprotein [37]. In contrast to DOX·HCl, the DOX-TAT-CCLMs bypassed the drug efflux pumps, thereby significantly increasing DOX accumulation in MCF-7/ADR cells.
value higher than 160 μg/mL, suggesting TAT plays an essential role in the anti-proliferation of multidrug-resistant MCF-7/ADR cells. Notably, the IC50 value of DOX-TAT-CCLMs against the MCF-7/ADR cells was about 4 times lower than that of DOX·HCl, which could be attributed to the greatly increased accumulation of DOX-TAT-CCLMs in cells revealed in the cellular uptake analysis. Collectively, TAT modification generated DOX-TAT-CCLMs exhibiting a remarkable antiproliferative effect in MCF-7/ADR cells.
3.7. In vitro cytotoxicity assays
As presented in Fig. 8A, after administration for 24 h, free DOX showed a rather low accumulation at the tumor site compared to both DOX-loaded micelles. This was mainly attributed to the enhanced permeability and retention effect of DOX-loaded micelles. Indeed, DOXTAT-CCLMs are more abundant in the tumor site than DOX-CCLMs, which could be attributed to the TAT exposure facilitated cellular internalization of nanomicelles. For in vivo antitumor efficacy, as shown in Fig. 8B, tumor volume was altered with time. On day 15, the tumor volumes of the groups injected with saline, TAT-CCLMs, DOX·HCl, DOX-CCLMs, and DOXTAT-CCLMs were 768 ± 105 mm3, 744 ± 84 mm3, 458 ± 42 mm3, 342 ± 39 mm3, and 172 ± 41 mm3, respectively (Fig. 8B). The free DOX group showed a certain degree of tumor growth suppression, but its inhibitory performance was inferior compared to both DOX-loaded micelles (Fig. 8C). This was mainly due to the low accumulation of small molecules (DOX) in the tumor. On day 15, the mean tumor weight of the groups injected with saline, TAT-CCLMs, DOX·HCl, DOX-CCLMs, and DOX-TAT-CCLMs were 0.80 ± 0.19 g, 0.78 ± 0.13 g, 0.47 ± 0.05 g, 0.37 ± 0.03 g, and 0.19 ± 0.05 g, respectively (Fig. 8D). Unequivocally, the DOX-TAT-CCLMs group demonstrated the best in vivo antitumor efficacy among three treatment groups, which could be attributed to the greatly increased accumulation of DOX-TATCCLMs in the tumor site as indicated in the biodistribution analysis. As shown in Fig. 8E, the body weight of the DOX HCl group decreased during the injection period, indicating severe systemic toxicity. However, the weight of the DOX-TAT-CCLMs group was relatively stable, demonstrating lower side effects and improved drug delivery efficiency.
3.8. In vivo biodistribution and antitumor efficacy
The above results demonstrated that DOX-TAT-CCLMs micelles could enhance DOX uptake, reduce drug efflux, and site-specifically release DOX in the acidic lysosomal environment. It is expected that DOX-TAT-CCLMs could exert high anti-proliferative efficacy against cancer cells. Firstly, an in vitro MTT assay was performed to determine the toxicity of the carriers. MCF-7/ADR and MCF-7 cells were treated with different concentrations of CCLMs and TAT-CCLMs for 72 h, respectively. As shown in Figs. S12B and 7 C, after treatment with blank micelles, the cell viability of MCF-7 as well as MCF-7/ADR cells was higher than 85 %, confirming the negligible toxicity of the carriers. Subsequently, the cytotoxicity of DOX-TAT-CCLMs was evaluated using DOX·HCl and DOX-CCLMs as controls. As shown in Fig. S12C, when the DOX concentration was lower than 2.5 μg/mL, the cytotoxicity of DOXTAT-CCLMs against MCF-7 cells was slightly inferior to that of DOX·HCl. However, when the DOX concentration increased to 5 μg/mL, the cytotoxicity of DOX-TAT-CCLMs was comparable to that of DOX·HCl. Furthermore, the IC50 values of DOX-TAT-CCLMs, DOX HCl, and DOX-CCLMs were 0.71 ± 0.04 μg/mL, 0.31 ± 0.05 μg/mL, and 4.12 ± 0.3 μg/mL, respectively. Collectively, DOX-TAT-CCLMs demonstrated superior in vitro antitumor activity compared to DOXCCLMs. Furthermore, compared with DOX and DOX-CCLMs, DOX-TATCCLMs showed higher cytotoxicity in MCF-7/ADR cells (Fig. 7D). After 72 h of incubation, the IC50 values of DOX-TAT-CCLMs and DOX·HCl against the MCF-7/ADR cells were 11.61 ± 0.95 μg/mL and 42.77 ± 1.93 μg/mL, respectively. DOX-CCLMs exhibited an IC50 8
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4. Conclusion [11]
In this study, TAT-modified core cross-linked nanomicelles with a redox-sheddable PEG shell were fabricated by the self-assembly of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT as the delivery carrier for DOX. The nanomicelles resisted excessive dilution and achieved lysosomal pH-triggered intracellular drug release due to the core cross-linking between Fe3+ and catechol. In vitro studies demonstrated that the multifunctional DOX-TAT-CCLMs significantly enhanced the cellular internalization and prominently reduced the drug efflux of DOX against the multidrug-resistant MCF-7/ADR cells. Compared with DOX·HCl, DOX-TAT-CCLMs exerted a higher antiproliferative effect on MCF-7/ ADR cells, with an IC50 value nearly 4 times lower than DOX HCl. Furthermore, the DOX-TAT-CCLMs showed good suppression of tumor growth in the 4T1 tumor-bearing mouse model. In summary, the DOXTAT-CCLMs have great potential to increase drug delivery efficiency and overcome multidrug resistance during cancer therapy.
[12] [13] [14]
[15]
[16]
[17]
[18]
Author contributions [19]
Yuliu Zhang designed the study, analysed the data, and drafted the manuscript. Yi Xiao and Yushu Huang conceived the study and revised the manuscript. Yuliu Zhang, Yushu Huang and Yang He performed the experiments. Yuliu Zhang, Yang He, Yanyun Xu and Wei Lu analysed the data. Jiahui Yu is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors reviewed the manuscript.
[20]
[21]
[22]
[23]
Declaration of Competing Interest [24]
None.
[25]
Acknowledgment [26]
The research work was supported by the National Natural Science Foundation of China (81871405, 51573050).
[27]
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.colsurfb.2020.110772.
