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Glutathione and pH-responsive chitosan-based nanogel as an efficient nanoplatform for controlled delivery of doxorubicin
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Glutathione and pH-responsive chitosan-based nanogel as an efficient nanoplatform for controlled delivery of doxorubicin Farideh Mahmoodzadeha, Marjan Ghorbanib,∗, Behrooz Jannata a b
Halal Research Center of IRI, FDA, Tehran, Iran Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Doxorubicin Smart responsive Nanogels Drug delivery Cancer
Incomplete delivery of drugs to the cancerous tissue and the drug resistance mechanisms limit the medical applications of anticancer drugs. Colloidal systems offers many advantages owing to the non-invasive way of drug administration as well as centralized delivery of anti-cancer drugs to tumor tissue. Nanogels (Ngs) as a part of these colloidal systems have a good perspective for their ability to incorporate and encapsulate low-molecularmass drugs, bio-macromolecules, and proteins. Therefore, we developed pH and redox-responsive Ngs to provide a hopeful prospect for targeted delivery of anti-cancer drugs in cancer cells. For this purpose, chitosan (CS) was first modified with a chain transfer agent (CTA) and then, the polymerization of 2-Hydroxyethyl methacrylate (HEMA) monomer occurred to create (CTS-g-PHEMA). Hydroxyl groups of HEMA reacted with maleic anhydride molecules to prepare CTS-g-PHEMA-maleic acid (MAc). Finally, the double bonds of MAc were used for the grafting of N, N′ bis(acryloyl) cystamine (BAC) as a crosslinker agent to prepare redox-sensitive Ngs. The biocompatibility, chemical structures, DOX loading capacity, content of the drug released and in-vitro cytotoxicity effects were also studied. As a result, it is expected that Ngs can be applied as a potential nanomedicine carrier for the treatment of cancer.
1. Introduction Cancer is a group of diseases with many possible causes worldwide, and cancer patients are most often treated with radiation or chemotherapy. Chemotherapy is one of the most common types of treatment, but like other treatments, it often causes side effects [1,2]. Scientists constantly work to develop drugs, drug combinations, and ways of giving treatment with fewer side effects [3]. For example, nano-sized drug carriers (NDC) in the area of nanomedicine have achieved considerable attention to overcome the problems arising from side effects of chemotherapy via the enhanced penetration effect (EPR) [4,5]. Among various types of NDC, three-dimensional polymeric networks named as nanogels (Ngs) have shown interesting potential owing to robustness and flexibility properties, nanoscale size and smart responsiveness to external stimuli [6–8]. The Ngs with smart responsiveness to endogenous or exogenous environmental changes (temperature, pH, and redox) can be a candidate for targeted and controlled-delivery of drugs to cancerous cells [9,10]. The advantage of redox-responsive Ngs is owing to the stability during their circulation in extracellular compartment, and degradation of Ngs is achieved by the high intracellular glutathione (GSH, a type of strong reducing agent inside cell)
∗
concentration inside cancer cells, leading to the fast drug release in the nucleus of cells [1,11,12]. Moreover, the pH in blood and healthy tissues is close to 7.4 while in tumors or inflammatory tissues it drops 0.5–1 units. Since the carriers first reach the internal cell structures from endosome (pH about 6) to lysosome with a pH of 4.5, so the pH gradient is observed during cellular uptake. According to this conditions, pH-sensitive NGs are also promising candidate for cancer therapy. Polymeric Ngs based on polysaccharides have been developed as the most promising candidate owing to their biocompatibility, biodegradability, functionality, low toxicity, and low cost [6,8,13]. In this regard, chitosan-based Ngs can be prepared via controlled radical polymerization as well as reversible addition of the fragmentation chain transfer (RAFT) polymerization technique owing to providing polymers with narrow dispersity, and controlled molecular weight [14,15]. The present study aimed to develop pH and redox-responsive Ngs based on N-phtaloyl chitosan-graft-poly (hydroxyl ethyl metacrylate)-graftmaleic acid-graft-N,N′ bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMAg-MAc-g-MBA)] for the triggered DOX release in cancer tissue. DOX was selected as the positively charged small molecule which can be easily penetrate small pores of the nanogels for conjugating to the negatively charged of the sites (maleic acid groups) on the developed nanogel.
Corresponding author. E-mail address: [email protected] (M. Ghorbani).
https://doi.org/10.1016/j.jddst.2019.101315 Received 16 August 2019; Received in revised form 5 October 2019; Accepted 7 October 2019 1773-2247/ © 2019 Published by Elsevier B.V.
Please cite this article as: Farideh Mahmoodzadeh, Marjan Ghorbani and Behrooz Jannat, Journal of Drug Delivery Science and Technology, https://doi.org/10.1016/j.jddst.2019.101315
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Scheme 1. Cell uptake mechanism for delivery of Doxorubicin (DOX) to the cancerous cell.
dried in a vacuum oven at 25 °C.
Moreover, DOX is a type of general anticancer drug called an anthracycline. But, resistance towards this drug is a general problem upon chemotherapy and like all chemotherapy drugs, DOX can cause side effects. In this regards, nanoformulation of DOX reduce the toxic side effects of drugs, increase their solubility and enable prolonged and controlled release of DOX. In the first step, functionalized N-phthaloyl-chitosan with 4-cyano, 4- [(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent) was prepared to provide macroinitiator. Then, HEMA was polymerized via the macroinitiator until the hydroxyl groups of HEMA could react with maleic anhydride (MAn) monomers to prepare CTS-g-PHEMA-g-MAc. In the third step, using the double bonds of MA, the N,N′ bis(acryloyl) cystamine (BAC) was grafted as a redox-sensitive monomer and a crosslinker agent to prepare novel dual-responsive Ngs. The designed Ngs were applied for loading DOX, and the improved drug’ anticancer activity was proved by in vitro assessments such as cellular uptake, cell imaging, DAPI staining and cell cycle analyses against cancer cells (Scheme 1).
2.3. Preparation of CTS-g-PHEMA-g-MAc The CTS-g-PHEMA-g-MAc was synthesized according to previously reported approach with minor modification [19]. Briefly, a dry polymerization ampoule was charged with CTS-g-PHEMA macroinitiator (1.00 g), and dried DMF (5 mL). After complete dissolution, 600 mg of MAn (6 mmol) was added to the solution under nitrogen atmosphere. After 24 h, the soluble products were dialyzed with dialysis bag against distilled water during 3 days (MWCO 12000) and lyophilized for further experiments. 2.4. Preparation of smart Ngs (CTS-g-PHEMA-g-MAc-g-BAC) Briefly, 40 mg of BAC (0.16 mmol), 2.65 mg of AIBN (0.016 mmol) and 80 mg of CTS-g-PHEMA-g-MAc were dissolved in 10 ml dried DMF solution at the round-bottom flask. After being purged with nitrogen atmosphere for 30 min, the flask was placed in an oil bath preset at 70 °C for 48. Finally, to remove the unreacted monomer and solvent, the solution was diluted with water (25 mL) and transferred into a dialysis bag (molecular weight cut off 1000 Da) and dialyzed during 3 days to achieve Ngs.
