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reduction-responsive polymeric conjugates for enhanced drug delivery to tumor
Materials Science & Engineering C 82 (2018) 234–243
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumor
MARK
Shudi Yanga, Ying Wanga, Zhaoxiang Renb, Mengtian Chena, Weiliang Chena, Xuenong Zhanga,⁎ a
Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou, People's Republic of China Jiangsu Key Laboratory for Translational Research and Therapy for Neuropsycho-disorders, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, People's Republic of China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Charge reversion Stimuli-responsive Smart Drug delivery Cancer therapy
In this research, a charge-conversional polymer, poly-L-lysine-lipoic acid (PLL-LA), was prepared by dimethylmaleic anhydride (DA) modification and applied as a carrier with enhanced cell internalization and intracellular pH- and reduction-triggered doxorubicin (Dox) release. The surface charge of dimethylmaleic anhydride-poly-L-lysine-lipoic acid micelles (DA-PLL-LA) was negative at physiological pH and reversed to positive at the extracellular and intracellular pH of cancer cells. At tumor extracellular pH of 6.8, the conjugates underwent a rapid charge-reversible process with almost 80% DA cleavage within 2 h, and then endocytosed into the endo/lysosomes more rapidly than at physiological pH of 7.4. The Dox/DA-PLL-LA micelles (Dox-micelles) demonstrated a sustained drug release in vitro under physiological condition, and rapid Dox release was triggered by both extracellular pH and high-concentration reducing glutathione. The Dox-micelles also exhibited enhanced internalization at extracellular pH, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Excellent tumor-penetrating efficacy was also found in A549 tumor spheroids and solid tumor slices. Moreover, the DA-PLL-LA micelles exhibited excellent tumor-targeting ability in tumor tissues and excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing mice. Therefore, the DA-PLL-LA micelles demonstrated great potential for targeted and efficient drug delivery in cancer treatments.
1. Introduction Extensive work has been done in the area of various targeted delivery carriers of chemotherapeutic anticancer drugs, such as polymeric nanoparticles, synthetic nonviral systems, supramolecular hydrogel, hybrid nanocarriers and biodegradable polymers [1–8]. Nowadays, multiple smart delivery systems for drugs and genes responded to temperature, light, pH and reactive oxygen species (ROS) were developed [9–11]. In recent years, stimuli-responsive nanoparticles have attracted high attention because of their effective delivery ability of anticancer drugs via recognizing the micro-environment of tumors [12]. The greatest advantages of nanoparticles were higher hydrophobic drugs solubility and lesser side effects. Nanoparticles also rapidly increased the accumulation in tumor tissue compared to normal tissue by the enhanced permeability and retention (EPR) effect [13–15]. However, the poor cell internalization as well as slow and incomplete intracellular drug release posed great challenges for efficient treatment; these phenomena diametrically resulted in low intracellular drug concentration below the required therapeutic level and unsatisfactory therapy efficacy [16–18]. Hence, smart stimuli-responsive ⁎
Corresponding author. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.msec.2017.08.079 Received 16 June 2017; Received in revised form 3 August 2017; Accepted 10 August 2017 0928-4931/ © 2017 Published by Elsevier B.V.
nanoparticles based on the special microenvironment of tumor tissues have been given particular attention. In the systemic circulation, nanoparticles bio-distribution, delivery and release of drugs or gene were significantly affected by the changes of physicochemical properties of nanoparticles responded to stimuli, such as size, zeta potential, solubility and structure [11], which were strongly associated with the delivery efficiency of drug [19–24]. Generally, the positively-charged nanoparticles with higher cellular internalization would be captured by the reticular endothelin system (RES) and have a strong force with the negatively-charged serum components, causing further aggregation and leading to a short blood circulation time [25–27]. To solve this contradiction, an ideal system based on a charge-reversal concept is desirable. Charge-switchable systems have been developed for prolonged circulation and improved cell uptake by the tumor microenvironment-triggered shell detachment based on the slight pH difference between extracellular acidity of tumor sites (pH ≈ 6.8) and normal tissues (pH ≈ 7.4) [28,29]. These systems could stay stable in bloodstream and avoid the non-special absorption by serum proteins. After the systems accumulated in tumor sites, the low-pH-triggered ionization and the detachment of protective moiety
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Scheme 1. Schematic illustration of the stepwise pH/reduction-responsive DA-PLLLA micelles.
methyl thiazolyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluorescamine was provided by J & K Scientific Ltd. (Beijing, China). GSH, 1-(3-dimethylaminopropyl)3-ethyl carbon carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were ordered from Aladdin Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and RPMI-1640 were purchased from Hyclone (Thermo-Fisher Biochemical Products Co., Ltd., Beijing, China). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (Dir) was provided by Baomanbio Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. Human lung adenocarcinoma cell line luc-A549 was provided from pharmaceutics teaching and research section (Suzhou, China). This cell line was maintained in RPMI-1640 media supplemented with 10% (v/ v) FBS under 5% CO2-humidified conditions at 37 °C. Female and male nude mice (BALB/C, nu/nu) (18 ± 2 g) were purchased from the Experimental Animal Center of Soochow University. All animals were raised in surroundings that obeyed the guidelines of the standard laboratory conditions of Soochow University. All animal process was performed following the protocols authorized by the Institutional Animal Care.
result in positive surface charge, which facilitates the intracellular endocytosis [30,31]. There is now a general consensus that there are significant differences between the microenvironments of tumor tissue and nearby normal cells, such as vascular distribution, pH and redox state, and numerous tumor targeted nanoparticles were exploited through recognizing them. Particularly, glutathione (GSH, 1–10 mM in tumor cell cytoplasm and 2–20 μM in blood fluids) has been extensively investigated [32,33]. Numerous studies have utilized reduction-sensitive nanocarriers containing disulfide bonds, such as dithiodipropionic acid and lipoic acid (LA). This method promoted sharp and sufficient drug release, wherein the disulfide bonds would break up in response to intracellular high concentration of GSH and further cause destabilization and cleavage of drug-loading nanocarriers. In this work, we reported a nanocarrier with high stability and sensitivity to tumor microenvironment that is sequentially activated at the extracellular and intracellular levels of tumor tissue by gradual transformation (Scheme 1). The mildly acidic microenvironment of tumor tissue was recognized by this nanoparticle and the surface charge of this nanoparticle was shifted to positive charge to strengthen cellular uptake. In addition, the nanoparticle has a chemically cross-linked core of poly-L-lysine (PLL) with excellent hydrophilicity and LA with a disulfide bond, which is cleaved of GSH, to enhance the accumulation of drugs in tumor sites at the intracellular level. The prepared stepwise pH/reduction-responsive polymeric micelles could achieve enhanced cell internalization and efficient intracellular drug release, leading to enhanced cytotoxicity [32]. In the present study, the stepwise responsive nanocarrier (DA-PLL-LA) copolymer was successfully synthesized, and doxorubicin (Dox)-loaded micelles (Dox-micelles) were prepared and characterized. Cell internalization, intracellular drug release, in vitro cytotoxicity, tumor-targeting ability, and penetrating efficacy, as well as in vivo antitumor activity of Dox-micelles, were comprehensively evaluated.
2.2. Synthesis and characterization 2.2.1. Synthesis of the PLL-LA copolymer To conjugate PLL to LA, 0.5 g of PLL was dissolved in 30 mL of distilled water at room temperature. Approximately 0.25 g of LA with 0.184 g of EDC·HCl and 0.11 g of NHS were added into 25 mL of methanol solution [34]. The mixture was allowed to react in a roundbottom flask for 12 h at 45 °C (Fig. 1A). The reaction product was dialyzed (MWCO: 1000 Da) with distilled water for 3 days and then lyophilized to obtain PLL-LA with approximately 95% yield. 2.2.2. Synthesis of the DA-PLL-LA copolymer A total of 0.1 g of PLL-LA was dissolved in phosphate buffer (pH 8.0, 20 mL), and 0.158 mg of DA were added [25]. After 6 h, the solution was dialyzed as mentioned; the pH was adjusted during 8.0–9.0 by 0.1 M of NaHCO3 solution. Finally, the solution was lyophilized to obtain DA-PLL-LA with approximately 95% yield.