[28]
References
[29]
[1] J. Liu, M. Li, Z. Luo, L. Dai, X. Guo, K. Cai, Design of nanocarriers based on complex biological barriers in vivo for tumor therapy, Nano Today 15 (2017) 56–90. [2] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nat. Biotechnol. 33 (2015) 941–951. [3] M. Gu, X. Wang, T.B. Toh, E.K. Chow, Applications of stimuli-responsive nanoscale drug delivery systems in translational research, Drug Discov. Today 23 (2018) 1043–1052. [4] T. Ramasamy, H.B. Ruttala, B. Gupta, B.K. Poudel, H.G. Choi, C.S. Yong, J.O. Kim, Smart chemistry-based nanosized drug delivery systems for systemic applications: a comprehensive review, J. Control. Release 258 (2017) 226–253. [5] M. Mohammadi, M. Ramezani, K. Abnous, M. Alibolandi, Biocompatible polymersomes-based cancer theranostics: towards multifunctional nanomedicine, Int. J. Pharm. 519 (2017) 287–303. [6] Y. Li, M. Kroger, W.K. Liu, Endocytosis of PEGylated nanoparticles accompanied by structural and free energy changes of the grafted polyethylene glycol, Biomaterials 35 (2014) 8467–8478. [7] L. Mo, J.G. Song, H. Lee, M. Zhao, H.Y. Kim, Y.J. Lee, H.W. Ko, H.K. Han, PEGylated hyaluronic acid-coated liposome for enhanced in vivo efficacy of sorafenib via active tumor cell targeting and prolonged systemic exposure, Nanomedicine 14 (2018) 557–567. [8] T. Lang, X. Dong, Z. Zheng, Y. Liu, G. Wang, Q. Yin, Y. Li, Tumor microenvironment-responsive docetaxel-loaded micelle combats metastatic breast cancer, Sci. Bull. 64 (2019) 91–100. [9] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress, J. Control. Release 161 (2012) 175–187. [10] H. Kim, M. Kitamatsu, T. Ohtsuki, Enhanced intracellular peptide delivery by
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
9
multivalent cell-penetrating peptide with bioreducible linkage, Bioorg. Med. Chem. Lett. 28 (2018) 378–381. W.B. Kauffman, T. Fuselier, J. He, W.C. Wimley, Mechanism matters: a taxonomy of cell penetrating peptides, Trends Biochem. Sci. 40 (2015) 749–764. F. Madani, S. Lindberg, U. Langel, S. Futaki, A. Graslund, Mechanisms of cellular uptake of cell-penetrating peptides, J. Biophys. 2011 (2011) 414729. J.D. Ramsey, N.H. Flynn, Cell-penetrating peptides transport therapeutics into cells, Pharmacol. Ther. 154 (2015) 78–86. J. Zhang, Y. Zheng, X. Xie, L. Wang, Z. Su, Y. Wang, K.W. Leong, M. Chen, Cleavable multifunctional targeting mixed micelles with sequential pH-triggered TAT peptide activation for improved antihepatocellular carcinoma efficacy, Mol. Pharm. 14 (2017) 3644–3659. L. Zhu, T. Wang, F. Perche, A. Taigind, V.P. Torchilin, Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cellpenetrating moiety, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 17047–17052. W.Y. Rui Kuai, Yao Qin, Huali Chen, Jie Tang, Mingqing Yuan, Zhirong Zhang, Qin He, Efficient delivery of payload into tumor cells in a controlled manner by TAT and thiolytic cleavable PEG Co-modified liposomes, Mol. Pharceut. 7 (2010) 1816–1826. E.S. Lee, Z. Gao, D. Kim, K. Park, I.C. Kwon, Y.H. Bae, Super pH-sensitive multifunctional polymeric micelle for tumor pH(e) specific TAT exposure and multidrug resistance, J. Control. Release 129 (2008) 228–236. S.M. Farkhani, A. Valizadeh, H. Karami, S. Mohammadi, N. Sohrabi, F. Badrzadeh, Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules, Peptides 57 (2014) 78–94. S.C. Owen, D.P.Y. Chan, M.S. Shoichet, Polymeric micelle stability, Nano Today 7 (2012) 53–65. M. Talelli, M. Barz, C.J. Rijcken, F. Kiessling, W.E. Hennink, T. Lammers, Corecrosslinked polymeric micelles: principles, preparation, biomedical applications and clinical translation, Nano Today 10 (2015) 93–117. Y.-B. Long, W.-X. Gu, C. Pang, J. Ma, H. Gao, Construction of coumarin-based crosslinked micelles with pH responsive hydrazone bond and tumor targeting moiety, J. Mater. Chem. B 4 (2016) 1480–1488. J. Ren, Y. Zhang, J. Zhang, H. Gao, G. Liu, R. Ma, Y. An, D. Kong, L. Shi, pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery, Biomacromolecules 14 (2013) 3434–3443. H. He, Y. Bai, J. Wang, Q. Deng, L. Zhu, F. Meng, Z. Zhong, L. Yin, Reversibly crosslinked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity, Biomacromolecules 16 (2015) 1390–1400. R. Mrowczynski, Polydopamine-based multifunctional (Nano)materials for cancer therapy, ACS Appl. Mater. Interfaces 10 (2018) 7541–7561. N. Holten-Andersen, M.J. Harrington, H. Birkedal, B.P. Lee, P.B. Messersmith, K.Y. Lee, J.H. Waite, pH-induced metal-ligand cross-links inspired by mussel yield self-healing polymer networks with near-covalent elastic moduli, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 2651–2655. B.J. Kim, H. Cheong, B.H. Hwang, H.J. Cha, Mussel-inspired protein nanoparticles containing Iron(III)-DOPA complexes for pH-responsive drug delivery, Angew. Chem. Int. Ed. Engl. 54 (2015) 7318–7322. Y. Huang, Y. Xu, Y. Wu, T. Chen, W. Lu, J. Yu, Bioinspired nanoplatform for enhanced delivery efficiency of doxorubicin into nucleus with fast endocytosis, lysosomal pH-triggered drug release, and reduced efflux, Colloids Surf. B Biointerfaces 183 (2019) 110413. H.L. Periannan Kuppusamy, Govindasamy Ilangovan, Arturo J. Cardounel, Jay L. Zweier, Kenichi Yamada, Murali C. Krishna, James B. Mitchell, Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels, Cancer Res. 62 (2002) 307–312. Y. Zhao, H. Su, L. Fang, T. Tan, Superabsorbent hydrogels from poly(aspartic acid) with salt-, temperature- and pH-responsiveness properties, Polymer 46 (2005) 5368–5376. K. Xin, M. Li, D. Lu, X. Meng, J. Deng, D. Kong, D. Ding, Z. Wang, Y. Zhao, Bioinspired coordination micelles integrating high stability, triggered cargo release, and magnetic resonance imaging, ACS Appl. Mater. Interfaces 9 (2017) 80–91. A. Ghadban, A.S. Ahmed, Y. Ping, R. Ramos, N. Arfin, B. Cantaert, R.V. Ramanujan, A. Miserez, Bioinspired pH and magnetic responsive catechol-functionalized chitosan hydrogels with tunable elastic properties, Chem. Commun. (Camb.) 52 (2016) 697–700. G.H. Hwang, K.H. Min, H.J. Lee, H.Y. Nam, G.H. Choi, B.J. Kim, S.Y. Jeong, S.C. Lee, pH-responsive robust polymer micelles with metal-ligand coordinated core cross-links, Chem. Commun. (Camb.) 50 (2014) 4351–4353. Ö. Topel, B.A. Çakır, L. Budama, N. Hoda, Determination of critical micelle concentration of polybutadiene-block-poly(ethyleneoxide) diblock copolymer by fluorescence spectroscopy and dynamic light scattering, J. Mol. Liq. 177 (2013) 40–43. N. Li, W. Lu, J. Yu, Y. Xiao, S. Liu, L. Gan, J. Huang, Rod-like cellulose nanocrystal/ cis-aconityl-doxorubicin prodrug: a fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug controlled release, Mater. Sci. Eng. C Mater. Biol. Appl. 91 (2018) 179–189. N. Li, H. Zhang, Y. Xiao, Y. Huang, M. Xu, D. You, W. Lu, J. Yu, Fabrication of cellulose-nanocrystal-based folate targeted nanomedicine via layer-by-layer assembly with lysosomal pH-controlled drug release into the nucleus, Biomacromolecules 20 (2019) 937–948. X. Wei, Y. Wang, X. Xiong, X. Guo, L. Zhang, X. Zhang, S. Zhou, Codelivery of a π-π stacked dual anticancer drug combination with nanocarriers for overcoming multidrug resistance and tumor metastasis, Adv. Funct. Mater. 26 (2016) 8266–8280. M.M. Gottesman, T. Fojo, S.E. Bates, Multidrug resistance in cancer: role of ATPdependent transporters, Nat. Rev. Cancer 2 (2002) 48–58.