2. Experimental 2.1. Materials HEMA, CS (medium molecular weight, extent of deacetylation 75–85%), GSH, BAC, MAn, dimethylaminopyridine (DMAP), dicyclohexyl carbodiimide (DCC), WST1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene Disulfonate), phosphate buffered saline (PBS), glutathione (GSH), 4-cyano, 4- [(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent), and phthalic anhydride were purchased from Sigma-Aldrich (Company Ltd., Dorset, UK). The initiator of 2, 2-azobisisobutyronitrile (AIBN; Switzerland) was purchased from Sigma-Aldrich.
2.5. Characterization Fourier transform infrared (FTIR) spectroscopy was performed using a Shimadzu 8101 M FTIR (Kyoto, Japan) in the pellet form with potassium bromide (KBr) powder at room temperature. Proton nuclear magnetic resonance (1HNMR) spectra were recorded on a Bruker spectrometer (Bruker, Ettlingen, Germany) with an operating frequency of 400 MHz at 25 °C. Transmission electron microscopy (TEM) was conducted on a Philips CM10-TH microscope (Phillips, Eindhoven, Netherlands). By photon correlation spectroscopy, the average diameter was analyzed (Instruments, Malvern, UK). The average hydrodynamic diameters of the samples were measured by photon correlation spectroscopy (PCS) (ZetasizerZS, Malvern, UK) equipped with a He–Ne laser at the scattering angle of 90 °C.
2.2. Preparation of CTS-g-PHEMA CTS macroinitiator was obtained from the esterification of Nphthaloyl-CS via RAFT agent [16–18]. In a typical procedure, 0.2 g of CTS, 2.0 mg of AIBN (12 μmol) and 0.5 g of HEMA (3 mmol) was dissolved in 6 ml dried DMF solution in the round-bottom flask. After being purged with argon gas, the flask was placed in an oil bath preset at 75 °C for 8 h. Lastly, the flask was cooled using an ice/water bath to quench the polymerization. To obtain the CTS-g-PHEMA, the product was diluted with 6 mL DMF, and then purred into a diethyl ether (300 mL). Finally, the product was filtered by Whatman filter paper and
2.6. Preparations of DOX-Ngs DOX-Ngs were achieved by using a membrane dialysis method [1] Briefly, 25 mg of smart Ngs was dispersed in 5 mL of DI water to achieve polymer solution. Then, 10 mg of DOX was added to the polymeric 2
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2.11. Cell cycle arrest study
solution and then stirred for 48 h After this time, the unloaded drug was separated from DOX-Ngs by using an Amicon® filter (molecular weight cutoff 100 kDa kDa, Millipore, UK), and then its concentration was investigated by UV–Vis spectrophotometer at 480 nm. The encapsulation efficiency (EE) and loading capacity (LC) of DOX were calculated by the following equations (12)
LC(%) =
milligram of drug in nanogels × 100 milligram of nanogels
EE(%) =
milligram of drug in nanogels × 100 milligram of initial added drug
The test is established on the stoichiometric binding of propidium iodide to cumulative amounts of DNA in different cell cycle phases (G0/ G1, S, and G2/M) by flow cytometry. The cells (4 × 105 cells/mL) were seeded, cultured and then treated with DOX, DOX-Ngs and nano-blank for 24 h. After that, the cells were trypsinized and the pellet of samples was washed twice with PBS, fixed and permeabilized using ethanol (70%). After 24 h, the incubated cells were centrifuged (300 ×g for 10 min at 4 °C), rinsed in phosphate-buffered saline (PBS), and next treated with RNAase. DNA was then quantitatively stained with propidium iodide for 1 h at 37 °C. Cell population were analyzed using a FACS calibur flow cytometer (Becton Dickinson) and FlowJo software (Treestar, Ashland, OR, USA).
2.7. In vitro release study
3. Results and discussion
10 mg of DOX-Ngs were suspended in phosphate buffered saline (30 ml, PBS) and then put into a dialysis bag [20]. To confirm the effective performance of DOX-Ngs, the release behavior of DOX-Ngs were assessed in both simulated tumor tissue (pH 5.3, 40 °C) and physiologic (pH 7.4, 37 °C) conditions. 1 mL of released drug were collected at predetermined time intervals to evaluation the released drug content using UV–vis spectrophotometer at 480 nm. It is essential to note, the aliquots were brought back into the flask to keep the same total solution volume.
Engineered DOX nanoparticles were prepared using BAC as a stabilizing and crosslinker agent. In this research, we provided a novel pH and redox-responsive nanoplatform for delivery of DOX based on chitosan. The brief synthesis protocols of DOX-Ngs are as follows: CS was first modified by a chain transfer agent (CTA) to provide CTS macroinitiator. Then, the grafting polymerization of the HEMA monomer was controlled via RAFT polymerization to generate CTS-gPHEMA. The Hydroxyl groups of HEMA were applied to react with MAn to prepare CTS-g-PHEMA-MA. To produce redox-responsive Ngs, the double bonds of MAn were used for polymerization of BAC as a redoxsensitive crosslinker agent. Last, the antitumor drug DOX was loaded via two ways: a) by physical entrapment in cavity of Ngs and b) electrostatic interaction with maleic acid groups (Scheme 2).
2.8. In vitro cell cytotoxicity The MDAMB-231 cells were obtained from Iranian National Cell Bank (Pasteur Institute, Tehran, Iran) and cultivated in RPMI1640. The cells were cultured into flasks before the addition of samples. To study the cytotoxicity effects of developed nanocarriers, the cells were treated with different concentrations of Ngs, DOX, and DOX-Ngs for 48 and 72 h. Finally, the culture medium was aspirated, and 200 μL cultivation medium containing 20 μL of WST1 solution was added to each well. Next, after incubation for 1 h, the optical density was recorded via a micro-plate reader at 450 nm (Elx808, Biotek, USA) [21–26].