2. Experimental section 2.1. Materials PLL with molecular weight (MW) of < 5000 was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). LA was acquired from Adamas Reagent Co., Ltd. (Switzerland). 2,3-DA and
2.2.3. Characterization of the copolymer The structures of the PLL, LA, PLL-LA, and DA-PLL-LA copolymer 235
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Fig. 1. (A) Synthesis route of DA-PLL-LA copolymer. (B) 1H NMR spectra of PLL (a), LA (b), PLL-LA (c), and DA-PLL-LA (d) in D2O. (C) The degradation of DA-PLL-LA at pH 7.4 and 6.8.
were characterized by 1H-nuclear magnetic resonance (1H NMR) spectroscopy (400 MHz, Varian, Palo Alto, CA, USA) using deuterium water (D2O) as solvent.
determined. The zeta potential of the blank DA-PLL-LA micelles was also monitored with the aforementioned medium after incubation for 1 h [36]. The absorbance of blank micelles at 630 nm wavelength was also detected using a microplate reader after incubated with different PBS conditions (pH 7.4, pH 6.8 or pH 6.8 + 10 mM GSH) for 2 h [37]. Doxloaded DA-PLL-LA micelles (1 mL) were prepared as described later. The micelles were incubated with different PBS conditions (pH 7.4, pH 6.8 or pH 6.8 + 10 mM GSH) for 2 h and the encapsulation efficiency of micelles was measured.
2.3. Degradation of DA Degradation of the DA-PLL-LA copolymers was measured by the fluorescamine method [35]. The copolymers were incubated with the buffer solutions of pH 6.8 and 7.4 at 37 °C. At a particular point in time (0, 0.25, 0.5, 1, 2, and 4 h), 100 mL of each pattern was mixed with 20 μL of fluorescamine solution (2 mg/mL in DMF) and incubated under the conditions of protection from light (25 °C, 10 min). Then, the fluorescence intensity (Fs) was estimated at 365 nm (excitation) and 475 nm (emission) by a full-wavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland). An average exposed amine (Fo) of 100% was calculated from the fluorescence of the sample after incubation in 0.1 M HCl for 24 h [35]. The fluorescence of blank buffer solution was considered to be 0% (Fc) as a negative control. The amount of exposed amine on behalf of the degradation rate of DA block, which was evaluated (Fs − Fc) / (Fo − Fc) × 100%.
2.4.2. Critical micelle concentration of micelles Critical micelle concentration was measured using the pyrene fluorescent probe method as previously reported [34]. Data were calculated and analyzed in the same way. 2.4.3. Adsorption of proteins on micelles The adsorption of proteins on micelles was carried out by incubating DA-PLL-LA micelles (1 mg/mL) with bovine serum albumin (pH 7.4 or pH 6.8, 37 °C 2 h) [38]. The mixture was centrifuged (12,000 rpm, 30 min) to separate free proteins. Afterward, the protein concentration and adsorption were measured.
2.4. Evaluation of copolymer
2.4.4. Serum stability study DA-PLL-LA micelles (1 mg/mL) were incubated with the same volume of FBS at 37 °C [37]. And then the absorbance of above mixture was measured at 630 nm with microplate reader at different time. The relative turbidity was calculated with the absorbance and the absorbance at t = 0 h of each group was normalized as 100%.
2.4.1. pH-triggered particle size and zeta potential charge To investigate whether the micrometers exhibit pH/reduction susceptibility in the simulated environment of cytoplasm and nucleus, the changes in the nanoparticle size and surface zeta potential of DA-PLLLA micelles responding to the trigger of GSH (10 mM) or pH from 7.4 to 6.8 were observed by dynamic light scattering (DLS) method at 25 °C using a ZEH 3600 (Malvern Instruments, Malvern, UK) [32]. Briefly, a blank DA-PLL-LA micelle solution (2 mg/mL) was prepared with the probe ultrasonic method. A 1 mL sample was incubated with 5 mL of phosphate-buffered solution (PBS) with or without GSH (10 mM) at different pH for a predesigned time, and then particle size was
2.5. Preparation and characterization of Dox-micelles Dox-micelles were prepared via the dialysis method. A total of 5 mL of blank micelles (2 mg/mL) were prepared with the abovementioned 236
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Fig. 2. (A) Size changes and zeta potential changes (B) of the blank micelles at different pH levels and 10 mM GSH. (C) Relative turbidities of DA-PLL-LA micelles with different PBS in different pH or GSH. (D) EE% of Dox in Dox-loaded micelles after incubation with PBS in different pH or GSH for 2 h. (E) The pH/reduction response mechanism of DA-PLL-LA in the presence of weak acid and GSH (10 mM).
Fig. 3. (A) CMC of micelles (a) and plot of I1/I3 and log C (b). (B) Protein adsorption and (C) serum stability of blank micelles at different pH values.
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with or without 10 mM GSH at different pH values and was stirred at 37 ± 0.5 °C with 100 rpm. A 1 mL sample of release medium was taken out at predetermined time points and replenished with equal volumes of the fresh medium [41]. The amount of Dox released from the Dox-micelles was determined using a full-wavelength microplate reader. 2.7. In vitro cellular uptake and distribution of Dox-micelles 2.7.1. Cellular uptake by confocal laser scanning microscopy (CLSM) A549 cells were seeded for 24 h on microscope slides in a 6-well plate with a density of 5 × 103 cells/well. The culture medium was replaced with serum-free medium containing Dox-micelles (pH 7.4 or 6.8, Dox concentration of 5 μg/mL) in comparison with Dox·HCl and Dox-micelles. After incubation for 2 or 6 h, the cells were rinsed with cold PBS (pH 7.4), and then fixed with 4% paraformaldehyde for 10 min. Cell nuclei were stained with Hoechst 33258 (10 μg/mL, 15 min). The intracellular fluorescence images were obtained using a confocal laser scanning microscope (CLSM, ZEISS710, Germany) [32]. 2.7.2. In vitro quantitative uptake detected by flow cytometry To quantitatively investigate the cellular uptake of Dox indifferent formulations by tumor cells, A549 cells were seeded on a 6-well plate at an intensity of 5 × 103 cells/well followed by 24 h of incubation. The cells were treated with Dox and Dox-micelles with the final Dox concentration of 5 μg/mL under pH 7.4 or 6.8 after incubation for 2 h or 6 h. The Fs of Dox was determined by flow cytometry (FC500, Beckman Coulter, USA). 2.7.3. Dynamic uptake observation A live cell station (Cell' R, Olympus) was used to observe the dynamic phagocytosis. MPMs were cultured in a glass-bottomed dish [34,42]. Cell nuclei were stained with 10 μg/mL Hoechst 33258 for 90 min, and then Dox-micelles were mixed into the dish slowly, then immobilized on the microscope stage of the live cell station to obtain the images of DIC, TRITC, and DAPI (captured every 15 s for 1.5 h) [34].
Fig. 4. (A) Size and TEM images of Dox-micelles in different medium. (B) In vitro Dox release from Dox-micelles at different pH values and reduction conditions.
2.8. Cytotoxicity study of Dox-micelles
method, followed by the addition of Dox·HCl solution (0.5 mL, 10 mg/ mL) and 0.5 mL of trimethylamine dropwise. The mixture was dialyzed against fresh distilled water for 8 h to remove the free Dox [39]. Then, Dox-micelles were obtained after filtering through 0.22-μm filter membranes. The micelle morphology of Dox-micelles was evaluated by transmission electron microscopy (TEM, JEOL Ltd., Japan). In addition, the size distribution and zeta potential of Dox-micelles were determined. The amounts of Dox encapsulated in micelles were measured using a full wavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland) with an excitation and emission wavelength of 484 and 598 nm, respectively. Drug encapsulation efficiency (EE%) and drug loading capacity (LC%) of the Dox-micelles were calculated by
w EE% = 0 × 100% w1
(1)
w0 × 100% w
(2)
LC% =
In vitro cytotoxicity of different formulations to A549 cells was evaluated by MTT assay. The cells were plated in 96-well plates (5 × 103 cells/well) and incubated for 24 h at 37 °C. The cells were then exposed to Dox and Dox-micelles, at various concentrations with medium in fresh (pH 7.4 or 6.8). After treatment for 48 h, MTT solution (5 mg/mL, 10 μL/well) was incubated for another 4 h. The plates with 100 μL of dimethyl sulfoxide were oscillated for 10 min preceded to detection of Cell viability (%) with a microplate reader (ELx808, BioTek, USA) as follows:
Cell Viability (%) =
A570(treated) × 100% A570(untreated)
(3)
where A570(treated) was the absorbance of Dox or Dox-micelle-treated and A570(untreated) was the absorbance of medium-treated cells at 570 nm, respectively. Meanwhile, we evaluated the cytotoxicity of blank micelles on the tumor cell A549 and mouse fibroblast L929 to determine vector safety.
where w0 and w1 are the amounts of loaded Dox and the total weight of Dox in the solution, respectively, and w is the total weight of Dox-micelles.