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Poly(ethylene glycol) shell-sheddable TAT-modified core cross-linked nanomicelles: TAT-enhanced cellular uptake and lysosomal pH-triggered doxorubicin release
T
Yuliu Zhanga, Yi Xiaob, Yushu Huanga, Yang Hea, Yanyun Xua, Wei Lua, Jiahui Yua,* a
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, PR China b Department of Radiology and Nuclear Medicine, Changzheng Hospital, Naval Medical University, Shanghai 200003, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: TAT-modified Enhanced endocytosis Core cross-linked nanomicelles Lysosomal pH-triggered doxorubicin release
This study aimed to develop sheddable polyethylene glycol (PEG) shells with TAT-modified core cross-linked nanomicelles as drug-delivery carriers of doxorubicin (DOX) to establish a programmed response against the tumor microenvironment, enhanced endocytosis, and lysosomal pH-triggered DOX release. First, poly(L-succinimide) (PSI) underwent a ring-opening reaction with ethylenediamine to generate poly(N-(2-aminoethyl)-Laspartamide) (P(ae-Asp)). Next, the thiolytic cleavable PEG, 3,4-dihydroxyphenylacetic acid, and TAT were grafted onto P(ae-Asp) to synthesize the amphiphilic graft copolymer of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. In aqueous solution, the amphiphilic polymer self-assembled into nanomicelles, encapsulating DOX into the hydrophobic core of micelles. TAT was shielded by the PEG corona during circulation to avoid non-specific transmembrane interaction with normal cells, while the tumor redox environment-responsive shedding of PEG could expose TAT to promote internalization of tumor cells. In order to improve the stability of nanomicelles and achieve pH-triggered drug release, a core cross-linking strategy based on the coordination of catechol and Fe3+ was adopted. In vitro studies demonstrated that core cross-linked nanomicelles maintained the nanostructure in 100 times dilution in pH 7.4 phosphate-buffered saline (PBS). Moreover, DOX release from DOX-loaded core cross-linked nanomicelles (DOX-TAT-CCLMs) was favored at simulated lysosomal conditions over simulated plasma conditions, indicating that these nanomicelles demonstrate characteristics of pH-triggered DOX release. The TAT modification considerably enhanced the mean fluorescence intensity of the nanomicelles endocytosed by MCF-7/ADR cells by 8 times, compared with DOX·HCl after 8 h of incubation. Notably, the IC50 value of nanomicelles (11.61 ± 0.95 μg/mL) was nearly 4 times lower than that of DOX·HCl against MCF-7/ADR cells, implying that the nanomicelles could overcome drug resistance observed in MCF-7/ADR cells. Furthermore, the DOX-TAT-CCLMs reported superior tumor growth suppression in a 4T1 tumor-bearing mouse model. Thus, the redox- and pH- stimuli stepwise-responsive novel nanomicelles fabricated from the mPEG-SS-g-P(ae-Asp)-MCADA-TAT graft copolymer exhibited multifunctionality and displayed great potential for drug delivery.
1. Introduction Polymeric nanomicelles and liposomes, have been rapidly developed to enhance the therapeutic efficacy of anticancer drugs and reduce the systemic toxicity associated with cancer therapy [1–4]. Notably, polymeric nanomicelles play an indispensable role in drug delivery systems due to the superior modification and biomedical potential [5]. Functional ligands are often modified to generate more intuitive nanomicelles. For example, modification of polyethylene glycol (PEG) on nanomicelles can lengthen the systemic circulation time, enhancing the
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cumulation of anticancer drugs in cancer tissues [6,7]. Furthermore, the modification of nanomicelles with targeting ligands possessing high binding affinity with overexpressed receptors on cancer cells can improve the internalization efficiency of nanomicelles [8,9]. Another way to improve the internalization efficiency is to modify the cell-penetrating peptides (CPP) on nanomicelles. CPP can efficiently carry nanomicelles or macromolecules into cells without destroying the integrity of the cell membranes [10,11]. The main advantage of CPP is that it can penetrate the cytomembrane at low micromolar concentrations in vivo and in vitro without using any other receptors [12]. TAT,
Corresponding author. E-mail address: [email protected] (J. Yu).
https://doi.org/10.1016/j.colsurfb.2020.110772 Received 28 October 2019; Received in revised form 2 January 2020; Accepted 3 January 2020 Available online 20 January 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved.
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2.1. Synthesis of mPEG-SS-g-P(ae-Asp)
one such CPP, was isolated from the HIV-1 protein by Green et al. [13] and has been considered an effective CPP to transport different molecules into various cell lines [14,15]. However, the non-selectivity of TAT against all kinds of cells limits its application in anticancer drug delivery. Notably, several attempts have been made to solve this problem. An effective method to avoid non-specific transmembrane interaction with normal cells involves the use of PEG as a shield before the nanodrugs reach the tumor tissue. Upon reaching tumor tissues, the PEG shell of nanodrugs is rescinded in response to the redox environment of tumor tissues (GSH 10 mM), thereby exposing TAT to mediate the endocytosis of nanodrugs with high efficiency [16–18]. Nevertheless, the extracellular shedding of PEG is always accompanied by some challenges, such as the disintegration of drug-loaded micelles and premature drug release. Additionally, the access of nanomicelles to tumor tissues is a prerequisite for TAT to exert its function [19]. As previously known, following in vivo administration, drugloaded micelles usually undergo excessive dilution in the systemic circulation. The disintegration of drug-loaded micelles during hemodilution invalidates the functional ligands installed in nanomicelles, resulting in premature drug leakage and serious side effects. Therefore, the structural integrity of nanomicelles is crucial to drug delivery efficiency and treatment efficacy. In order to address these problems, core cross-linking has been utilized as an effective strategy to maintain the integrity of polymer micelles [20]. Several core cross-linking methods [21–23], including the use of reduction- or pH-sensitive chemical bonds to form cross-linked structures, have been reported. Among these strategies, the complexation between Fe3+ and catechol has attracted significant attention due to its dynamic response to pH [24–26]. This pH-dependent cross-linking strategy can store the drug in polymer micelles during systemic circulation, releasing the drug through controlled behavior at the lower pH value in lysosomes [27]. In this study, a new strategy to prepare PEG shell-sheddable TATmodified core cross-linked nanomicelles as the vector of doxorubicin (DOX) was proposed to enhance endocytosis and pH-triggered drug release. P(ae-Asp), which is rich in amino groups, was selected as the framework material. Here, P(ae-Asp) was modified with thiolytic cleavable PEG, 3,4-dihydroxyphenylacetic acid, and TAT. DOX was embedded in the hydrophobic core of the nanomicelles obtained from self-assembly of the amphiphilic polymer in aqueous solution. Since the low concentration of glutathione (GSH) in plasma (2−20 μM) is insufficient to separate PEG from nanomicelles, TAT can be shielded by the steric resistance of PEG during systemic circulation to avoid nonspecific interaction with normal cells. The GSH content in tumor tissues is four times higher compared with normal tissues [28]. Therefore, the PEG shells of DOX-TAT-CCLMs were removed in response to a high concentration of GSH in tumor tissues, and the exposed TAT efficiently mediated the uptake of DOX-TAT-CCLMs. After core cross-linking was achieved with the addition of Fe3+, the DOX-TAT-CCLMs were stable enough to maintain the nanostructure following PEG detachment by the redox tumor environment. Once the micelles were internalized into tumor cells, the coordination between catechol and Fe3+ was modified from bis-complexes to mono-complexes under acidic conditions in lysosomes, thus releasing DOX accurately (Fig. 1).