3.1. Characterization of synthesized CS-based polymers The chemical composition of CS-based polymers was characterized by FTIR analysis (Fig. 1). The stretching vibrations of amide carbonyl group at 1652 cm-1, the aliphatic C–H band at 2925–2881 cm−1, the C–O group at 1072 cm−1, and the C–N at 1388 cm−1 were shown in the FTIR spectrum of CS [27]. The synthesis of CTS was specified by the presence of stretching vibrations of the aliphatic and aromatic C–H band at 3100–2850 cm−1, the stretching vibrations of carbonyl groups at 1710 cm−1, and γ (C–H) in the aromatic ring at 873 and 721 cm−1. The FTIR spectrum of CTS displays the stretching vibrations of the cyanide and carbonyl groups of CTA moiety as new bands at 2250 and 1728 cm−1, respectively [16]. Moreover, FTIR clearly confirms the preparation of CTS-g-PHEMA copolymer, which showed two strong absorption peaks at approximately 1730 cm−1 and 3400 cm−1. These bonds could be attributed to the carbonyl and hydroxyl groups of HEMA. The ring opening reaction of MAn groups was approved by increasing the intensity of band at 1730 cm−1, which is attributed to the formation of carbonyl groups (steric groups) of MAc [1,28]. The presence of new bands in the spectrum of Ngs approved the successful synthesis of Ngs. The band at 1682 cm−1 could correspond to the new amide I (C = O stretching), which was attributed to the amide group of BAC. The stretching vibration of the (C–N) group of BAC appeared at 1280 cm−1, and the absorption peaks of (C–O) in HEMA and Mac were observed at approximately 1126 and 1187 cm−1, respectively. The synthesized polymers were further characterized using 1HNMR spectroscopy (Fig. 2). Some characteristic chemical shifts of the CS backbone were overlapped by the DMSO chemical resonance as the NMR solvent impurity. The chemical shift of CTS aromatic protons appeared at 7.6–7.9 related to the benzene and phthaloyl rings [16]. The 1HNMR spectrum of the CTS-g-PHEMA (in DMSO) showed the peaks at: 4.1 (2H, - CH2–CH2–OH of PHEMA), 3.90 (2H, –COO–CH2- of PHEMA), 1.60–2.10 (2H, –CH2- C(CH3)(COO)- of PHEMA), and 0.95–1.2 (3H, eCH2C(CH3)(COO)e of PHEMA). The signals at 6.28 and 6.86 ppm were attributed to the methane protons of the MA groups, confirming
2.9. DAPI staining MDAMB-231 cells were seeded on glass cover slips in 6-well plates with fresh RPMI1640 containing 10% FBS and 1% penicillin-streptomycin at 37 °C for 24 h [1,22]. Briefly, DOX-Ngs, Ngs and DOX in fresh culture medium were added to substitute the primary media and incubated with cells (5 × 105) for 1 day. After the treatment, cells were washed three times with cold PBS and fixed in 2 mL 4% paraformaldehyde for 10 min. After that, the cells were stained with DAPI following the manufacturer's instructions and observed using an inverted fluorescence microscopy (Bh2-RFCA, Olympus, Japan), and the typical photographs were captured. 2.10. Cellular uptake study Cellular uptake evaluation of MDAMB-231 cells was measured by Flow Cytometry [23]. The cells were seeded in six-well plates at a density of 3 × 105 cells per well and cultured at 37 °C for 24 h. For cellular internalization assay, cells were treated by free DOX or DOXNgs at an equivalent DOX concentration of 5 μg/mL. After incubation at 37 °C for 3 h, the original medium was removed and the cells were washed three times with 1 mL of cold PBS solution. Digestion of cells were performed by adding 0.5 mL of trypsin and centrifuged at 1000 rpm for 5 min. The pelletes were washed twice with cold PBS, followed by centrifugation. Finally, the cell were resuspended in 500 μL PBS and the fluorescence of DOX trapped by cells was analyzed using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). 3
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Scheme 2. The method for the synthesis of N-phtaloyl chitosan-graft-poly (hydroxyl ethyl metacrylate)-graft-maleic acid-graft- N,N′-bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMA-g-MAc-g-MBA)].
environment-modulated zeta potential of the surfaces. Therefore, the zeta potential of Ngs was determined to be −24 (mV) that was enough to absorb the DOX drug on the surface of Ngs (Fig. 3c). (Fig. 3c). After the coupling of DOX on the surface of Ngs, the zeta potential was decreased to −13 (mV) (Fig. 3d).
the successful gafting of MAc onto the backbone of the CTS-g-PHEMA. Additional confirmation data for the successful synthesis of the CTSgPHEMA-g-MAc with narrow dispersity were estimated from the GPC analysis (Fig. 2b). The average molecular weight and polydispersity index were 285,000 g/mol and 1.42, respectively. Good control during the polymerization was determined with dispersity (Ð) values below 1.6 [29]. As Fig. 3a shows, the resulting Ngs were first carefully considered by DLS in PBS. The particle size and PDI of nanocarrier systems are the main physicochemical features influencing the endocytosis-dependent cellular uptake [30]. The most nanocarriers larger than 100–150 nm can be taken up by phagocytes or remain in these tissues for an extended time.Thus, we measured the nano-ranged size of the Ngs using TEM observation and PCS measurements. TEM observation showed uniformly spherical shape of Ngs with a diameter of 60–70 nm and demonstrated well dispersed Ngs, being in agreement with the PCS measurements (Fig. 3e). As this Figure shows, no obviously detectable aggregates could be observed in the DOX-Ngs sample. Moreover, by PCS measurement, the average size of the Ngs were recorded to be 91.22 nm. Furthermore, as Fig. 3b demonstrates, the values of the hydrodynamic diameter was reduced to < 10 nm at GSH 10 Mm, since GSH was a strong reducing agent, which can diffuse into the Ngs network and break the -S-S- bonds into 2 mercapto groups (-SH) of polymer chains. The variance between DLS and TEM can be related to the hydration water layer outside the Ngs, which could not be observed by TEM due to the different mechanism of each procedure. It should be noted that the size distribution of the Ngs was very narrow (PDI~0.199). Surface properties play a key role in the colloidal stability of nanoparticles. Ngs stabilized with the repulsive interactions are considerably to be mostly electrostatic and controlled by the
3.2. Encapsulation and in vitro release behavior study of DOX-Ngs MAc has been widely used in pH-responsive nanoparticles owing to the presence of reversibly carboxylic groups. Thus, in this work, the prepared Ngs showed some pHsensitive properties due to the carboxylic groups of MAc. DOX in salt form, an anticancer hydrophilic drug was chosen as a model drug to be loaded into the corresponding Ngs. The corresponding resulting drug loading capacities and efficiencies demonstrated good loading capacity (14%) and efficiency (76%). Since DOX is a positively charged drug in physiological pH condition (pH 7.4), DOX loading can be achieved through the electrostatic interaction. Considering the lower pH value in tumor cells, this pH-responsive property of Ngs contributes to acceleration of the drug release from Ngs. Fig. 4 presents the drug release profiles of DOX-Ngs in PBS (pH 7.4 and 5.3). Owing to the existence of disulfide bonds and carboxylic groups on the structure of Ngs that contributed to realize the quick and controllable release of DOX, the drug release behavior would be affected by both GSH and pH values. From the size profile of DOX-Ngs after treating with 10 mM GSH, It could be found that the Ngs disappeared for 48 h, confirming that DOX could be completely released as 7 stimulated by GSH. While, the cumulative release rate of DOX was < 20% at a physiologic condition (pH 7.4, 37 °C, 2 μm) after 48 h in the absence of GSH or low content of GSH (2 μm). A faster release in higher content of GSH (10 mM) reaching 71.0% or 82% was observed at 4
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Fig. 1. FTIR spectra of chitosan (CS), N-phthaloyl-CS, CS-chain transfer agent (CTS), CS-g-poly(2-Hydroxyethyl methacrylate (HEMA) [(CTS-g-PHEMA)], CTS-g-PHEMA-maleic acid (MAc)[(CTS-g-PHEMA-g-MAc)], (CTS-g-PHEMA-gMAc)] -g-N,N′-bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMA-g-MAc-g-MBA)].