2.9. Dox distribution in tumor spheroids and solid tumors 2.6. In vitro release of Dox from Dox-micelles To prepare three-dimensional tumor spheroids [32], A549 cells were seeded on 96-well plates coated with 2% agarose at a density of 600 cells/200 μL. After cultured for 5–7 days, the tumor spheroids were transferred to a glass-bottomed Petri dish and treated with Dox and Dox-micelles at a Dox concentration of 5 μg/mL. After 2 h, the
The in vitro release features of Dox from Dox-micelles were investigated with the dialysis membrane method [40]. Briefly, 5.0 mL of Dox-micelle solution was placed in a dialysis bag (MWCO = 1000), and then transferred into a bottle containing 100 mL of release medium 238
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Fig. 5. (A) Cellular uptake of Dox in different formulations at pH 7.4 or 6.8 with different incubation time: ((a) 2 h and (b) 6 h) by a CLSM. (B) Dynamic uptake images of micelles, where red and blue areas correspond to Dox and cell nucleus, respectively. Kinetics of fluorescence intensities ((a, c) pH 6.8 and (b, d) pH 7.4). (E) Quantitative uptake of different formulations as detected by a flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescent intensity was monitored by CLSM. The frozen slices of tumor tissues were obtained 24 h after administration with Dox or Doxmicelles to investigate Dox distribution in solid tumor tissues. Cell nuclei were observed in Hoechst 33258 staining, and then solid tumor tissues slices were prepared for observation by CLSM. The frozen slices of tumor tissues were obtained 24 h after administration with Dox-micelles to investigate Dox distribution in solid tumor tissues. Solid tumor tissues slices were prepared for observation under a fluorescence microscope (20 ×) subsequent to stain cell nuclei with Hoechst 33258.
2.11. In vivo antitumor growth efficiency study BALB/c mice subcutaneous A549 tumor model was produced as described (Section 2.10). The mice were randomly divided into three groups (six mice in each group): physiological saline, Dox, and Doxmicelles. The mice in each group were given intravenous administration of Dox or Dox-micelles at a dose of 5 mg/kg through the tail vein every 4 days since the tumor volume reached approximately 80 mm3. The day before the first injection was set as day 0. The body weight and tumor volume were recorded every 2 days. The volume of tumor tissue was calculated using the formula V = 0.5 LS2, where L and S represented the longest (mm) and the shortest (mm) diameters, respectively, which were measured by an electronic Vernier caliper (ASONE, Japan). The animals were sacrificed 5 days after the last injection, and the tumor tissues were collected and weighed [44].
2.10. In vivo tumor-targeted imaging The in vivo tumor-targeting ability of polymeric micelles was evaluated by using Dir as a fluorescent dye [43]. Briefly, a subcutaneous lung tumor mice model was established through subcutaneous injection of prepared A549 cells. Then, the mice were intravenously administrated with Dir-loaded micelles at a dose of 300 μg/kg via the tail vein when the tumor volume reached 100 ± 30 mm3. Fluorescence distribution imaging was conducted using IVIS Spectrum (Caliper Life Science) with an excitation wavelength of 710 nm and an emission wavelength of 745 nm. After 24 h, the mice were sacrificed and collected the major organs and tumor tissues, followed by immediate imaging under the condition described above.
2.12. Statistical analysis Experimental data in this work are expressed as means ± standard deviation (SD) of three experiments at least, which were analyzed by ANOVA. Student's t-test was applied for paired comparisons (SPSS 16; SPSS, Chicago, IL). Statistical significance levels were stood as *p < 0.05, **p < 0.01, and ***p < 0.001.
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Fig. 6. (A) Cytotoxic effect of various formulations of Dox at different pH against A549 cells. (B) Cytotoxicity of two blank micelles against A549 and L929 cells. Values are presented as mean ± SD (n = 3). (C) Penetrating evaluation: fluorescence distribution of A549 tumor spheroids after incubation with different formulations at pH 7.4 for 2 h: (a) Dox and (b) Doxmicelles. (D) Fluorescence distribution of Dox in the deep region of tumor slices 24 h post-injection.
3. Results and discussion
As shown in Fig. 1C, the cleavage of coupled DA in DA-PLL-LA rapidly reached approximately 50% within 30 min at pH 6.8 and then increased to almost 80% with another 2 h incubation. By contrast, the hydrolysis profile was relatively less and slow at pH 7.4, only 32% DA release in 4 h [35], indicating a desirable pH-responsive detachment of DA as the shielding group.
3.1. Synthesis and characterization of DA-PLL-LA copolymer As shown in Fig. 1A, the PLL-LA copolymer was synthesized via the acylation reaction between the primary amines of PLL and carboxyl groups of LA activated by EDC·HCl and NHS. Finally, the DA-PLL-LA copolymer was obtained by reacting PLL-LA with DA in basic aqueous solution. The copolymer was more sensitive to mildly acid conditions that would hydrolyze at mildly acidic conditions due to the acid-labile β-carboxylic amide in DA [45]. Byproducts could be easily removed by dialysis and filtration. The DA-PLL-LA copolymer and its intermediates were characterized by 1H NMR (Fig. 1B). The 1H NMR spectrum signals of PLL appeared at δ = 2.9–3.3 ppm, 1.6–2.1 ppm, and δ = 1.4 ppm (peptide unit). The signals at δ = 2.31, 2.67, and 3.18 ppm were attributed to the specific proton peaks of penta-heterocyclic in LA, which suggested the successful grafting of LA to PLL. The proton peaks at δ = 1.8 ppm in the spectrum of DA-PLL-LA conjugates confirmed the introduction of DA [28]. The graft ratios of LA and DA were 42.17% and 14.52%, respectively, which were calculated from the 1H NMR spectrum.
3.2. Surface charge conversion and pH/reduction responsiveness To demonstrate the surface charge-conversion property of the DAPLL-LA copolymer in response to tumor site pH, the size changes and zeta potential of DA-PLL-LA micelles incubated under different conditions were monitored. Fig. 2A revealed that DA-PLL-LA micelles exhibited a constant average size of 117.0 nm at pH 7.4 over time. As the surrounding pH decreased to 6.8, no significant swell of DA-PLL-LA micelles was found; 10 mM GSH caused rapid dissociation of polymeric micelles to > 1000 nm, which became remarkable with prolonged incubation time. The zeta potential of DA-PLL-LA micelles reversed from − 18.2 mV to 10.4 mV as the surrounding pH value changed from 7.4 to 6.8 after incubation for 1 h and up to 13.2 mV under 10 mM GSH (Fig. 2B). The turbidity of the DA-PLL-LA micelles was measured by 240
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Fig. 7. Targeting property and antitumor effects of micelles toward A549 tumors in vivo. (A) Real-time NIR fluorescence images of subcutaneously transplanted A549 tumors treated with Dox-micelles. (B) Variation in the bioluminescence of subcutaneous tumor under the treatment of various Dox formulations in vivo. (C) Tumor weight, (D) tumor volume, and (E) body weights of tumor-bearing mice during the 20-day treatment (**p < 0.01, and ***p < 0.005).
As shown in Fig. 3B, at pH 7.4, the negatively charged DA-PLL-LA micelles showed the poor protein adsorption ability at pH 7.4. However, at pH 6.8, the zeta potential of DA-PLL-LA changed from negative to positive, thereby leading to a remarkable adsorption of protein. Furthermore, at physiological condition, the negatively charged DAPLL-LA micelles demonstrated no significant aggregation in FBS (Fig. 3C), which suggested that they possessed an excellent serum stability. The results indicated that the negative zeta potential micelles, DA-PLL-LA, would avoid nonspecific adsorption with negatively charged serum protein and display a remarkable stability during blood circulation, which was beneficial to the passive target efficacy to solid tumor tissues in vivo via the EPR effect [47,48].
microplate reader at 630 nm to evaluate the absorbance. As shown in Fig. 2C, the DA-PLL-LA micelles treated with acidic PBS (pH 6.8) and GSH (10 mM) rapidly turned highly turbid and the relative turbidities increased to > 500% within 10 min. A decrease in the encapsulation efficiency of Dox was also monitored after treatment with weak acid and GSH, which could have resulted from severe aggregation (Fig. 2D). Consequently, the DA-PLL-LA micelles maintained clear after 5 min treatment with pH 6.8 or pH 7.4 PBS (Fig. 2E). In contrast, the micelles were rapidly get turbid within 10 min incubated with pH 6.8 PBS and 10 mM GSH (Fig. 2E). Moreover, the decrease in the surrounding pH resulted in further increase in positive charge of the DA-PLL-LA micelles. Accordingly, the charge-reversal property of DA-PLL-LA micelles was mainly attributed to the acid-labile β-carboxylic amide of DA with more sensitive to mildly acidic conditions such as pH 6.8. The de-cross-linking of micelles under reductive conditions leads to cleavage of disulfide cross-links into eSSe groups [40]. Therefore, DA-PLL-LA micelles with a negative charge under physiological conditions may have better stability in blood circulation, with less nonspecific interaction with serum proteins compared with positively-charged copolymer, moreover, were unstable under a reductive condition simulating the environment of cancer cells [46].