P(ae-Asp) was obtained from the complete ring-opening of PSI with ethylenediamine, where PSI was synthesized as previously described [29]. mPEG-SS-COOH was synthesized from mPEG-NH2 and 3,3'-dithiodipropionic acid. Details have been provided in the supplementary information. mPEG-SS-COOH was activated to produce mPEG-SS-NHS to improve the reaction efficiency between mPEG-SS-COOH and P(aeAsp). Briefly, a mixture of mPEG-SS-COOH (1.0 g, 0.4 mmol), NHS (0.23 g, 2 mmol), and EDC·HCl (0.38 g, 2 mmol) in DCM (5 mL) was stirred for 12 h at 25℃. When the reaction was completed, the reaction solution was concentrated under vacuum to remove DCM. Subsequently, P(ae-Asp) (0.25 g, 1.6 mmol) and the obtained residue was dissolved in distilled water (5 mL) and stirred for 12 h. Then, the obtained mixture was dialyzed with distilled water for three days (molecular weight cutoff (MWCO) 5000 Da). Finally, mPEG-SS-g-P(ae-Asp) was obtained from the dialysate by filtration and freeze-drying. 2.2. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA 6-Maleimidocaproic acid (MCA) (0.11 g, 0.5 mmol) was dissolved in DMF (5 mL). Next, NHS (0.07 g, 0.6 mmol) and EDC·HCl (0.12 g, 0.6 mmol) were added to the above solution and stirred for 2 h under nitrogen atmosphere. To the resulting solution, mPEG-SS-g-P(ae-Asp) (0.5 g, 1 mmol) was added and then stirred for another 12 h at 25℃. Next, the resulting mixture was dialyzed with distilled water for three days (MWCO 5000 Da), and then filtered and freeze-dried to obtain mPEG-SS-g-P(ae-Asp)-MCA. 2.3. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA-DA mPEG-SS-g-P(ae-Asp)-MCA (0.47 g, 0.5 mmol) was dissolved in DMF (5 mL). Next, 3,4-dihydroxyphenylacetic acid (DOPA) (0.17 g, 1 mmol) and EDC·HCl (0.12 g, 1 mmol) was added to the above solution and stirred using a magnetic stirrer for 12 h under nitrogen atmosphere. The resulting mixture was dialyzed with distilled water for three days (MWCO 5000 Da), and then filtered and freeze-dried to obtain mPEGSS-g-P(ae-Asp)-MCA-DA. 2.4. Synthesis of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT A mixture of TAT (12 mg, 7 mmol) and mPEG-SS-g-P(ae-Asp)-MCADA (0.2 g, 1.3 mmol) in DMF (8 mL) was slowly stirred at 25℃ for 12 h in a sealed flask. The resultant solution was purified by dialysis with distilled water for three days (MWCO 5000 Da), and freeze-dried to obtain mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. 2.5. Preparation of micelles For the preparation of micelles, mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT (20 mg) was thoroughly dissolved in DMSO (0.6 mL). Then, the solution was slowly added to stirring Milli-Q water (5 mL). After stirring for another 1 h, the solution was purified by dialysis with deionized water (MWCO 3500 Da). Finally, the uncross-linked micellar (TAT-UCLMs) solution was obtained. The core cross-linked micelles (TAT-CCLMs) were prepared by adding the appropriate 40 mM FeCl3 solution into the TAT-UCLMs solution (molar ratio of catechol:Fe3+ = 2:1). Next, the pH of the mixture was adjusted to 7.4 using the phosphate buffer (2 M) to induce Fe3+-catechol bis-complex formation [30–32]. After 2 h of reaction, the mixture was dialyzed to remove the extra ferric ions. Ultimately, the TAT-UCLMs solution and the TAT-CCLMs solution were reduced to the same concentration (2 mg/mL). Referring to the preparation method of TAT-CCLMs, the core crosslinked micelles without penetrating peptide (CCLMs) were prepared as the control group using mPEG-SS-g-P(ae-Asp)-MCA-DA.
2. Experimental L-aspartic acid, diisopropyl azodiformate (DIAD), N-hydroxysuccinimide (NHS), GSH, 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride (EDC·HCl), and 3,3'-dithiodipropionic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Methoxy polyethylene glycol (mPEG, molecular weight: 2000 Da) was obtained from Vita Chemical Reagent Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) were purified prior to use. TAT (CYGRKKRRQRRR) (95 %) was purchased from ChinaPeptides Co., Ltd. (Suzhou, China). 2
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Fig. 1. Illustration of DOX-TAT-CCLMs self-assembled by mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT and lysosomal pH-triggered drug release.
after the removal of the PEG shell. Therefore, we added GSH to simulate the reductive environment of the tumor tissue. To achieve this, the TATCCLMs solution was mixed with GSH (10 mM) in PBS buffer and vibrated on a shaker at 37℃. The nanoparticle size changes of micelles were measured by DLS at different time points.
2.6. Characterization of micelles We characterized the micelle morphology using transmission electron microscopy (TEM) after the samples were dried on the copper mesh. Dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Limited, UK) was employed to measure the average size and the critical aggregation concentration (CAC) of the micelles [33].
2.9. Release profiles of DOX from micelles in vitro
2.7. Fabrication of DOX-loaded nanomicelles
The release characteristics of DOX-loaded micelles in vitro were evaluated in phosphate buffers (pH 5.0 or pH 7.4). Firstly, each dialysis tube (MWCO 1000 Da) was filled with 1 mL of DOX-TAT-CCLMs solution. Next, they were immersed in a 20 mL release medium and shaken at 37°C. At a preset time point, 200 μL medium was removed for the measurement of fluorescence intensity, and 200 μL of fresh phosphate buffer was added to the release medium [34]. Finally, a microplate reader (SpectraMax M5, Molecular Devices) was employed to determine the concentration of DOX in the samples at 595 nm.
mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT (20 mg) was completely dissolved in DMSO (0.6 mL). To this solution, DOX·HCl and triethylamine (40 μL) were added and stirred for 4 h. Then, the above solution was slowly added to the stirred ultra-pure water (6 mL). After stirring for another 1 h, the solution was dialyzed with deionized water to remove DMSO and extra DOX·HCl (MWCO 3500 Da). Next, the DOX-loaded uncross-linked micelle (DOX-TAT-UCLMs) solution was obtained. The DOX-loaded core cross-linked micelle (DOX-TAT-CCLMs) solution was prepared by adding the appropriate 40 mM FeCl3 solution to the DOXTAT-UCLMs solution (molar ratio of catechol: Fe3+ = 2:1). Then, the pH of the mixture was adjusted to 7.4 using the phosphate buffer (2 M) to induce Fe3+-catechol bis-complex formation. After 2 h of reaction, the mixture was dialyzed to remove extra Fe3+. Ultimately, the DOXTAT-CCLMs solution was obtained. Using the preparation method of DOX-TAT-CCLMs, the DOX-loaded micelles group without penetrating peptide (DOX-CCLMs) was prepared as the control. UV–vis spectroscopy was used to determine the drug-loading content (DLC) and drug-loading efficiency (DLE). First, the DOX-TATCCLMs and DOX-CCLMs solutions were lyophilized to obtain a solid red powder. Then, the solid red powder was dissolved in DMSO and the absorbance at 480 nm was determined using UV–vis spectroscopy. A fitting calibration curve was obtained by dissolving different formulations of DOX in DMSO. DLC and DLE were calculated using formulas (1) and (2).