Fig. 2. 1HNMR spectra of CTS-g-poly(2-Hydroxyethyl methacrylate (HEMA) [(CTS-g-PHEMA)], CTS-g-PHEMA-maleic acid (MAc)[(CTS-g-PHEMA-g-MAc)] (a) and GPC chromatogram of CTS-g-PHEMA-g-MAc (b).
pH 7.4 and 5.3, respectively. Therefore, at different Ph values, the drug release rate at pH 5.0 was higher than that of pH 7.4. In conclusion, owing to a significant difference between tumor tissue and normal physiologic condition, the low release rate at pH 7.4 and low content of GSH was promising to keep the Ngs stable and thus improving the anticancer effects.
the cell viability considerably decreased when the cells were incubated with DOX and DOX-Ngs during 72 h even at 4 μg/mL and 16 μg/mL for DOX-Ngs and DOX. Correspondingly, an IC50 value of 9.26 μg/mL, 7.24 μg/mL and 3.75 μg/mL was obtained for DOX-Ngs during 24, 48, and 72 h, respectively. Overall, these results proved that the released DOX from the Ngs could still exhibit high anticancer activity in most concentrations. It should be noted since the EPR effect can only occur in in vivo tests, in in vitro studies, we could not clearly observe the superiority of drugloaded nanocarriers compared to free drugs. Thus, the cytotoxicity of DOX-Ngs is slightly higher than that of free DOX at all the studied concentrations. Moreover, since the blank Ngs did not show any cytotoxicity, the therapeutic efficacy was only owing to the DOX-
3.3. In vitro cytotoxicity The effects of the concentration of the Ngs on the proliferation of MDAMB-231 cells were investigated. Fig. 5 shows that no signs of cytotoxicity on cells were observed upon treatment with the blank-Ngs for 24, 48 and 72 h. This was further confirmed that all the free Ngs were low-cytotoxic even after 72 h in high concentrations. On the contrary, 5
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Fig. 3. Size profiles of Doxorubicin-loaded nanogels (DOX-Ngs) in pH 7.4, GSH 2 μM (a) and DOX-Ngs in pH 5.3, GSH 10 mM (b), Zeta potentials of Ngs (b) and DOXNgs (c), and Transmission electron microscopy (TEM) of DOX-Ngs (e).
Fig. 6. DAPI staining of MDAMB-231 cells (control), treated with DOX, DOXNgs and Ngs. Fig. 4. In vitro Doxorubicin (DOX) release profiles under different pH (pH = 7.4; and 5.0) and GSH concentrations from the DOX-Ngs (data are presented as mean ± standard deviation, n = 3).
nuclei and condensation of chromatin in nuclear via both DOX and DOX-Ngs. As Fig. 6 shows, the rate of these morphological changes named as apoptosis for cells was more in the cells treated with DOX-Ngs than those treated with free DOX. Finally, it could be concluded that the DNA damage was in more stages in DOX-Ngs compared to free DOX, suggesting that this developed nanocarrier may be a promising delivery
loaded Ngs. In another study, the apoptosis properties of the DOX-Ngs were analyzed and compared to free DOX against MDAMB-231 cells [31,32]. The morphological changes were detected as fragmentation of
Fig. 5. The cell viability study of Doxorubicin (DOX), DOX-Ngs and Ngs with different concentrations in the time period of 24 h (a), 48 h (b) and 72 h (c) against human breast adenocarcinoma (MDAMB-231) cells. The results was calculated as the mean ± standard deviation (n = 3). *P < .05, **P < .01. 6
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Fig. 7. Cellular uptake study of human breast adenocarcinoma (MDAMB-231) cells after incubation with fluorescein labeled Doxorubicin (DOX)-nanogels (DOX-Ngs) after 1 and 3 h.
treatment with DOX-MP-Au/Ngs from 2.3% to 21.18% for DOX and DOX-Ngs, respectively. Finally, there was no effect on the cell cycle arrest identified in the cells treated with the untreated group.
system for chemotherapeutic agents. 3.4. Cell internalization study
4. Conclusion
It is extremely important that nanocarriers should be efficiently internalized in cells and be able to deliver the drug there to use the required therapeutic efficacy [33]. Since DOX is a fluorescent molecule, its cell uptake by MDAMB-231 cells could be observed by a fluorescent microscope. As Fig. 7 depicts, the results indicated that cells treated with DOX-Ngs presented obvious stronger intensity in DOX-Ngs than DOX alone. The enhanced cell uptake ability may be associated with the expected mild drug resistance of DOX alone. In this regard, as the cell viability profiles show, DOX-Ngs exhibited higher or similar cytotoxicity against cancer cells as compared to the free DOX. Furthermore, the low fluorescence intensity was observed after 1 h, which could be related to the low amount of DOX released from the Ngs. While, the fluorescence intensity was rather enhanced after 3 h, approving that a larger amount of DOX was released. These results proved that the Ngs could effectively deliver DOX into the cell nuclei.
In summary, we designed the stimuli-responsive chitosan-based Ngs as a new smart drug delivery nanosystem via the RAFT polymerization, in which DOX was coupled into MAc groups of Ngs. The redox-responsiveness of Ngs were successfully achieved by polymerization of BAC as a disulfide crosslinker agent. The prepared (CTS-g-PHEMA-gMA-g-BAC) Ngs had a suitable loading capacity of DOX (14%) and uniform sphere shape in aqueous solution. The developed DOX-Ngs demonstrated a smart and quickly release behavior in response to thiol reducing agents such as GSH, and pH in simulated cancerous tissue medium. Experiments in MDAMB-231 cells exposed that the Ngs were compatible with these cell lines. DOX-Ng's showed a high cytotoxicity in comparing with free DOX due to the delivery of DOX into the cells, as shown by inverted fluorescence microscopy. This new designing concept may be promising to help the development of a variety of new Ngs for other chemotherapeutic agents, which may offer favorable and novel prospect in cancer therapy.