3.4. Evaluation of Dox-micelles Dox, which was a widely used and hydrophobic antitumor drug, was telescoped in the core of self-assembled DA-PLL-LA micelles via the dialysis method. The mean particle size of Dox-micelles in pH 7.4 was (112.8 ± 4.02) nm while > 1000 nm in pH 6.8 and 10 mM GSH (PDI < 0.1; Fig. 4A). TEM micrograph showed uniformly spherical morphology with smaller average diameters compared with the results determined by DLS, which might be due to the loss of the hydrated film. In addition, Dox-micelles showed a negative charge under the physiological condition; the EE% and LC% were (93.6 ± 2.26)% and (22.4 ± 2.52)%, respectively. In vitro release of Dox from Dox/DA-PLL-LA micelles was carried out in PBS, which mimics the microenvironment of blood or tumor tissues, to estimate the pH- and/or reduction-triggered drug release behavior of micelles. Dox-micelles presented a sustained and slow drug release at physiological pH (pH 7.4) with the final cumulative release rate of approximately 32% for over 48 h (Fig. 4B). The drug release rate was
3.3. Evaluation of DA-PLL-LA copolymer As shown in Fig. 3A(a), CMC was measured within the DA-PLL-LA concentration range of 1 μg/mL to 1 mg/mL. The value of CMC was calculated to be 3.98 μg/mL according to the plots of I1/I3 and log C (Fig. 3A(b)). Thus, the DA-PLL-LA polymeric micelles showed high stability and revealed great potential application as a suitable drug carrier. 241
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This finding demonstrated the desirable safety of the DA-PLL-LA micelles (Fig. 6B).
almost unchanged at pH 6.8. Moreover, the reductive surrounding (10 mM GSH + pH 6.8) remarkably accelerated the release of Dox (approximately 60%) over 24 h. The fastest and almost complete release was observed at pH 5.3 in the presence of 10 mM GSH, in which approximately 80%, due to the acid-labile β-carboxylic amide of DA would hydrolyze at mildly acidic conditions [49], as well as the break of disulfide bonds in DA-PLL-LA copolymer responding to 10 mM of GSH stimuli, might cause a synergistic effect on the swelling and destabilization of DA-PLL-LA micelles. This effect was consistent with the outcomes of the size change study described in Section 3.2. These results revealed that the DA-PLL-LA micelles performed an on-demand drug release manner with little Dox released in the blood circulation but a rapid Dox release within tumor cells, which might lead to a lower systemic toxicity and an enhanced antitumor effect.
3.7. Tumor penetrability evaluation The targeting evaluation reflected the accumulation of DA-PLL-LA micelles in the tumor site but did not ensure further distribution in solid tumor. Therefore, the distribution of Dox and Dox-micelles in A549 tumor spheroids and solid tumor slices was further investigated via CLSM. After a 2-h pretreatment, the Dox-micelles group exhibited the strongest Dox fluorescence and showed no dramatic decrease from the edge to the deep section of tumor spheroids (Fig. 6C). These results indicate that the Dox-micelles exerted excellent penetrating effects. To further evaluate the tumor penetration ability, we illustrated the distribution of the micelles in subcutaneous tumor-bearing mice, which were injected with Dox in different formulations (5 mg/kg) via the tail vein. As shown in Fig. 6D, the Dox-micelles exhibited remarkably high distributions in the deep area. Thus, the Dox-micelles exhibited not only a better accumulation in the tumor site but also an enhanced penetrating ability into the internal region of the solid tumor.
3.5. Cell uptake 3.5.1. Subcellular distribution of micelles In Fig. 5A, A549 cells were incubated with Dox-micelles at pH 7.4 or 6.8 to investigate whether pH-triggered charge-reversal and reductionsensitive properties could facilitate cellular uptake and improve intracellular drug release. The uptake of Dox-micelles was enhanced as the surrounding pH decreased to 6.8, which benefited from the chargereversal property responding to the lower pH. Dox-micelles incubated for 2 h were mainly located in the cytoplasm, and the Fs of Dox increased with time. In other words, micelles gradually released Dox to cytoplasm then diffused to nuclei. These results indicated that targeted intracellular release of Dox from the dually pH/reduction-responsive Dox-micelles could be triggered by the microenvironment within tumor cells. This phenomenon allows for the co-location of Dox and nuclei and an enhanced apoptosis of A549 cells.
3.8. Targeting efficiency and antitumor effect in vivo
3.5.3. Quantitative flow cytometry analysis Results of the quantitative flow cytometry analysis, indicated that cells incubated with pH/reduction-responsive Dox-micelles in pH 6.8 exhibited a higher uptake than in pH 7.4 or Dox·HCl solution (Fig. 5C). Moreover, the fluorescence of Dox-micelles gradually increased with time.
Dir, a near-infrared (NIR) probe, was trapped into the micelles to explore the targeting effect of Dir/DA-PLL-LA in vivo by NIR fluorescence imaging. As shown in Fig. 7A, mice treated with Dir/DA-PLL-LA exhibited significantly high fluorescence intensities in the liver within 12 h because of the rapid uptake of micelles by the RES. At 24-h postinjection, more Dox was located in tumor tissue compared with adjacent normal tissues and organs. Hence, this result demonstrated that the micelles could targeted-delivery drugs to cancer cells via the EPR effect after systemic circulation. Thus, the stepwise pH/reduction-responsive polymeric micelles exerted an excellent target effect on the tumor. The fluorescence of major organs also confirmed the targeteddelivery ability of the micelles. To investigate the synergistic antitumor activity of the Dox-micelles in vivo, lung-tumor-bearing BALB/c mice were administrated with multiple formulations to detected the antitumor effect (Fig. 7B). The Dox-micelles exhibited stronger antitumor activity than saline and Dox and the body weight of mice in Dox alone treatment group showed notable decreased without tumor suppression effect. Furthermore, this body weight decrease exhibited in DOX group due to the systemic toxicity of Dox. After 11 days treatment, the tumor volume in the Dox group couldn't be inhibited but increased rapidly. Drug resistance may contribute to these failure anti-cancer effects. From the above, the Doxmicelles could exert a strong tumor suppressive effect in vivo. In general, the anticancer efficiency experiment in vivo confirmed that the stepwise pH/reduction-responsive micelles could simultaneously improve the therapeutic effect of Dox in vivo.
3.6. Cytotoxicity of Dox-micelles in vitro
4. Conclusion
The in vitro cytotoxicity of the blank micelles and Dox in different formulations was evaluated by MTT assay. Dox formulations, including Dox·HCl and Dox-micelles, exhibited dose-dependent cytotoxicity against A549 cell lines (Fig. 6A). The Dox-micelles showed higher cytotoxicity against A549 cells at pH 6.8 than at pH 7.4, which was further demonstrated by the IC50 value shown in Fig. 4B. The superior cytotoxicity of Dox-micelles at pH 6.8 was attributed to the enhanced cell internalization. Dox·HCl with low cellular uptake exhibited obvious cytotoxicity against A549 cells, which might be due to the rapid colocation with the cell nuclei and the induced cell apoptosis. These results demonstrated that the stepwise pH/reduction-responsive DA-PLLLA micelles showed high cytotoxicity to A549 cancer cells in a pH-dependent way. Of the cells, 90.11% and 91.78% survived after treatment with the micelles for 48 h against L929 and A549 cells, respectively.
In this study, we successfully developed a novel drug delivery system with enhanced cellular internalization and efficient drug release based on the stepwise pH/reduction-responsive DA-PLL-LA micelles. The charge-convertible cross-linked copolymer micelles with stable structure and negative surface charge in the blood stream exhibited high tumor-targeting ability via EPR effect. The internalized Dox-micelles exhibited fast and complete intracellular Dox release response to both low extracellular pH and high concentration of GSH in cytoplasm, which resulted in an enhanced cytotoxicity against A549 tumor cells. Therefore, the Dox-micelles showed excellent antitumor effects with lesser side effects in vivo. In summary, the stepwise pH/reduction-responsive DA-PLL-LA micelles were promising for the development of functional drug delivery systems for a safe and efficient therapy of lung cancer. These special tumor microenvironment and stimuli-responsive
3.5.2. Dynamic phagocytosis of micelles Dynamic uptake of Dox-micelles in A549 cells was detected by the live-cell station (Fig. 5B(a, b)). The results of Dox fluorescence in different formulations were according to Section 3.4. Moreover, the Doxmicelles under showed rapid uptake at pH 6.8 than at pH 7.4 within 1.5 h after administration. Several areas in the cytoplasm after Doxmicelles treatments were selected as ROIs to monitor the fluorescence intensities (Fig. 5B(c, d)). Cells treated with Dox-micelles in pH 6.8 exhibited a speedy accumulative uptake within 1.5 h than the cells treated with Dox-micelles in pH 7.4.