DLC% =
weight of loaded drug × 100% weight of drug loaded micelle
(1)
DLE% =
weight of loaded drug × 100% weight of drug in feed
(2)
2.10. Cellular uptake evaluation and DOX efflux assay MCF-7 cells and MCF-7/ADR cells were grown in 24-well plates (1 × 105 cells/well) for 24 h, respectively. Afterward, the medium was drained and replaced with fresh medium (with or without 10 mM GSH) containing DOX-loaded micelles or DOX·HCl (DOX concentration: 1 μg/ mL). After 4 h of incubation, the medium was drained, and the plates were washed with PBS for 3 times. Finally, an inverted fluorescence microscope (Olympus, TH4-200 with Olympus UHGLGPS) was employed to capture the fluorescence pictures. The cellular uptake efficiency of DOX-TAT-CCLMs was measured with a fluorescence detector (SpectraMax M5, Molecular Devices) [35,36]. The specific process is as follows: firstly, MCF-7/ADR cells and MCF-7 cells were grown in 6-well plates (2 × 105 cells/well), respectively. After 24 h of incubation, replace the old medium with fresh medium (with 10 mM GSH) containing DOX-loaded micelles or DOX·HCl (DOX concentration: 5 μg/mL). After incubation for another 2, 4, or 8 h, for quantitative analysis of cellular uptake efficiency, cell lysis buffer (0.01 M HCl, 10 % sodium dodecyl sulfonate and 5 % isobutyl alcohol) was added to break the cell structure after removing DOXloaded micelles and DOX·HCl solutions. The fluorescence emission intensity at each time point was determined at 595 nm with a microplate reader (SpectraMax M5, Molecular Devices) to calculate cellular uptake efficiency. In the case of DOX efflux assays, MCF-7/ADR cells were grown in 24-well plates. Twenty-four hours later, fresh medium (with 10 mM
2.8. Reduction-sensitized micelles in vitro According to the design, TAT in nanomicelles can only function 3
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aspartic acid. Subsequently, the five-membered rings of PSI were completely opened with ethylenediamine, which was confirmed by the disappearance of the peak at 5.25 ppm (Fig. S3). As shown in Fig. S4A, the appearance of the peak at 3.23 ppm indicated the successful synthesis of polymer mPEG-SS-g-P(ae-Asp). According to the following formula (4), the grafting degree (DG) of mPEG was 20 %.
GSH) containing DOX-TAT-CCLMs or DOX HCl (DOX concentration: 10 μg/mL) replaced the old medium. After incubation for 4 h, the DOXTAT-CCLMs and DOX·HCl solutions were drained out and then washed with PBS. Subsequently, fresh medium was added to each well and incubated for another 1 and 4 h. To quantify the DOX mass pumped from the cells at each time point, a fluorescence detector (SpectraMax M5, Molecular Devices) was used to determine the fluorescence emission intensity of the medium at 595 nm. The DOX mass accumulated inside the cells was determined following incubation with the corresponding dose of DOX.
DG = S1/(3S2)
In the formula, S2 is the integral area of 4.34–4.75 ppm assigned to methylene protons in the aspartic acid unit, and S1 is the integral area of terminal methyl protons at 3.22–3.25 ppm in PEG. Fig. S4B shows the 1H-NMR spectrum of mPEG-SS-g-P(ae-Asp)MCA-DA. The appearance of peaks at 7.0 ppm and 6.43–6.68 ppm was attributed to the maleimide groups of MCA and the protons of catechols, respectively. The DG of DOPA and MCA were 7 % and 14 %, respectively, determined using the following formula:
2.11. In vitro cytotoxicity assays To perform the in vitro cytotoxicity assays, MCF-7/ADR cells and MCF-7 cells were grown in 96-well plates (5000 cells/well), respectively. Twenty-four hours later, the culture medium was removed and 180 μL of fresh medium (with 10 mM GSH) was added. Subsequently, the culture medium containing different concentrations of DOX-loaded micelles (20 μL) was filled into the wells and incubated for 72 h. Then, 10 μL of MTT solution (5 mg/mL) was added to each well after the removal of 80 μL of culture media. After 4 h of incubation, a pyrolysis solution (50 mL/well) was added to dissolve the crystals. An automatic BIO-TEK microplate reader (Powerwave XS, USA) was employed to measure the absorbance of each well. The cells incubated with 200 μL medium were employed as a control (ODcontrol). The culture media without cells was employed as a blank (ODblank). Cell viability was calculated according to formula (3).
Cell viability% =
ODsample - ODblank × 100% ODcontrol - ODblank
(4)
DG = S3/(2S2)
(5)
DG = S4/(2S2)
(6)
where S3 is the integral area of the maleimide groups of MCA, and S4 is the integral area of the protons of catechols. Finally, TAT was grafted onto mPEG-SS-g-P(ae-Asp)-MCA-DA via the Michael addition reaction. Fig. S4C shows the 1H-NMR spectrum of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT. The appearance of TAT characteristic peaks indicated that TAT was successfully grafted onto the copolymers. The 1H-NMR spectrum of TAT is shown in Fig. S2B. 3.2. Fabrication and characterization of nanomicelles
(3)
DLS and TEM were utilized for further analysis of the particle size and observation of the morphology of TAT-UCLMs and TAT-CCLMs. Fig. S5A and S5C demonstrate that the TAT-UCLMs were exhibited as nano-spheres with an average size of 188.7 nm (PDI: 0.166). As shown in Fig. S5B, the TAT-CCLMs morphology was observed as spherical nanomicelles with enhanced contrast in the center of the TAT-CCLMs, which could be attributed to the high atomic amount of iron. As shown in Fig. S5D, the average size of TAT-CCLMs was 180.1 nm (PDI: 0.156). The particle size measured by DLS was larger than that measured by TEM, which may be attributed to the shrinkage of the hydrophilic PEG shell during TEM sample preparation. It is well known that CAC plays an important role in micellization. Polymer micelles as drug carriers should possess lower CAC values to resist extensive blood dilution in the systemic circulation. Here, DLS was employed to determine the CAC values. The CAC value of mPEGSS-g-P(ae-Asp)-MCA-DA-TAT micelles was determined as 1.41 × 10−2 mg/mL (Fig. S6). The lower CAC value indicated that the nanomicelles have the potential to maintain the integrity of nanostructures during blood circulation. In addition, sample dilution experiments were also performed. As shown in Fig. S7, the size distribution of TAT-UCLMs and TAT-CCLMs diluted 100 times with PBS. Fig. S7A demonstrates that the diluted TAT-UCLMs sample was unsuitable for DLS measurement. In contrast, the average size of TAT-CCLMs was 238.7 nm (Fig. S7B), and its size distribution was relatively narrow (PDI: 0.355). This suggested that TAT-CCLMs have the potential to withstand dilution to avoid disassembly of nanomicelles and premature drug release during in vivo circulation.
2.12. In vivo biodistribution For biodistribution analysis, the tumor model was established by subcutaneously injecting about 1.0 × 106 4T1 cells into the right upper armpit of female BALB/C mice (4–6 weeks old, weighing approximately 20 g). When tumor volumes reached about 400–500 mm3, the tumorbearing mice were randomly divided into three groups (n = 3) and intravenously administered DOX-TAT-CCLMs, DOX-CCLMs, and free DOX at a dose of 5 mg DOX/kg body weight per mouse. The mice were executed post 24 h of administration. Tumors and main organs, including heart, liver, spleen, lung, and kidney were excised carefully and washed with saline, weighed after drying, and homogenized in 1 mL mixture of water and DMSO (V/V: 1/1). The supernatant fractions were collected, and the concentration of DOX in the samples was determined using a microplate reader (SpectraMax M5, Molecular Devices) at 595 nm. 2.13. In vivo antitumor efficacy To further evaluate the anticancer efficacy of the DOX-TAT-CCLMs in vivo, 4T1 cells were subcutaneously injected into the right upper armpit of female BALB/C mice (1 × 106 cells per mouse). When tumors reached to a volume of approximately 30 mm3 (determined as day 0), mice were randomly divided into five groups (n = 4). Then 4T1 tumor bearing mice were injected through the tail vein with saline, TATCCLMs, DOX-CCLMs, DOX-TAT-CCLMs, or free DOX at 3-day intervals (0, 3, 6, and 9 days, and DOX dosage: 3 mg/kg).