3.5. Cell cycle arrest analysis To explore the potential of DOX-Ngs to induce the cell growth inhibition, the effect of Ngs, DOX and DOX-Ngs on cell cycle distribution was tested [34] (Fig. 8). For this 9 purpose, the cell populations of dead cells are quantified by flow cytometry analysis. To determine the distribution in different phases of the cell cycle, the DNA content in the cells was detected by propidium iodide (PI) staining on the cells incubated with DOX, DOX-Ngs and Ngs for 24 h. The results showed that DOX-Ngs induced a significant increase in the percentage of the cells in the sub-G0/G1 phase. Therefore, an increase in the portion of apoptotic cells by accumulation of cells in the sub-G0/G1 region enhanced after
Declaration of competing interest The authors confirm that there are no known conflicts of interest associated with this publication and there has been no financial support for this work that could have influenced its outcome. Acknowledgment The financial support from the Stem Cell Research Center, Tabriz 7
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Fig. 8. Effects of the DOX, Ngs (without drugs) and drug loaded Ngs (DOX-Ngs) on cell cycle distribution of MCF-7 cells (human breast epithelial adenocarcinoma cell line).
University of Medical Sciences was gratefully acknowledged.
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Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Glutathione and pH-responsive chitosan-based nanogel as an efficient nanoplatform for controlled delivery of doxorubicin Farideh Mahmoodzadeha, Marjan Ghorbanib,∗, Behrooz Jannata a b
Halal Research Center of IRI, FDA, Tehran, Iran Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Doxorubicin Smart responsive Nanogels Drug delivery Cancer
Incomplete delivery of drugs to the cancerous tissue and the drug resistance mechanisms limit the medical applications of anticancer drugs. Colloidal systems offers many advantages owing to the non-invasive way of drug administration as well as centralized delivery of anti-cancer drugs to tumor tissue. Nanogels (Ngs) as a part of these colloidal systems have a good perspective for their ability to incorporate and encapsulate low-molecularmass drugs, bio-macromolecules, and proteins. Therefore, we developed pH and redox-responsive Ngs to provide a hopeful prospect for targeted delivery of anti-cancer drugs in cancer cells. For this purpose, chitosan (CS) was first modified with a chain transfer agent (CTA) and then, the polymerization of 2-Hydroxyethyl methacrylate (HEMA) monomer occurred to create (CTS-g-PHEMA). Hydroxyl groups of HEMA reacted with maleic anhydride molecules to prepare CTS-g-PHEMA-maleic acid (MAc). Finally, the double bonds of MAc were used for the grafting of N, N′ bis(acryloyl) cystamine (BAC) as a crosslinker agent to prepare redox-sensitive Ngs. The biocompatibility, chemical structures, DOX loading capacity, content of the drug released and in-vitro cytotoxicity effects were also studied. As a result, it is expected that Ngs can be applied as a potential nanomedicine carrier for the treatment of cancer.
1. Introduction Cancer is a group of diseases with many possible causes worldwide, and cancer patients are most often treated with radiation or chemotherapy. Chemotherapy is one of the most common types of treatment, but like other treatments, it often causes side effects [1,2]. Scientists constantly work to develop drugs, drug combinations, and ways of giving treatment with fewer side effects [3]. For example, nano-sized drug carriers (NDC) in the area of nanomedicine have achieved considerable attention to overcome the problems arising from side effects of chemotherapy via the enhanced penetration effect (EPR) [4,5]. Among various types of NDC, three-dimensional polymeric networks named as nanogels (Ngs) have shown interesting potential owing to robustness and flexibility properties, nanoscale size and smart responsiveness to external stimuli [6–8]. The Ngs with smart responsiveness to endogenous or exogenous environmental changes (temperature, pH, and redox) can be a candidate for targeted and controlled-delivery of drugs to cancerous cells [9,10]. The advantage of redox-responsive Ngs is owing to the stability during their circulation in extracellular compartment, and degradation of Ngs is achieved by the high intracellular glutathione (GSH, a type of strong reducing agent inside cell)
∗
concentration inside cancer cells, leading to the fast drug release in the nucleus of cells [1,11,12]. Moreover, the pH in blood and healthy tissues is close to 7.4 while in tumors or inflammatory tissues it drops 0.5–1 units. Since the carriers first reach the internal cell structures from endosome (pH about 6) to lysosome with a pH of 4.5, so the pH gradient is observed during cellular uptake. According to this conditions, pH-sensitive NGs are also promising candidate for cancer therapy. Polymeric Ngs based on polysaccharides have been developed as the most promising candidate owing to their biocompatibility, biodegradability, functionality, low toxicity, and low cost [6,8,13]. In this regard, chitosan-based Ngs can be prepared via controlled radical polymerization as well as reversible addition of the fragmentation chain transfer (RAFT) polymerization technique owing to providing polymers with narrow dispersity, and controlled molecular weight [14,15]. The present study aimed to develop pH and redox-responsive Ngs based on N-phtaloyl chitosan-graft-poly (hydroxyl ethyl metacrylate)-graftmaleic acid-graft-N,N′ bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMAg-MAc-g-MBA)] for the triggered DOX release in cancer tissue. DOX was selected as the positively charged small molecule which can be easily penetrate small pores of the nanogels for conjugating to the negatively charged of the sites (maleic acid groups) on the developed nanogel.
Corresponding author. E-mail address: [email protected] (M. Ghorbani).
https://doi.org/10.1016/j.jddst.2019.101315 Received 16 August 2019; Received in revised form 5 October 2019; Accepted 7 October 2019 1773-2247/ © 2019 Published by Elsevier B.V.
Please cite this article as: Farideh Mahmoodzadeh, Marjan Ghorbani and Behrooz Jannat, Journal of Drug Delivery Science and Technology, https://doi.org/10.1016/j.jddst.2019.101315
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Scheme 1. Cell uptake mechanism for delivery of Doxorubicin (DOX) to the cancerous cell.
dried in a vacuum oven at 25 °C.
Moreover, DOX is a type of general anticancer drug called an anthracycline. But, resistance towards this drug is a general problem upon chemotherapy and like all chemotherapy drugs, DOX can cause side effects. In this regards, nanoformulation of DOX reduce the toxic side effects of drugs, increase their solubility and enable prolonged and controlled release of DOX. In the first step, functionalized N-phthaloyl-chitosan with 4-cyano, 4- [(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent) was prepared to provide macroinitiator. Then, HEMA was polymerized via the macroinitiator until the hydroxyl groups of HEMA could react with maleic anhydride (MAn) monomers to prepare CTS-g-PHEMA-g-MAc. In the third step, using the double bonds of MA, the N,N′ bis(acryloyl) cystamine (BAC) was grafted as a redox-sensitive monomer and a crosslinker agent to prepare novel dual-responsive Ngs. The designed Ngs were applied for loading DOX, and the improved drug’ anticancer activity was proved by in vitro assessments such as cellular uptake, cell imaging, DAPI staining and cell cycle analyses against cancer cells (Scheme 1).