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumor
MARK
Shudi Yanga, Ying Wanga, Zhaoxiang Renb, Mengtian Chena, Weiliang Chena, Xuenong Zhanga,⁎ a
Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou, People's Republic of China Jiangsu Key Laboratory for Translational Research and Therapy for Neuropsycho-disorders, Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, People's Republic of China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Charge reversion Stimuli-responsive Smart Drug delivery Cancer therapy
In this research, a charge-conversional polymer, poly-L-lysine-lipoic acid (PLL-LA), was prepared by dimethylmaleic anhydride (DA) modification and applied as a carrier with enhanced cell internalization and intracellular pH- and reduction-triggered doxorubicin (Dox) release. The surface charge of dimethylmaleic anhydride-poly-L-lysine-lipoic acid micelles (DA-PLL-LA) was negative at physiological pH and reversed to positive at the extracellular and intracellular pH of cancer cells. At tumor extracellular pH of 6.8, the conjugates underwent a rapid charge-reversible process with almost 80% DA cleavage within 2 h, and then endocytosed into the endo/lysosomes more rapidly than at physiological pH of 7.4. The Dox/DA-PLL-LA micelles (Dox-micelles) demonstrated a sustained drug release in vitro under physiological condition, and rapid Dox release was triggered by both extracellular pH and high-concentration reducing glutathione. The Dox-micelles also exhibited enhanced internalization at extracellular pH, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Excellent tumor-penetrating efficacy was also found in A549 tumor spheroids and solid tumor slices. Moreover, the DA-PLL-LA micelles exhibited excellent tumor-targeting ability in tumor tissues and excellent antitumor efficacy and low systemic toxicity in breast tumor-bearing mice. Therefore, the DA-PLL-LA micelles demonstrated great potential for targeted and efficient drug delivery in cancer treatments.
1. Introduction Extensive work has been done in the area of various targeted delivery carriers of chemotherapeutic anticancer drugs, such as polymeric nanoparticles, synthetic nonviral systems, supramolecular hydrogel, hybrid nanocarriers and biodegradable polymers [1–8]. Nowadays, multiple smart delivery systems for drugs and genes responded to temperature, light, pH and reactive oxygen species (ROS) were developed [9–11]. In recent years, stimuli-responsive nanoparticles have attracted high attention because of their effective delivery ability of anticancer drugs via recognizing the micro-environment of tumors [12]. The greatest advantages of nanoparticles were higher hydrophobic drugs solubility and lesser side effects. Nanoparticles also rapidly increased the accumulation in tumor tissue compared to normal tissue by the enhanced permeability and retention (EPR) effect [13–15]. However, the poor cell internalization as well as slow and incomplete intracellular drug release posed great challenges for efficient treatment; these phenomena diametrically resulted in low intracellular drug concentration below the required therapeutic level and unsatisfactory therapy efficacy [16–18]. Hence, smart stimuli-responsive ⁎
Corresponding author. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.msec.2017.08.079 Received 16 June 2017; Received in revised form 3 August 2017; Accepted 10 August 2017 0928-4931/ © 2017 Published by Elsevier B.V.
nanoparticles based on the special microenvironment of tumor tissues have been given particular attention. In the systemic circulation, nanoparticles bio-distribution, delivery and release of drugs or gene were significantly affected by the changes of physicochemical properties of nanoparticles responded to stimuli, such as size, zeta potential, solubility and structure [11], which were strongly associated with the delivery efficiency of drug [19–24]. Generally, the positively-charged nanoparticles with higher cellular internalization would be captured by the reticular endothelin system (RES) and have a strong force with the negatively-charged serum components, causing further aggregation and leading to a short blood circulation time [25–27]. To solve this contradiction, an ideal system based on a charge-reversal concept is desirable. Charge-switchable systems have been developed for prolonged circulation and improved cell uptake by the tumor microenvironment-triggered shell detachment based on the slight pH difference between extracellular acidity of tumor sites (pH ≈ 6.8) and normal tissues (pH ≈ 7.4) [28,29]. These systems could stay stable in bloodstream and avoid the non-special absorption by serum proteins. After the systems accumulated in tumor sites, the low-pH-triggered ionization and the detachment of protective moiety
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Scheme 1. Schematic illustration of the stepwise pH/reduction-responsive DA-PLLLA micelles.
methyl thiazolyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluorescamine was provided by J & K Scientific Ltd. (Beijing, China). GSH, 1-(3-dimethylaminopropyl)3-ethyl carbon carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were ordered from Aladdin Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and RPMI-1640 were purchased from Hyclone (Thermo-Fisher Biochemical Products Co., Ltd., Beijing, China). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (Dir) was provided by Baomanbio Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade. Human lung adenocarcinoma cell line luc-A549 was provided from pharmaceutics teaching and research section (Suzhou, China). This cell line was maintained in RPMI-1640 media supplemented with 10% (v/ v) FBS under 5% CO2-humidified conditions at 37 °C. Female and male nude mice (BALB/C, nu/nu) (18 ± 2 g) were purchased from the Experimental Animal Center of Soochow University. All animals were raised in surroundings that obeyed the guidelines of the standard laboratory conditions of Soochow University. All animal process was performed following the protocols authorized by the Institutional Animal Care.
result in positive surface charge, which facilitates the intracellular endocytosis [30,31]. There is now a general consensus that there are significant differences between the microenvironments of tumor tissue and nearby normal cells, such as vascular distribution, pH and redox state, and numerous tumor targeted nanoparticles were exploited through recognizing them. Particularly, glutathione (GSH, 1–10 mM in tumor cell cytoplasm and 2–20 μM in blood fluids) has been extensively investigated [32,33]. Numerous studies have utilized reduction-sensitive nanocarriers containing disulfide bonds, such as dithiodipropionic acid and lipoic acid (LA). This method promoted sharp and sufficient drug release, wherein the disulfide bonds would break up in response to intracellular high concentration of GSH and further cause destabilization and cleavage of drug-loading nanocarriers. In this work, we reported a nanocarrier with high stability and sensitivity to tumor microenvironment that is sequentially activated at the extracellular and intracellular levels of tumor tissue by gradual transformation (Scheme 1). The mildly acidic microenvironment of tumor tissue was recognized by this nanoparticle and the surface charge of this nanoparticle was shifted to positive charge to strengthen cellular uptake. In addition, the nanoparticle has a chemically cross-linked core of poly-L-lysine (PLL) with excellent hydrophilicity and LA with a disulfide bond, which is cleaved of GSH, to enhance the accumulation of drugs in tumor sites at the intracellular level. The prepared stepwise pH/reduction-responsive polymeric micelles could achieve enhanced cell internalization and efficient intracellular drug release, leading to enhanced cytotoxicity [32]. In the present study, the stepwise responsive nanocarrier (DA-PLL-LA) copolymer was successfully synthesized, and doxorubicin (Dox)-loaded micelles (Dox-micelles) were prepared and characterized. Cell internalization, intracellular drug release, in vitro cytotoxicity, tumor-targeting ability, and penetrating efficacy, as well as in vivo antitumor activity of Dox-micelles, were comprehensively evaluated.
2.2. Synthesis and characterization 2.2.1. Synthesis of the PLL-LA copolymer To conjugate PLL to LA, 0.5 g of PLL was dissolved in 30 mL of distilled water at room temperature. Approximately 0.25 g of LA with 0.184 g of EDC·HCl and 0.11 g of NHS were added into 25 mL of methanol solution [34]. The mixture was allowed to react in a roundbottom flask for 12 h at 45 °C (Fig. 1A). The reaction product was dialyzed (MWCO: 1000 Da) with distilled water for 3 days and then lyophilized to obtain PLL-LA with approximately 95% yield. 2.2.2. Synthesis of the DA-PLL-LA copolymer A total of 0.1 g of PLL-LA was dissolved in phosphate buffer (pH 8.0, 20 mL), and 0.158 mg of DA were added [25]. After 6 h, the solution was dialyzed as mentioned; the pH was adjusted during 8.0–9.0 by 0.1 M of NaHCO3 solution. Finally, the solution was lyophilized to obtain DA-PLL-LA with approximately 95% yield.