3.3. pH-Sensitive coordination between catechol and Fe3+ 3. Results and discussion The pH-sensitive coordination of catechol and Fe3+ was determined by UV–vis spectroscopy under different pH values. As shown in Fig. 3A, the absorption peak at 470 nm was attributed to the formation of triscomplexes between catechol and ferric ions at pH 10.0. The UV absorption peak shifted to 540 nm and 590 nm when the pH values were modified to 7.4 and 5.0, indicating that catechol and Fe3+ formed bis-
3.1. Synthesis and characterization of mPEG-SS-g-P(ae-Asp)-MCA-DATAT Fig. 2 represents the detailed synthetic route of mPEG-SS-g-P(aeAsp)-MCA-DA-TAT. Firstly, PSI was synthesized by condensation of L4
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Fig. 2. The detailed synthetic route of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT.
fracture. Additionally, another size distribution of 100−200 nm was assigned to the core cross-linked TAT-CCLMs after the addition of GSH in nanomicelles. The above results implied that the PEG shell of nanomicelles can be removed in the redox environment of tumor tissue, while TAT-CCLMs could still exist stably due to the cross-linking of catechol with Fe3+.
complexes and mono-complexes, respectively. In addition, the pH-responsive variations of coordination between catechol and Fe3+ were further confirmed by obvious color changes of the complex (Fig. 3B). The color of the TAT-CCLMs solution was orange at pH 10.0, changed to violet at pH 7.4, and pale yellow at pH 5.0. 3.4. Reduction-sensitivity of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT micelles
3.5. Drug encapsulation and drug release in vitro The disulfide bonds in mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT were susceptible to the tumor redox environment. This is helpful for the tumor tissue-specific detachment of the PEG shell and the exposure of TAT. DLS was employed to measure the size change in nanomicelles in response to GSH (10 mM) in PBS buffer (pH 7.4). As shown in Fig. 3C, nanoparticles with a diameter of about 20 nm appeared after the addition of GSH to the nanomicelles for 20 min. After 200 min, the small particles increased slightly. The appearance of small particles can probably be attributed to the peeling of PEG shells due to disulfide bond
DOX was loaded in TAT-CCLMs via the dialysis method. According to the design, the synthesized amphiphilic polymer self-assembled in water. Owing to the hydrophobic interaction between the DA segment and hydrophobic DOX, DOX was enfolded in the hydrophobic core of nanomicelles. DLC and DLE of DOX were measured as 10.18 ± 0.24 % and 61.16 ± 1.65 %, respectively. The TEM image shows the spherical shape of DOX-TAT-CCLMs (Fig. S8A). As shown in Fig. S8B, the particle size of DOX-TAT-CCLMs was 251.7 nm, with a narrow PDI of 0.297. It is
Fig. 3. (A) Determination of pH-responsive coordination between catechol with Fe3+ by UV–vis. (B) Color changes under diverse pH values. (C) Size change of TATCCLMs in response to GSH (10 mM). 5
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Fig. 4. (A) Drug release curve of DOX-TAT-CCLMs at different pH values. (B) Drug release curve of DOX-TAT-UCLMs and DOX-TAT-CCLMs at pH 7.4.
S10, in MCF-7/ADR and MCF-7 cells, no differences were observed between the cellular internalization of DOX-CCLMs and DOX-TATCCLMs without the addition of GSH. This result validated that the cellpenetration effect of TAT could be effectively shielded by the PEG shell with a grafting ratio of 20 %, which is in line with results reported by Kuai et al. [16]. Conversely, as demonstrated in Fig. 6, the fluorescent intensity of DOX in MCF-7/ADR cells treated with DOX-TAT-CCLMs and 10 mM GSH was much higher than that without GSH. This phenomenon could be due to the removal of the PEG shell in DOX-TATCCLMs in response to GSH, contributing to the exposure of TAT and subsequent facilitated cellular internalization of nanomicelles. Furthermore, it is worth noting that the addition of GSH or not had no influence on the cellular internalization of DOX·HCl. Similar cellular uptake results of DOX·HCl and DOX-TAT-CCLMs were observed in MCF7 cells (Fig. S11). The cellular internalization of DOX·HCl, DOX-CCLMs, and DOX-TAT-CCLMs was further quantified by the fluorescent intensity measurement of DOX. As shown in Fig. 7A, compared with DOX·HCl and DOX-CCLMs, DOX-TAT-CCLMs were abundantly internalized into MCF-7/ADR cells. Specifically, after incubation for 8 h, the mean fluorescence intensity of DOX-TAT-CCLMs in MCF-7/ADR cells was 8 times higher than that of DOX·HCl. These results verified that the enhancement of TAT can remarkably increase the tumor cell intracellular accumulation of DOX. Similar to MCF-7/ADR cells, the DOXTAT-CCLMs also showed the highest mean fluorescence intensity of DOX in MCF-7 cells compared with DOX·HCl and DOX-CCLMs (Fig. S12A). In order to demonstrate that the low fluorescent intensity of
obvious that the particle diameter of DOX-TAT-CCLMs was larger than TAT-CCLMs, resulting from the encapsulation of DOX in micelles. The stability of DOX-TAT-CCLMs was determined by DLS measurement after dilution in fetal bovine serum (FBS; HyClone, Logan, UT) at 37℃. As shown in Fig. S9, the size of DOX-TAT-CCLMs was not significantly altered within 4 days. The results showed that DOX-TAT-CCLMs was relatively stable in FBS for 4 days. The in vitro drug release analysis of DOX-TAT-CCLMs was performed by the dialysis method. It has been anticipated that the core crosslinked DOX-TAT-CCLMs could minimize premature leakage of DOX in blood circulation while accomplishing pH-triggered DOX release in cancer cells. As shown in Fig. 4A, the core cross-linked DOX-TATCCLMs exhibited a pH-dependent drug release profile. Since the coordination ratio between catechol and ferric ions reduced from 2 (at pH 7.4) to 1 (at pH 5.0), DOX release was more favorable at pH 5.0 than at pH 7.4. Furthermore, the drug release behavior of DOX-TAT-UCLMs at pH 7.4 was evaluated. Fig. 4B demonstrates the rapid release of DOX from the DOX-TAT-UCLMs compared with DOX-TAT-CCLMs. All these results demonstrated that the DOX-TAT-CCLMs could effectively constrain DOX in the cross-linked core of nanomicelles during systemic circulation, while effectively releasing DOX in the acidic lysosomal environment. 3.6. Cellular uptake and DOX efflux assay DOX emits red fluorescence when stimulated, which can be utilized to measure the cellular uptake efficiency. As exhibited in Figs. 5 and
Fig. 5. Fluorescence images of the MCF-7/ADR cells incubated with DOX-CCLMs and DOX-TAT-CCLMs without GSH treatment (incubation for 4 h, bar represents 50 μm). 6
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Fig. 6. Fluorescence images of the MCF-7/ADR cells incubated with DOX-TAT-CCLMs and DOX·HCl with or without GSH treatment (incubation for 4 h, bar represents 50 μm).
Fig. 7. (A) Mean fluorescence intensity of MCF-7/ADR cells incubated with DOX·HCl, DOX-TAT-CCLMs, and DOX-CCLMs at 2, 4, and 8 h. (B) DOX efflux percentage of MCF-7/ADR cells incubated with DOX·HCl and DOX-TATCCLMs. (C) Survival rate of MCF-7/ADR cells cultured in different concentrations of blank micelles for 72 h. (D) Inhibitory activity on MCF-7/ADR cells in different groups (DOX·HCl, DOX-TAT-CCLMs, DOX-CCLMs). Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
7
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Fig. 8. (A) Quantitative evaluation of in vivo biodistribution of different formulations in tumors and main organs normalized with organ or tumor weights (post 24 h of administration). (B) 4T1 tumor growth curves after intravenous administration of five types of formulations. (C) Photos of tumor tissues of five groups on day 15 after intravenous injection. (D) The quantitative summary of tumor weights after treatment with five different formulations on day 15. (E) The body weight variations of 4T1 tumor-bearing mice during treatment. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
DOX·HCl in MCF-7/ADR cells was caused by drug efflux, DOX efflux analysis was performed with 10 μg/mL DOX. As shown in Fig. 7B, the DOX efflux against MCF-7/ADR cells was performed in a time-dependent manner and the DOX efflux ratio increased to 25 % within 4 h. However, the DOX-TAT-CCLMs showed a rather low DOX efflux ratio of 4.5 %. These results indicated that the DOX·HCl failed to efficiently enter into multidrug-resistant MCF-7/ADR cells owing to the high expression of drug efflux pumps such as P-glycoprotein [37]. In contrast to DOX·HCl, the DOX-TAT-CCLMs bypassed the drug efflux pumps, thereby significantly increasing DOX accumulation in MCF-7/ADR cells.
value higher than 160 μg/mL, suggesting TAT plays an essential role in the anti-proliferation of multidrug-resistant MCF-7/ADR cells. Notably, the IC50 value of DOX-TAT-CCLMs against the MCF-7/ADR cells was about 4 times lower than that of DOX·HCl, which could be attributed to the greatly increased accumulation of DOX-TAT-CCLMs in cells revealed in the cellular uptake analysis. Collectively, TAT modification generated DOX-TAT-CCLMs exhibiting a remarkable antiproliferative effect in MCF-7/ADR cells.