2.3. Preparation of CTS-g-PHEMA-g-MAc The CTS-g-PHEMA-g-MAc was synthesized according to previously reported approach with minor modification [19]. Briefly, a dry polymerization ampoule was charged with CTS-g-PHEMA macroinitiator (1.00 g), and dried DMF (5 mL). After complete dissolution, 600 mg of MAn (6 mmol) was added to the solution under nitrogen atmosphere. After 24 h, the soluble products were dialyzed with dialysis bag against distilled water during 3 days (MWCO 12000) and lyophilized for further experiments. 2.4. Preparation of smart Ngs (CTS-g-PHEMA-g-MAc-g-BAC) Briefly, 40 mg of BAC (0.16 mmol), 2.65 mg of AIBN (0.016 mmol) and 80 mg of CTS-g-PHEMA-g-MAc were dissolved in 10 ml dried DMF solution at the round-bottom flask. After being purged with nitrogen atmosphere for 30 min, the flask was placed in an oil bath preset at 70 °C for 48. Finally, to remove the unreacted monomer and solvent, the solution was diluted with water (25 mL) and transferred into a dialysis bag (molecular weight cut off 1000 Da) and dialyzed during 3 days to achieve Ngs.
2. Experimental 2.1. Materials HEMA, CS (medium molecular weight, extent of deacetylation 75–85%), GSH, BAC, MAn, dimethylaminopyridine (DMAP), dicyclohexyl carbodiimide (DCC), WST1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene Disulfonate), phosphate buffered saline (PBS), glutathione (GSH), 4-cyano, 4- [(phenylcarbothioyl) sulfanyl] pentanoic acid (RAFT agent), and phthalic anhydride were purchased from Sigma-Aldrich (Company Ltd., Dorset, UK). The initiator of 2, 2-azobisisobutyronitrile (AIBN; Switzerland) was purchased from Sigma-Aldrich.
2.5. Characterization Fourier transform infrared (FTIR) spectroscopy was performed using a Shimadzu 8101 M FTIR (Kyoto, Japan) in the pellet form with potassium bromide (KBr) powder at room temperature. Proton nuclear magnetic resonance (1HNMR) spectra were recorded on a Bruker spectrometer (Bruker, Ettlingen, Germany) with an operating frequency of 400 MHz at 25 °C. Transmission electron microscopy (TEM) was conducted on a Philips CM10-TH microscope (Phillips, Eindhoven, Netherlands). By photon correlation spectroscopy, the average diameter was analyzed (Instruments, Malvern, UK). The average hydrodynamic diameters of the samples were measured by photon correlation spectroscopy (PCS) (ZetasizerZS, Malvern, UK) equipped with a He–Ne laser at the scattering angle of 90 °C.
2.2. Preparation of CTS-g-PHEMA CTS macroinitiator was obtained from the esterification of Nphthaloyl-CS via RAFT agent [16–18]. In a typical procedure, 0.2 g of CTS, 2.0 mg of AIBN (12 μmol) and 0.5 g of HEMA (3 mmol) was dissolved in 6 ml dried DMF solution in the round-bottom flask. After being purged with argon gas, the flask was placed in an oil bath preset at 75 °C for 8 h. Lastly, the flask was cooled using an ice/water bath to quench the polymerization. To obtain the CTS-g-PHEMA, the product was diluted with 6 mL DMF, and then purred into a diethyl ether (300 mL). Finally, the product was filtered by Whatman filter paper and
2.6. Preparations of DOX-Ngs DOX-Ngs were achieved by using a membrane dialysis method [1] Briefly, 25 mg of smart Ngs was dispersed in 5 mL of DI water to achieve polymer solution. Then, 10 mg of DOX was added to the polymeric 2
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2.11. Cell cycle arrest study
solution and then stirred for 48 h After this time, the unloaded drug was separated from DOX-Ngs by using an Amicon® filter (molecular weight cutoff 100 kDa kDa, Millipore, UK), and then its concentration was investigated by UV–Vis spectrophotometer at 480 nm. The encapsulation efficiency (EE) and loading capacity (LC) of DOX were calculated by the following equations (12)
LC(%) =
milligram of drug in nanogels × 100 milligram of nanogels
EE(%) =
milligram of drug in nanogels × 100 milligram of initial added drug
The test is established on the stoichiometric binding of propidium iodide to cumulative amounts of DNA in different cell cycle phases (G0/ G1, S, and G2/M) by flow cytometry. The cells (4 × 105 cells/mL) were seeded, cultured and then treated with DOX, DOX-Ngs and nano-blank for 24 h. After that, the cells were trypsinized and the pellet of samples was washed twice with PBS, fixed and permeabilized using ethanol (70%). After 24 h, the incubated cells were centrifuged (300 ×g for 10 min at 4 °C), rinsed in phosphate-buffered saline (PBS), and next treated with RNAase. DNA was then quantitatively stained with propidium iodide for 1 h at 37 °C. Cell population were analyzed using a FACS calibur flow cytometer (Becton Dickinson) and FlowJo software (Treestar, Ashland, OR, USA).
2.7. In vitro release study
3. Results and discussion
10 mg of DOX-Ngs were suspended in phosphate buffered saline (30 ml, PBS) and then put into a dialysis bag [20]. To confirm the effective performance of DOX-Ngs, the release behavior of DOX-Ngs were assessed in both simulated tumor tissue (pH 5.3, 40 °C) and physiologic (pH 7.4, 37 °C) conditions. 1 mL of released drug were collected at predetermined time intervals to evaluation the released drug content using UV–vis spectrophotometer at 480 nm. It is essential to note, the aliquots were brought back into the flask to keep the same total solution volume.
Engineered DOX nanoparticles were prepared using BAC as a stabilizing and crosslinker agent. In this research, we provided a novel pH and redox-responsive nanoplatform for delivery of DOX based on chitosan. The brief synthesis protocols of DOX-Ngs are as follows: CS was first modified by a chain transfer agent (CTA) to provide CTS macroinitiator. Then, the grafting polymerization of the HEMA monomer was controlled via RAFT polymerization to generate CTS-gPHEMA. The Hydroxyl groups of HEMA were applied to react with MAn to prepare CTS-g-PHEMA-MA. To produce redox-responsive Ngs, the double bonds of MAn were used for polymerization of BAC as a redoxsensitive crosslinker agent. Last, the antitumor drug DOX was loaded via two ways: a) by physical entrapment in cavity of Ngs and b) electrostatic interaction with maleic acid groups (Scheme 2).
2.8. In vitro cell cytotoxicity The MDAMB-231 cells were obtained from Iranian National Cell Bank (Pasteur Institute, Tehran, Iran) and cultivated in RPMI1640. The cells were cultured into flasks before the addition of samples. To study the cytotoxicity effects of developed nanocarriers, the cells were treated with different concentrations of Ngs, DOX, and DOX-Ngs for 48 and 72 h. Finally, the culture medium was aspirated, and 200 μL cultivation medium containing 20 μL of WST1 solution was added to each well. Next, after incubation for 1 h, the optical density was recorded via a micro-plate reader at 450 nm (Elx808, Biotek, USA) [21–26].