2. Experimental section 2.1. Materials PLL with molecular weight (MW) of < 5000 was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). LA was acquired from Adamas Reagent Co., Ltd. (Switzerland). 2,3-DA and
2.2.3. Characterization of the copolymer The structures of the PLL, LA, PLL-LA, and DA-PLL-LA copolymer 235
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Fig. 1. (A) Synthesis route of DA-PLL-LA copolymer. (B) 1H NMR spectra of PLL (a), LA (b), PLL-LA (c), and DA-PLL-LA (d) in D2O. (C) The degradation of DA-PLL-LA at pH 7.4 and 6.8.
were characterized by 1H-nuclear magnetic resonance (1H NMR) spectroscopy (400 MHz, Varian, Palo Alto, CA, USA) using deuterium water (D2O) as solvent.
determined. The zeta potential of the blank DA-PLL-LA micelles was also monitored with the aforementioned medium after incubation for 1 h [36]. The absorbance of blank micelles at 630 nm wavelength was also detected using a microplate reader after incubated with different PBS conditions (pH 7.4, pH 6.8 or pH 6.8 + 10 mM GSH) for 2 h [37]. Doxloaded DA-PLL-LA micelles (1 mL) were prepared as described later. The micelles were incubated with different PBS conditions (pH 7.4, pH 6.8 or pH 6.8 + 10 mM GSH) for 2 h and the encapsulation efficiency of micelles was measured.
2.3. Degradation of DA Degradation of the DA-PLL-LA copolymers was measured by the fluorescamine method [35]. The copolymers were incubated with the buffer solutions of pH 6.8 and 7.4 at 37 °C. At a particular point in time (0, 0.25, 0.5, 1, 2, and 4 h), 100 mL of each pattern was mixed with 20 μL of fluorescamine solution (2 mg/mL in DMF) and incubated under the conditions of protection from light (25 °C, 10 min). Then, the fluorescence intensity (Fs) was estimated at 365 nm (excitation) and 475 nm (emission) by a full-wavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland). An average exposed amine (Fo) of 100% was calculated from the fluorescence of the sample after incubation in 0.1 M HCl for 24 h [35]. The fluorescence of blank buffer solution was considered to be 0% (Fc) as a negative control. The amount of exposed amine on behalf of the degradation rate of DA block, which was evaluated (Fs − Fc) / (Fo − Fc) × 100%.
2.4.2. Critical micelle concentration of micelles Critical micelle concentration was measured using the pyrene fluorescent probe method as previously reported [34]. Data were calculated and analyzed in the same way. 2.4.3. Adsorption of proteins on micelles The adsorption of proteins on micelles was carried out by incubating DA-PLL-LA micelles (1 mg/mL) with bovine serum albumin (pH 7.4 or pH 6.8, 37 °C 2 h) [38]. The mixture was centrifuged (12,000 rpm, 30 min) to separate free proteins. Afterward, the protein concentration and adsorption were measured.
2.4. Evaluation of copolymer
2.4.4. Serum stability study DA-PLL-LA micelles (1 mg/mL) were incubated with the same volume of FBS at 37 °C [37]. And then the absorbance of above mixture was measured at 630 nm with microplate reader at different time. The relative turbidity was calculated with the absorbance and the absorbance at t = 0 h of each group was normalized as 100%.
2.4.1. pH-triggered particle size and zeta potential charge To investigate whether the micrometers exhibit pH/reduction susceptibility in the simulated environment of cytoplasm and nucleus, the changes in the nanoparticle size and surface zeta potential of DA-PLLLA micelles responding to the trigger of GSH (10 mM) or pH from 7.4 to 6.8 were observed by dynamic light scattering (DLS) method at 25 °C using a ZEH 3600 (Malvern Instruments, Malvern, UK) [32]. Briefly, a blank DA-PLL-LA micelle solution (2 mg/mL) was prepared with the probe ultrasonic method. A 1 mL sample was incubated with 5 mL of phosphate-buffered solution (PBS) with or without GSH (10 mM) at different pH for a predesigned time, and then particle size was
2.5. Preparation and characterization of Dox-micelles Dox-micelles were prepared via the dialysis method. A total of 5 mL of blank micelles (2 mg/mL) were prepared with the abovementioned 236
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Fig. 2. (A) Size changes and zeta potential changes (B) of the blank micelles at different pH levels and 10 mM GSH. (C) Relative turbidities of DA-PLL-LA micelles with different PBS in different pH or GSH. (D) EE% of Dox in Dox-loaded micelles after incubation with PBS in different pH or GSH for 2 h. (E) The pH/reduction response mechanism of DA-PLL-LA in the presence of weak acid and GSH (10 mM).
Fig. 3. (A) CMC of micelles (a) and plot of I1/I3 and log C (b). (B) Protein adsorption and (C) serum stability of blank micelles at different pH values.
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with or without 10 mM GSH at different pH values and was stirred at 37 ± 0.5 °C with 100 rpm. A 1 mL sample of release medium was taken out at predetermined time points and replenished with equal volumes of the fresh medium [41]. The amount of Dox released from the Dox-micelles was determined using a full-wavelength microplate reader. 2.7. In vitro cellular uptake and distribution of Dox-micelles 2.7.1. Cellular uptake by confocal laser scanning microscopy (CLSM) A549 cells were seeded for 24 h on microscope slides in a 6-well plate with a density of 5 × 103 cells/well. The culture medium was replaced with serum-free medium containing Dox-micelles (pH 7.4 or 6.8, Dox concentration of 5 μg/mL) in comparison with Dox·HCl and Dox-micelles. After incubation for 2 or 6 h, the cells were rinsed with cold PBS (pH 7.4), and then fixed with 4% paraformaldehyde for 10 min. Cell nuclei were stained with Hoechst 33258 (10 μg/mL, 15 min). The intracellular fluorescence images were obtained using a confocal laser scanning microscope (CLSM, ZEISS710, Germany) [32]. 2.7.2. In vitro quantitative uptake detected by flow cytometry To quantitatively investigate the cellular uptake of Dox indifferent formulations by tumor cells, A549 cells were seeded on a 6-well plate at an intensity of 5 × 103 cells/well followed by 24 h of incubation. The cells were treated with Dox and Dox-micelles with the final Dox concentration of 5 μg/mL under pH 7.4 or 6.8 after incubation for 2 h or 6 h. The Fs of Dox was determined by flow cytometry (FC500, Beckman Coulter, USA). 2.7.3. Dynamic uptake observation A live cell station (Cell' R, Olympus) was used to observe the dynamic phagocytosis. MPMs were cultured in a glass-bottomed dish [34,42]. Cell nuclei were stained with 10 μg/mL Hoechst 33258 for 90 min, and then Dox-micelles were mixed into the dish slowly, then immobilized on the microscope stage of the live cell station to obtain the images of DIC, TRITC, and DAPI (captured every 15 s for 1.5 h) [34].
Fig. 4. (A) Size and TEM images of Dox-micelles in different medium. (B) In vitro Dox release from Dox-micelles at different pH values and reduction conditions.
2.8. Cytotoxicity study of Dox-micelles
method, followed by the addition of Dox·HCl solution (0.5 mL, 10 mg/ mL) and 0.5 mL of trimethylamine dropwise. The mixture was dialyzed against fresh distilled water for 8 h to remove the free Dox [39]. Then, Dox-micelles were obtained after filtering through 0.22-μm filter membranes. The micelle morphology of Dox-micelles was evaluated by transmission electron microscopy (TEM, JEOL Ltd., Japan). In addition, the size distribution and zeta potential of Dox-micelles were determined. The amounts of Dox encapsulated in micelles were measured using a full wavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland) with an excitation and emission wavelength of 484 and 598 nm, respectively. Drug encapsulation efficiency (EE%) and drug loading capacity (LC%) of the Dox-micelles were calculated by
w EE% = 0 × 100% w1
(1)
w0 × 100% w
(2)
LC% =
In vitro cytotoxicity of different formulations to A549 cells was evaluated by MTT assay. The cells were plated in 96-well plates (5 × 103 cells/well) and incubated for 24 h at 37 °C. The cells were then exposed to Dox and Dox-micelles, at various concentrations with medium in fresh (pH 7.4 or 6.8). After treatment for 48 h, MTT solution (5 mg/mL, 10 μL/well) was incubated for another 4 h. The plates with 100 μL of dimethyl sulfoxide were oscillated for 10 min preceded to detection of Cell viability (%) with a microplate reader (ELx808, BioTek, USA) as follows:
Cell Viability (%) =
A570(treated) × 100% A570(untreated)
(3)
where A570(treated) was the absorbance of Dox or Dox-micelle-treated and A570(untreated) was the absorbance of medium-treated cells at 570 nm, respectively. Meanwhile, we evaluated the cytotoxicity of blank micelles on the tumor cell A549 and mouse fibroblast L929 to determine vector safety.
where w0 and w1 are the amounts of loaded Dox and the total weight of Dox in the solution, respectively, and w is the total weight of Dox-micelles.