3.7. In vitro cytotoxicity assays
As presented in Fig. 8A, after administration for 24 h, free DOX showed a rather low accumulation at the tumor site compared to both DOX-loaded micelles. This was mainly attributed to the enhanced permeability and retention effect of DOX-loaded micelles. Indeed, DOXTAT-CCLMs are more abundant in the tumor site than DOX-CCLMs, which could be attributed to the TAT exposure facilitated cellular internalization of nanomicelles. For in vivo antitumor efficacy, as shown in Fig. 8B, tumor volume was altered with time. On day 15, the tumor volumes of the groups injected with saline, TAT-CCLMs, DOX·HCl, DOX-CCLMs, and DOXTAT-CCLMs were 768 ± 105 mm3, 744 ± 84 mm3, 458 ± 42 mm3, 342 ± 39 mm3, and 172 ± 41 mm3, respectively (Fig. 8B). The free DOX group showed a certain degree of tumor growth suppression, but its inhibitory performance was inferior compared to both DOX-loaded micelles (Fig. 8C). This was mainly due to the low accumulation of small molecules (DOX) in the tumor. On day 15, the mean tumor weight of the groups injected with saline, TAT-CCLMs, DOX·HCl, DOX-CCLMs, and DOX-TAT-CCLMs were 0.80 ± 0.19 g, 0.78 ± 0.13 g, 0.47 ± 0.05 g, 0.37 ± 0.03 g, and 0.19 ± 0.05 g, respectively (Fig. 8D). Unequivocally, the DOX-TAT-CCLMs group demonstrated the best in vivo antitumor efficacy among three treatment groups, which could be attributed to the greatly increased accumulation of DOX-TATCCLMs in the tumor site as indicated in the biodistribution analysis. As shown in Fig. 8E, the body weight of the DOX HCl group decreased during the injection period, indicating severe systemic toxicity. However, the weight of the DOX-TAT-CCLMs group was relatively stable, demonstrating lower side effects and improved drug delivery efficiency.
3.8. In vivo biodistribution and antitumor efficacy
The above results demonstrated that DOX-TAT-CCLMs micelles could enhance DOX uptake, reduce drug efflux, and site-specifically release DOX in the acidic lysosomal environment. It is expected that DOX-TAT-CCLMs could exert high anti-proliferative efficacy against cancer cells. Firstly, an in vitro MTT assay was performed to determine the toxicity of the carriers. MCF-7/ADR and MCF-7 cells were treated with different concentrations of CCLMs and TAT-CCLMs for 72 h, respectively. As shown in Figs. S12B and 7 C, after treatment with blank micelles, the cell viability of MCF-7 as well as MCF-7/ADR cells was higher than 85 %, confirming the negligible toxicity of the carriers. Subsequently, the cytotoxicity of DOX-TAT-CCLMs was evaluated using DOX·HCl and DOX-CCLMs as controls. As shown in Fig. S12C, when the DOX concentration was lower than 2.5 μg/mL, the cytotoxicity of DOXTAT-CCLMs against MCF-7 cells was slightly inferior to that of DOX·HCl. However, when the DOX concentration increased to 5 μg/mL, the cytotoxicity of DOX-TAT-CCLMs was comparable to that of DOX·HCl. Furthermore, the IC50 values of DOX-TAT-CCLMs, DOX HCl, and DOX-CCLMs were 0.71 ± 0.04 μg/mL, 0.31 ± 0.05 μg/mL, and 4.12 ± 0.3 μg/mL, respectively. Collectively, DOX-TAT-CCLMs demonstrated superior in vitro antitumor activity compared to DOXCCLMs. Furthermore, compared with DOX and DOX-CCLMs, DOX-TATCCLMs showed higher cytotoxicity in MCF-7/ADR cells (Fig. 7D). After 72 h of incubation, the IC50 values of DOX-TAT-CCLMs and DOX·HCl against the MCF-7/ADR cells were 11.61 ± 0.95 μg/mL and 42.77 ± 1.93 μg/mL, respectively. DOX-CCLMs exhibited an IC50 8
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4. Conclusion [11]
In this study, TAT-modified core cross-linked nanomicelles with a redox-sheddable PEG shell were fabricated by the self-assembly of mPEG-SS-g-P(ae-Asp)-MCA-DA-TAT as the delivery carrier for DOX. The nanomicelles resisted excessive dilution and achieved lysosomal pH-triggered intracellular drug release due to the core cross-linking between Fe3+ and catechol. In vitro studies demonstrated that the multifunctional DOX-TAT-CCLMs significantly enhanced the cellular internalization and prominently reduced the drug efflux of DOX against the multidrug-resistant MCF-7/ADR cells. Compared with DOX·HCl, DOX-TAT-CCLMs exerted a higher antiproliferative effect on MCF-7/ ADR cells, with an IC50 value nearly 4 times lower than DOX HCl. Furthermore, the DOX-TAT-CCLMs showed good suppression of tumor growth in the 4T1 tumor-bearing mouse model. In summary, the DOXTAT-CCLMs have great potential to increase drug delivery efficiency and overcome multidrug resistance during cancer therapy.
[12] [13] [14]
[15]
[16]
[17]
[18]
Author contributions [19]
Yuliu Zhang designed the study, analysed the data, and drafted the manuscript. Yi Xiao and Yushu Huang conceived the study and revised the manuscript. Yuliu Zhang, Yushu Huang and Yang He performed the experiments. Yuliu Zhang, Yang He, Yanyun Xu and Wei Lu analysed the data. Jiahui Yu is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors reviewed the manuscript.
[20]
[21]
[22]
[23]
Declaration of Competing Interest [24]
None.
[25]
Acknowledgment [26]
The research work was supported by the National Natural Science Foundation of China (81871405, 51573050).
[27]
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.colsurfb.2020.110772.