3.1. Characterization of synthesized CS-based polymers The chemical composition of CS-based polymers was characterized by FTIR analysis (Fig. 1). The stretching vibrations of amide carbonyl group at 1652 cm-1, the aliphatic C–H band at 2925–2881 cm−1, the C–O group at 1072 cm−1, and the C–N at 1388 cm−1 were shown in the FTIR spectrum of CS [27]. The synthesis of CTS was specified by the presence of stretching vibrations of the aliphatic and aromatic C–H band at 3100–2850 cm−1, the stretching vibrations of carbonyl groups at 1710 cm−1, and γ (C–H) in the aromatic ring at 873 and 721 cm−1. The FTIR spectrum of CTS displays the stretching vibrations of the cyanide and carbonyl groups of CTA moiety as new bands at 2250 and 1728 cm−1, respectively [16]. Moreover, FTIR clearly confirms the preparation of CTS-g-PHEMA copolymer, which showed two strong absorption peaks at approximately 1730 cm−1 and 3400 cm−1. These bonds could be attributed to the carbonyl and hydroxyl groups of HEMA. The ring opening reaction of MAn groups was approved by increasing the intensity of band at 1730 cm−1, which is attributed to the formation of carbonyl groups (steric groups) of MAc [1,28]. The presence of new bands in the spectrum of Ngs approved the successful synthesis of Ngs. The band at 1682 cm−1 could correspond to the new amide I (C = O stretching), which was attributed to the amide group of BAC. The stretching vibration of the (C–N) group of BAC appeared at 1280 cm−1, and the absorption peaks of (C–O) in HEMA and Mac were observed at approximately 1126 and 1187 cm−1, respectively. The synthesized polymers were further characterized using 1HNMR spectroscopy (Fig. 2). Some characteristic chemical shifts of the CS backbone were overlapped by the DMSO chemical resonance as the NMR solvent impurity. The chemical shift of CTS aromatic protons appeared at 7.6–7.9 related to the benzene and phthaloyl rings [16]. The 1HNMR spectrum of the CTS-g-PHEMA (in DMSO) showed the peaks at: 4.1 (2H, - CH2–CH2–OH of PHEMA), 3.90 (2H, –COO–CH2- of PHEMA), 1.60–2.10 (2H, –CH2- C(CH3)(COO)- of PHEMA), and 0.95–1.2 (3H, eCH2C(CH3)(COO)e of PHEMA). The signals at 6.28 and 6.86 ppm were attributed to the methane protons of the MA groups, confirming
2.9. DAPI staining MDAMB-231 cells were seeded on glass cover slips in 6-well plates with fresh RPMI1640 containing 10% FBS and 1% penicillin-streptomycin at 37 °C for 24 h [1,22]. Briefly, DOX-Ngs, Ngs and DOX in fresh culture medium were added to substitute the primary media and incubated with cells (5 × 105) for 1 day. After the treatment, cells were washed three times with cold PBS and fixed in 2 mL 4% paraformaldehyde for 10 min. After that, the cells were stained with DAPI following the manufacturer's instructions and observed using an inverted fluorescence microscopy (Bh2-RFCA, Olympus, Japan), and the typical photographs were captured. 2.10. Cellular uptake study Cellular uptake evaluation of MDAMB-231 cells was measured by Flow Cytometry [23]. The cells were seeded in six-well plates at a density of 3 × 105 cells per well and cultured at 37 °C for 24 h. For cellular internalization assay, cells were treated by free DOX or DOXNgs at an equivalent DOX concentration of 5 μg/mL. After incubation at 37 °C for 3 h, the original medium was removed and the cells were washed three times with 1 mL of cold PBS solution. Digestion of cells were performed by adding 0.5 mL of trypsin and centrifuged at 1000 rpm for 5 min. The pelletes were washed twice with cold PBS, followed by centrifugation. Finally, the cell were resuspended in 500 μL PBS and the fluorescence of DOX trapped by cells was analyzed using a FACS caliber flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). 3
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Scheme 2. The method for the synthesis of N-phtaloyl chitosan-graft-poly (hydroxyl ethyl metacrylate)-graft-maleic acid-graft- N,N′-bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMA-g-MAc-g-MBA)].
environment-modulated zeta potential of the surfaces. Therefore, the zeta potential of Ngs was determined to be −24 (mV) that was enough to absorb the DOX drug on the surface of Ngs (Fig. 3c). (Fig. 3c). After the coupling of DOX on the surface of Ngs, the zeta potential was decreased to −13 (mV) (Fig. 3d).
the successful gafting of MAc onto the backbone of the CTS-g-PHEMA. Additional confirmation data for the successful synthesis of the CTSgPHEMA-g-MAc with narrow dispersity were estimated from the GPC analysis (Fig. 2b). The average molecular weight and polydispersity index were 285,000 g/mol and 1.42, respectively. Good control during the polymerization was determined with dispersity (Ð) values below 1.6 [29]. As Fig. 3a shows, the resulting Ngs were first carefully considered by DLS in PBS. The particle size and PDI of nanocarrier systems are the main physicochemical features influencing the endocytosis-dependent cellular uptake [30]. The most nanocarriers larger than 100–150 nm can be taken up by phagocytes or remain in these tissues for an extended time.Thus, we measured the nano-ranged size of the Ngs using TEM observation and PCS measurements. TEM observation showed uniformly spherical shape of Ngs with a diameter of 60–70 nm and demonstrated well dispersed Ngs, being in agreement with the PCS measurements (Fig. 3e). As this Figure shows, no obviously detectable aggregates could be observed in the DOX-Ngs sample. Moreover, by PCS measurement, the average size of the Ngs were recorded to be 91.22 nm. Furthermore, as Fig. 3b demonstrates, the values of the hydrodynamic diameter was reduced to < 10 nm at GSH 10 Mm, since GSH was a strong reducing agent, which can diffuse into the Ngs network and break the -S-S- bonds into 2 mercapto groups (-SH) of polymer chains. The variance between DLS and TEM can be related to the hydration water layer outside the Ngs, which could not be observed by TEM due to the different mechanism of each procedure. It should be noted that the size distribution of the Ngs was very narrow (PDI~0.199). Surface properties play a key role in the colloidal stability of nanoparticles. Ngs stabilized with the repulsive interactions are considerably to be mostly electrostatic and controlled by the
3.2. Encapsulation and in vitro release behavior study of DOX-Ngs MAc has been widely used in pH-responsive nanoparticles owing to the presence of reversibly carboxylic groups. Thus, in this work, the prepared Ngs showed some pHsensitive properties due to the carboxylic groups of MAc. DOX in salt form, an anticancer hydrophilic drug was chosen as a model drug to be loaded into the corresponding Ngs. The corresponding resulting drug loading capacities and efficiencies demonstrated good loading capacity (14%) and efficiency (76%). Since DOX is a positively charged drug in physiological pH condition (pH 7.4), DOX loading can be achieved through the electrostatic interaction. Considering the lower pH value in tumor cells, this pH-responsive property of Ngs contributes to acceleration of the drug release from Ngs. Fig. 4 presents the drug release profiles of DOX-Ngs in PBS (pH 7.4 and 5.3). Owing to the existence of disulfide bonds and carboxylic groups on the structure of Ngs that contributed to realize the quick and controllable release of DOX, the drug release behavior would be affected by both GSH and pH values. From the size profile of DOX-Ngs after treating with 10 mM GSH, It could be found that the Ngs disappeared for 48 h, confirming that DOX could be completely released as 7 stimulated by GSH. While, the cumulative release rate of DOX was < 20% at a physiologic condition (pH 7.4, 37 °C, 2 μm) after 48 h in the absence of GSH or low content of GSH (2 μm). A faster release in higher content of GSH (10 mM) reaching 71.0% or 82% was observed at 4
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Fig. 1. FTIR spectra of chitosan (CS), N-phthaloyl-CS, CS-chain transfer agent (CTS), CS-g-poly(2-Hydroxyethyl methacrylate (HEMA) [(CTS-g-PHEMA)], CTS-g-PHEMA-maleic acid (MAc)[(CTS-g-PHEMA-g-MAc)], (CTS-g-PHEMA-gMAc)] -g-N,N′-bis(acryloyl) cystamine (BAC) [(CTS-g-PHEMA-g-MAc-g-MBA)].