2.9. Dox distribution in tumor spheroids and solid tumors 2.6. In vitro release of Dox from Dox-micelles To prepare three-dimensional tumor spheroids [32], A549 cells were seeded on 96-well plates coated with 2% agarose at a density of 600 cells/200 μL. After cultured for 5–7 days, the tumor spheroids were transferred to a glass-bottomed Petri dish and treated with Dox and Dox-micelles at a Dox concentration of 5 μg/mL. After 2 h, the
The in vitro release features of Dox from Dox-micelles were investigated with the dialysis membrane method [40]. Briefly, 5.0 mL of Dox-micelle solution was placed in a dialysis bag (MWCO = 1000), and then transferred into a bottle containing 100 mL of release medium 238
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Fig. 5. (A) Cellular uptake of Dox in different formulations at pH 7.4 or 6.8 with different incubation time: ((a) 2 h and (b) 6 h) by a CLSM. (B) Dynamic uptake images of micelles, where red and blue areas correspond to Dox and cell nucleus, respectively. Kinetics of fluorescence intensities ((a, c) pH 6.8 and (b, d) pH 7.4). (E) Quantitative uptake of different formulations as detected by a flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescent intensity was monitored by CLSM. The frozen slices of tumor tissues were obtained 24 h after administration with Dox or Doxmicelles to investigate Dox distribution in solid tumor tissues. Cell nuclei were observed in Hoechst 33258 staining, and then solid tumor tissues slices were prepared for observation by CLSM. The frozen slices of tumor tissues were obtained 24 h after administration with Dox-micelles to investigate Dox distribution in solid tumor tissues. Solid tumor tissues slices were prepared for observation under a fluorescence microscope (20 ×) subsequent to stain cell nuclei with Hoechst 33258.
2.11. In vivo antitumor growth efficiency study BALB/c mice subcutaneous A549 tumor model was produced as described (Section 2.10). The mice were randomly divided into three groups (six mice in each group): physiological saline, Dox, and Doxmicelles. The mice in each group were given intravenous administration of Dox or Dox-micelles at a dose of 5 mg/kg through the tail vein every 4 days since the tumor volume reached approximately 80 mm3. The day before the first injection was set as day 0. The body weight and tumor volume were recorded every 2 days. The volume of tumor tissue was calculated using the formula V = 0.5 LS2, where L and S represented the longest (mm) and the shortest (mm) diameters, respectively, which were measured by an electronic Vernier caliper (ASONE, Japan). The animals were sacrificed 5 days after the last injection, and the tumor tissues were collected and weighed [44].
2.10. In vivo tumor-targeted imaging The in vivo tumor-targeting ability of polymeric micelles was evaluated by using Dir as a fluorescent dye [43]. Briefly, a subcutaneous lung tumor mice model was established through subcutaneous injection of prepared A549 cells. Then, the mice were intravenously administrated with Dir-loaded micelles at a dose of 300 μg/kg via the tail vein when the tumor volume reached 100 ± 30 mm3. Fluorescence distribution imaging was conducted using IVIS Spectrum (Caliper Life Science) with an excitation wavelength of 710 nm and an emission wavelength of 745 nm. After 24 h, the mice were sacrificed and collected the major organs and tumor tissues, followed by immediate imaging under the condition described above.
2.12. Statistical analysis Experimental data in this work are expressed as means ± standard deviation (SD) of three experiments at least, which were analyzed by ANOVA. Student's t-test was applied for paired comparisons (SPSS 16; SPSS, Chicago, IL). Statistical significance levels were stood as *p < 0.05, **p < 0.01, and ***p < 0.001.
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Fig. 6. (A) Cytotoxic effect of various formulations of Dox at different pH against A549 cells. (B) Cytotoxicity of two blank micelles against A549 and L929 cells. Values are presented as mean ± SD (n = 3). (C) Penetrating evaluation: fluorescence distribution of A549 tumor spheroids after incubation with different formulations at pH 7.4 for 2 h: (a) Dox and (b) Doxmicelles. (D) Fluorescence distribution of Dox in the deep region of tumor slices 24 h post-injection.
3. Results and discussion
As shown in Fig. 1C, the cleavage of coupled DA in DA-PLL-LA rapidly reached approximately 50% within 30 min at pH 6.8 and then increased to almost 80% with another 2 h incubation. By contrast, the hydrolysis profile was relatively less and slow at pH 7.4, only 32% DA release in 4 h [35], indicating a desirable pH-responsive detachment of DA as the shielding group.
3.1. Synthesis and characterization of DA-PLL-LA copolymer As shown in Fig. 1A, the PLL-LA copolymer was synthesized via the acylation reaction between the primary amines of PLL and carboxyl groups of LA activated by EDC·HCl and NHS. Finally, the DA-PLL-LA copolymer was obtained by reacting PLL-LA with DA in basic aqueous solution. The copolymer was more sensitive to mildly acid conditions that would hydrolyze at mildly acidic conditions due to the acid-labile β-carboxylic amide in DA [45]. Byproducts could be easily removed by dialysis and filtration. The DA-PLL-LA copolymer and its intermediates were characterized by 1H NMR (Fig. 1B). The 1H NMR spectrum signals of PLL appeared at δ = 2.9–3.3 ppm, 1.6–2.1 ppm, and δ = 1.4 ppm (peptide unit). The signals at δ = 2.31, 2.67, and 3.18 ppm were attributed to the specific proton peaks of penta-heterocyclic in LA, which suggested the successful grafting of LA to PLL. The proton peaks at δ = 1.8 ppm in the spectrum of DA-PLL-LA conjugates confirmed the introduction of DA [28]. The graft ratios of LA and DA were 42.17% and 14.52%, respectively, which were calculated from the 1H NMR spectrum.
3.2. Surface charge conversion and pH/reduction responsiveness To demonstrate the surface charge-conversion property of the DAPLL-LA copolymer in response to tumor site pH, the size changes and zeta potential of DA-PLL-LA micelles incubated under different conditions were monitored. Fig. 2A revealed that DA-PLL-LA micelles exhibited a constant average size of 117.0 nm at pH 7.4 over time. As the surrounding pH decreased to 6.8, no significant swell of DA-PLL-LA micelles was found; 10 mM GSH caused rapid dissociation of polymeric micelles to > 1000 nm, which became remarkable with prolonged incubation time. The zeta potential of DA-PLL-LA micelles reversed from − 18.2 mV to 10.4 mV as the surrounding pH value changed from 7.4 to 6.8 after incubation for 1 h and up to 13.2 mV under 10 mM GSH (Fig. 2B). The turbidity of the DA-PLL-LA micelles was measured by 240
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Fig. 7. Targeting property and antitumor effects of micelles toward A549 tumors in vivo. (A) Real-time NIR fluorescence images of subcutaneously transplanted A549 tumors treated with Dox-micelles. (B) Variation in the bioluminescence of subcutaneous tumor under the treatment of various Dox formulations in vivo. (C) Tumor weight, (D) tumor volume, and (E) body weights of tumor-bearing mice during the 20-day treatment (**p < 0.01, and ***p < 0.005).
As shown in Fig. 3B, at pH 7.4, the negatively charged DA-PLL-LA micelles showed the poor protein adsorption ability at pH 7.4. However, at pH 6.8, the zeta potential of DA-PLL-LA changed from negative to positive, thereby leading to a remarkable adsorption of protein. Furthermore, at physiological condition, the negatively charged DAPLL-LA micelles demonstrated no significant aggregation in FBS (Fig. 3C), which suggested that they possessed an excellent serum stability. The results indicated that the negative zeta potential micelles, DA-PLL-LA, would avoid nonspecific adsorption with negatively charged serum protein and display a remarkable stability during blood circulation, which was beneficial to the passive target efficacy to solid tumor tissues in vivo via the EPR effect [47,48].
microplate reader at 630 nm to evaluate the absorbance. As shown in Fig. 2C, the DA-PLL-LA micelles treated with acidic PBS (pH 6.8) and GSH (10 mM) rapidly turned highly turbid and the relative turbidities increased to > 500% within 10 min. A decrease in the encapsulation efficiency of Dox was also monitored after treatment with weak acid and GSH, which could have resulted from severe aggregation (Fig. 2D). Consequently, the DA-PLL-LA micelles maintained clear after 5 min treatment with pH 6.8 or pH 7.4 PBS (Fig. 2E). In contrast, the micelles were rapidly get turbid within 10 min incubated with pH 6.8 PBS and 10 mM GSH (Fig. 2E). Moreover, the decrease in the surrounding pH resulted in further increase in positive charge of the DA-PLL-LA micelles. Accordingly, the charge-reversal property of DA-PLL-LA micelles was mainly attributed to the acid-labile β-carboxylic amide of DA with more sensitive to mildly acidic conditions such as pH 6.8. The de-cross-linking of micelles under reductive conditions leads to cleavage of disulfide cross-links into eSSe groups [40]. Therefore, DA-PLL-LA micelles with a negative charge under physiological conditions may have better stability in blood circulation, with less nonspecific interaction with serum proteins compared with positively-charged copolymer, moreover, were unstable under a reductive condition simulating the environment of cancer cells [46].