[28]
References
[29]
[1] J. Liu, M. Li, Z. Luo, L. Dai, X. Guo, K. Cai, Design of nanocarriers based on complex biological barriers in vivo for tumor therapy, Nano Today 15 (2017) 56–90. [2] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nat. Biotechnol. 33 (2015) 941–951. [3] M. Gu, X. Wang, T.B. Toh, E.K. Chow, Applications of stimuli-responsive nanoscale drug delivery systems in translational research, Drug Discov. Today 23 (2018) 1043–1052. [4] T. Ramasamy, H.B. Ruttala, B. Gupta, B.K. Poudel, H.G. Choi, C.S. Yong, J.O. Kim, Smart chemistry-based nanosized drug delivery systems for systemic applications: a comprehensive review, J. Control. Release 258 (2017) 226–253. [5] M. Mohammadi, M. Ramezani, K. Abnous, M. Alibolandi, Biocompatible polymersomes-based cancer theranostics: towards multifunctional nanomedicine, Int. J. Pharm. 519 (2017) 287–303. [6] Y. Li, M. Kroger, W.K. Liu, Endocytosis of PEGylated nanoparticles accompanied by structural and free energy changes of the grafted polyethylene glycol, Biomaterials 35 (2014) 8467–8478. [7] L. Mo, J.G. Song, H. Lee, M. Zhao, H.Y. Kim, Y.J. Lee, H.W. Ko, H.K. Han, PEGylated hyaluronic acid-coated liposome for enhanced in vivo efficacy of sorafenib via active tumor cell targeting and prolonged systemic exposure, Nanomedicine 14 (2018) 557–567. [8] T. Lang, X. Dong, Z. Zheng, Y. Liu, G. Wang, Q. Yin, Y. Li, Tumor microenvironment-responsive docetaxel-loaded micelle combats metastatic breast cancer, Sci. Bull. 64 (2019) 91–100. [9] T. Lammers, F. Kiessling, W.E. Hennink, G. Storm, Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress, J. Control. Release 161 (2012) 175–187. [10] H. Kim, M. Kitamatsu, T. Ohtsuki, Enhanced intracellular peptide delivery by
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
9
multivalent cell-penetrating peptide with bioreducible linkage, Bioorg. Med. Chem. Lett. 28 (2018) 378–381. W.B. Kauffman, T. Fuselier, J. He, W.C. Wimley, Mechanism matters: a taxonomy of cell penetrating peptides, Trends Biochem. Sci. 40 (2015) 749–764. F. Madani, S. Lindberg, U. Langel, S. Futaki, A. Graslund, Mechanisms of cellular uptake of cell-penetrating peptides, J. Biophys. 2011 (2011) 414729. J.D. Ramsey, N.H. Flynn, Cell-penetrating peptides transport therapeutics into cells, Pharmacol. Ther. 154 (2015) 78–86. J. Zhang, Y. Zheng, X. Xie, L. Wang, Z. Su, Y. Wang, K.W. Leong, M. Chen, Cleavable multifunctional targeting mixed micelles with sequential pH-triggered TAT peptide activation for improved antihepatocellular carcinoma efficacy, Mol. Pharm. 14 (2017) 3644–3659. L. Zhu, T. Wang, F. Perche, A. Taigind, V.P. Torchilin, Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cellpenetrating moiety, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 17047–17052. W.Y. Rui Kuai, Yao Qin, Huali Chen, Jie Tang, Mingqing Yuan, Zhirong Zhang, Qin He, Efficient delivery of payload into tumor cells in a controlled manner by TAT and thiolytic cleavable PEG Co-modified liposomes, Mol. Pharceut. 7 (2010) 1816–1826. E.S. Lee, Z. Gao, D. Kim, K. Park, I.C. Kwon, Y.H. Bae, Super pH-sensitive multifunctional polymeric micelle for tumor pH(e) specific TAT exposure and multidrug resistance, J. Control. Release 129 (2008) 228–236. S.M. Farkhani, A. Valizadeh, H. Karami, S. Mohammadi, N. Sohrabi, F. Badrzadeh, Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules, Peptides 57 (2014) 78–94. S.C. Owen, D.P.Y. Chan, M.S. Shoichet, Polymeric micelle stability, Nano Today 7 (2012) 53–65. M. Talelli, M. Barz, C.J. Rijcken, F. Kiessling, W.E. Hennink, T. Lammers, Corecrosslinked polymeric micelles: principles, preparation, biomedical applications and clinical translation, Nano Today 10 (2015) 93–117. Y.-B. Long, W.-X. Gu, C. Pang, J. Ma, H. Gao, Construction of coumarin-based crosslinked micelles with pH responsive hydrazone bond and tumor targeting moiety, J. Mater. Chem. B 4 (2016) 1480–1488. J. Ren, Y. Zhang, J. Zhang, H. Gao, G. Liu, R. Ma, Y. An, D. Kong, L. Shi, pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery, Biomacromolecules 14 (2013) 3434–3443. H. He, Y. Bai, J. Wang, Q. Deng, L. Zhu, F. Meng, Z. Zhong, L. Yin, Reversibly crosslinked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity, Biomacromolecules 16 (2015) 1390–1400. R. Mrowczynski, Polydopamine-based multifunctional (Nano)materials for cancer therapy, ACS Appl. Mater. Interfaces 10 (2018) 7541–7561. N. Holten-Andersen, M.J. Harrington, H. Birkedal, B.P. Lee, P.B. Messersmith, K.Y. Lee, J.H. Waite, pH-induced metal-ligand cross-links inspired by mussel yield self-healing polymer networks with near-covalent elastic moduli, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 2651–2655. B.J. Kim, H. Cheong, B.H. Hwang, H.J. Cha, Mussel-inspired protein nanoparticles containing Iron(III)-DOPA complexes for pH-responsive drug delivery, Angew. Chem. Int. Ed. Engl. 54 (2015) 7318–7322. Y. Huang, Y. Xu, Y. Wu, T. Chen, W. Lu, J. Yu, Bioinspired nanoplatform for enhanced delivery efficiency of doxorubicin into nucleus with fast endocytosis, lysosomal pH-triggered drug release, and reduced efflux, Colloids Surf. B Biointerfaces 183 (2019) 110413. H.L. Periannan Kuppusamy, Govindasamy Ilangovan, Arturo J. Cardounel, Jay L. Zweier, Kenichi Yamada, Murali C. Krishna, James B. Mitchell, Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels, Cancer Res. 62 (2002) 307–312. Y. Zhao, H. Su, L. Fang, T. Tan, Superabsorbent hydrogels from poly(aspartic acid) with salt-, temperature- and pH-responsiveness properties, Polymer 46 (2005) 5368–5376. K. Xin, M. Li, D. Lu, X. Meng, J. Deng, D. Kong, D. Ding, Z. Wang, Y. Zhao, Bioinspired coordination micelles integrating high stability, triggered cargo release, and magnetic resonance imaging, ACS Appl. Mater. Interfaces 9 (2017) 80–91. A. Ghadban, A.S. Ahmed, Y. Ping, R. Ramos, N. Arfin, B. Cantaert, R.V. Ramanujan, A. Miserez, Bioinspired pH and magnetic responsive catechol-functionalized chitosan hydrogels with tunable elastic properties, Chem. Commun. (Camb.) 52 (2016) 697–700. G.H. Hwang, K.H. Min, H.J. Lee, H.Y. Nam, G.H. Choi, B.J. Kim, S.Y. Jeong, S.C. Lee, pH-responsive robust polymer micelles with metal-ligand coordinated core cross-links, Chem. Commun. (Camb.) 50 (2014) 4351–4353. Ö. Topel, B.A. Çakır, L. Budama, N. Hoda, Determination of critical micelle concentration of polybutadiene-block-poly(ethyleneoxide) diblock copolymer by fluorescence spectroscopy and dynamic light scattering, J. Mol. Liq. 177 (2013) 40–43. N. Li, W. Lu, J. Yu, Y. Xiao, S. Liu, L. Gan, J. Huang, Rod-like cellulose nanocrystal/ cis-aconityl-doxorubicin prodrug: a fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug controlled release, Mater. Sci. Eng. C Mater. Biol. Appl. 91 (2018) 179–189. N. Li, H. Zhang, Y. Xiao, Y. Huang, M. Xu, D. You, W. Lu, J. Yu, Fabrication of cellulose-nanocrystal-based folate targeted nanomedicine via layer-by-layer assembly with lysosomal pH-controlled drug release into the nucleus, Biomacromolecules 20 (2019) 937–948. X. Wei, Y. Wang, X. Xiong, X. Guo, L. Zhang, X. Zhang, S. Zhou, Codelivery of a π-π stacked dual anticancer drug combination with nanocarriers for overcoming multidrug resistance and tumor metastasis, Adv. Funct. Mater. 26 (2016) 8266–8280. M.M. Gottesman, T. Fojo, S.E. Bates, Multidrug resistance in cancer: role of ATPdependent transporters, Nat. Rev. Cancer 2 (2002) 48–58.