3.4. Evaluation of Dox-micelles Dox, which was a widely used and hydrophobic antitumor drug, was telescoped in the core of self-assembled DA-PLL-LA micelles via the dialysis method. The mean particle size of Dox-micelles in pH 7.4 was (112.8 ± 4.02) nm while > 1000 nm in pH 6.8 and 10 mM GSH (PDI < 0.1; Fig. 4A). TEM micrograph showed uniformly spherical morphology with smaller average diameters compared with the results determined by DLS, which might be due to the loss of the hydrated film. In addition, Dox-micelles showed a negative charge under the physiological condition; the EE% and LC% were (93.6 ± 2.26)% and (22.4 ± 2.52)%, respectively. In vitro release of Dox from Dox/DA-PLL-LA micelles was carried out in PBS, which mimics the microenvironment of blood or tumor tissues, to estimate the pH- and/or reduction-triggered drug release behavior of micelles. Dox-micelles presented a sustained and slow drug release at physiological pH (pH 7.4) with the final cumulative release rate of approximately 32% for over 48 h (Fig. 4B). The drug release rate was
3.3. Evaluation of DA-PLL-LA copolymer As shown in Fig. 3A(a), CMC was measured within the DA-PLL-LA concentration range of 1 μg/mL to 1 mg/mL. The value of CMC was calculated to be 3.98 μg/mL according to the plots of I1/I3 and log C (Fig. 3A(b)). Thus, the DA-PLL-LA polymeric micelles showed high stability and revealed great potential application as a suitable drug carrier. 241
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This finding demonstrated the desirable safety of the DA-PLL-LA micelles (Fig. 6B).
almost unchanged at pH 6.8. Moreover, the reductive surrounding (10 mM GSH + pH 6.8) remarkably accelerated the release of Dox (approximately 60%) over 24 h. The fastest and almost complete release was observed at pH 5.3 in the presence of 10 mM GSH, in which approximately 80%, due to the acid-labile β-carboxylic amide of DA would hydrolyze at mildly acidic conditions [49], as well as the break of disulfide bonds in DA-PLL-LA copolymer responding to 10 mM of GSH stimuli, might cause a synergistic effect on the swelling and destabilization of DA-PLL-LA micelles. This effect was consistent with the outcomes of the size change study described in Section 3.2. These results revealed that the DA-PLL-LA micelles performed an on-demand drug release manner with little Dox released in the blood circulation but a rapid Dox release within tumor cells, which might lead to a lower systemic toxicity and an enhanced antitumor effect.
3.7. Tumor penetrability evaluation The targeting evaluation reflected the accumulation of DA-PLL-LA micelles in the tumor site but did not ensure further distribution in solid tumor. Therefore, the distribution of Dox and Dox-micelles in A549 tumor spheroids and solid tumor slices was further investigated via CLSM. After a 2-h pretreatment, the Dox-micelles group exhibited the strongest Dox fluorescence and showed no dramatic decrease from the edge to the deep section of tumor spheroids (Fig. 6C). These results indicate that the Dox-micelles exerted excellent penetrating effects. To further evaluate the tumor penetration ability, we illustrated the distribution of the micelles in subcutaneous tumor-bearing mice, which were injected with Dox in different formulations (5 mg/kg) via the tail vein. As shown in Fig. 6D, the Dox-micelles exhibited remarkably high distributions in the deep area. Thus, the Dox-micelles exhibited not only a better accumulation in the tumor site but also an enhanced penetrating ability into the internal region of the solid tumor.
3.5. Cell uptake 3.5.1. Subcellular distribution of micelles In Fig. 5A, A549 cells were incubated with Dox-micelles at pH 7.4 or 6.8 to investigate whether pH-triggered charge-reversal and reductionsensitive properties could facilitate cellular uptake and improve intracellular drug release. The uptake of Dox-micelles was enhanced as the surrounding pH decreased to 6.8, which benefited from the chargereversal property responding to the lower pH. Dox-micelles incubated for 2 h were mainly located in the cytoplasm, and the Fs of Dox increased with time. In other words, micelles gradually released Dox to cytoplasm then diffused to nuclei. These results indicated that targeted intracellular release of Dox from the dually pH/reduction-responsive Dox-micelles could be triggered by the microenvironment within tumor cells. This phenomenon allows for the co-location of Dox and nuclei and an enhanced apoptosis of A549 cells.
3.8. Targeting efficiency and antitumor effect in vivo
3.5.3. Quantitative flow cytometry analysis Results of the quantitative flow cytometry analysis, indicated that cells incubated with pH/reduction-responsive Dox-micelles in pH 6.8 exhibited a higher uptake than in pH 7.4 or Dox·HCl solution (Fig. 5C). Moreover, the fluorescence of Dox-micelles gradually increased with time.
Dir, a near-infrared (NIR) probe, was trapped into the micelles to explore the targeting effect of Dir/DA-PLL-LA in vivo by NIR fluorescence imaging. As shown in Fig. 7A, mice treated with Dir/DA-PLL-LA exhibited significantly high fluorescence intensities in the liver within 12 h because of the rapid uptake of micelles by the RES. At 24-h postinjection, more Dox was located in tumor tissue compared with adjacent normal tissues and organs. Hence, this result demonstrated that the micelles could targeted-delivery drugs to cancer cells via the EPR effect after systemic circulation. Thus, the stepwise pH/reduction-responsive polymeric micelles exerted an excellent target effect on the tumor. The fluorescence of major organs also confirmed the targeteddelivery ability of the micelles. To investigate the synergistic antitumor activity of the Dox-micelles in vivo, lung-tumor-bearing BALB/c mice were administrated with multiple formulations to detected the antitumor effect (Fig. 7B). The Dox-micelles exhibited stronger antitumor activity than saline and Dox and the body weight of mice in Dox alone treatment group showed notable decreased without tumor suppression effect. Furthermore, this body weight decrease exhibited in DOX group due to the systemic toxicity of Dox. After 11 days treatment, the tumor volume in the Dox group couldn't be inhibited but increased rapidly. Drug resistance may contribute to these failure anti-cancer effects. From the above, the Doxmicelles could exert a strong tumor suppressive effect in vivo. In general, the anticancer efficiency experiment in vivo confirmed that the stepwise pH/reduction-responsive micelles could simultaneously improve the therapeutic effect of Dox in vivo.
3.6. Cytotoxicity of Dox-micelles in vitro
4. Conclusion
The in vitro cytotoxicity of the blank micelles and Dox in different formulations was evaluated by MTT assay. Dox formulations, including Dox·HCl and Dox-micelles, exhibited dose-dependent cytotoxicity against A549 cell lines (Fig. 6A). The Dox-micelles showed higher cytotoxicity against A549 cells at pH 6.8 than at pH 7.4, which was further demonstrated by the IC50 value shown in Fig. 4B. The superior cytotoxicity of Dox-micelles at pH 6.8 was attributed to the enhanced cell internalization. Dox·HCl with low cellular uptake exhibited obvious cytotoxicity against A549 cells, which might be due to the rapid colocation with the cell nuclei and the induced cell apoptosis. These results demonstrated that the stepwise pH/reduction-responsive DA-PLLLA micelles showed high cytotoxicity to A549 cancer cells in a pH-dependent way. Of the cells, 90.11% and 91.78% survived after treatment with the micelles for 48 h against L929 and A549 cells, respectively.
In this study, we successfully developed a novel drug delivery system with enhanced cellular internalization and efficient drug release based on the stepwise pH/reduction-responsive DA-PLL-LA micelles. The charge-convertible cross-linked copolymer micelles with stable structure and negative surface charge in the blood stream exhibited high tumor-targeting ability via EPR effect. The internalized Dox-micelles exhibited fast and complete intracellular Dox release response to both low extracellular pH and high concentration of GSH in cytoplasm, which resulted in an enhanced cytotoxicity against A549 tumor cells. Therefore, the Dox-micelles showed excellent antitumor effects with lesser side effects in vivo. In summary, the stepwise pH/reduction-responsive DA-PLL-LA micelles were promising for the development of functional drug delivery systems for a safe and efficient therapy of lung cancer. These special tumor microenvironment and stimuli-responsive
3.5.2. Dynamic phagocytosis of micelles Dynamic uptake of Dox-micelles in A549 cells was detected by the live-cell station (Fig. 5B(a, b)). The results of Dox fluorescence in different formulations were according to Section 3.4. Moreover, the Doxmicelles under showed rapid uptake at pH 6.8 than at pH 7.4 within 1.5 h after administration. Several areas in the cytoplasm after Doxmicelles treatments were selected as ROIs to monitor the fluorescence intensities (Fig. 5B(c, d)). Cells treated with Dox-micelles in pH 6.8 exhibited a speedy accumulative uptake within 1.5 h than the cells treated with Dox-micelles in pH 7.4.
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