Fig. 2. 1HNMR spectra of CTS-g-poly(2-Hydroxyethyl methacrylate (HEMA) [(CTS-g-PHEMA)], CTS-g-PHEMA-maleic acid (MAc)[(CTS-g-PHEMA-g-MAc)] (a) and GPC chromatogram of CTS-g-PHEMA-g-MAc (b).
pH 7.4 and 5.3, respectively. Therefore, at different Ph values, the drug release rate at pH 5.0 was higher than that of pH 7.4. In conclusion, owing to a significant difference between tumor tissue and normal physiologic condition, the low release rate at pH 7.4 and low content of GSH was promising to keep the Ngs stable and thus improving the anticancer effects.
the cell viability considerably decreased when the cells were incubated with DOX and DOX-Ngs during 72 h even at 4 μg/mL and 16 μg/mL for DOX-Ngs and DOX. Correspondingly, an IC50 value of 9.26 μg/mL, 7.24 μg/mL and 3.75 μg/mL was obtained for DOX-Ngs during 24, 48, and 72 h, respectively. Overall, these results proved that the released DOX from the Ngs could still exhibit high anticancer activity in most concentrations. It should be noted since the EPR effect can only occur in in vivo tests, in in vitro studies, we could not clearly observe the superiority of drugloaded nanocarriers compared to free drugs. Thus, the cytotoxicity of DOX-Ngs is slightly higher than that of free DOX at all the studied concentrations. Moreover, since the blank Ngs did not show any cytotoxicity, the therapeutic efficacy was only owing to the DOX-
3.3. In vitro cytotoxicity The effects of the concentration of the Ngs on the proliferation of MDAMB-231 cells were investigated. Fig. 5 shows that no signs of cytotoxicity on cells were observed upon treatment with the blank-Ngs for 24, 48 and 72 h. This was further confirmed that all the free Ngs were low-cytotoxic even after 72 h in high concentrations. On the contrary, 5
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Fig. 3. Size profiles of Doxorubicin-loaded nanogels (DOX-Ngs) in pH 7.4, GSH 2 μM (a) and DOX-Ngs in pH 5.3, GSH 10 mM (b), Zeta potentials of Ngs (b) and DOXNgs (c), and Transmission electron microscopy (TEM) of DOX-Ngs (e).
Fig. 6. DAPI staining of MDAMB-231 cells (control), treated with DOX, DOXNgs and Ngs. Fig. 4. In vitro Doxorubicin (DOX) release profiles under different pH (pH = 7.4; and 5.0) and GSH concentrations from the DOX-Ngs (data are presented as mean ± standard deviation, n = 3).
nuclei and condensation of chromatin in nuclear via both DOX and DOX-Ngs. As Fig. 6 shows, the rate of these morphological changes named as apoptosis for cells was more in the cells treated with DOX-Ngs than those treated with free DOX. Finally, it could be concluded that the DNA damage was in more stages in DOX-Ngs compared to free DOX, suggesting that this developed nanocarrier may be a promising delivery
loaded Ngs. In another study, the apoptosis properties of the DOX-Ngs were analyzed and compared to free DOX against MDAMB-231 cells [31,32]. The morphological changes were detected as fragmentation of
Fig. 5. The cell viability study of Doxorubicin (DOX), DOX-Ngs and Ngs with different concentrations in the time period of 24 h (a), 48 h (b) and 72 h (c) against human breast adenocarcinoma (MDAMB-231) cells. The results was calculated as the mean ± standard deviation (n = 3). *P < .05, **P < .01. 6
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Fig. 7. Cellular uptake study of human breast adenocarcinoma (MDAMB-231) cells after incubation with fluorescein labeled Doxorubicin (DOX)-nanogels (DOX-Ngs) after 1 and 3 h.
treatment with DOX-MP-Au/Ngs from 2.3% to 21.18% for DOX and DOX-Ngs, respectively. Finally, there was no effect on the cell cycle arrest identified in the cells treated with the untreated group.
system for chemotherapeutic agents. 3.4. Cell internalization study
4. Conclusion
It is extremely important that nanocarriers should be efficiently internalized in cells and be able to deliver the drug there to use the required therapeutic efficacy [33]. Since DOX is a fluorescent molecule, its cell uptake by MDAMB-231 cells could be observed by a fluorescent microscope. As Fig. 7 depicts, the results indicated that cells treated with DOX-Ngs presented obvious stronger intensity in DOX-Ngs than DOX alone. The enhanced cell uptake ability may be associated with the expected mild drug resistance of DOX alone. In this regard, as the cell viability profiles show, DOX-Ngs exhibited higher or similar cytotoxicity against cancer cells as compared to the free DOX. Furthermore, the low fluorescence intensity was observed after 1 h, which could be related to the low amount of DOX released from the Ngs. While, the fluorescence intensity was rather enhanced after 3 h, approving that a larger amount of DOX was released. These results proved that the Ngs could effectively deliver DOX into the cell nuclei.
In summary, we designed the stimuli-responsive chitosan-based Ngs as a new smart drug delivery nanosystem via the RAFT polymerization, in which DOX was coupled into MAc groups of Ngs. The redox-responsiveness of Ngs were successfully achieved by polymerization of BAC as a disulfide crosslinker agent. The prepared (CTS-g-PHEMA-gMA-g-BAC) Ngs had a suitable loading capacity of DOX (14%) and uniform sphere shape in aqueous solution. The developed DOX-Ngs demonstrated a smart and quickly release behavior in response to thiol reducing agents such as GSH, and pH in simulated cancerous tissue medium. Experiments in MDAMB-231 cells exposed that the Ngs were compatible with these cell lines. DOX-Ng's showed a high cytotoxicity in comparing with free DOX due to the delivery of DOX into the cells, as shown by inverted fluorescence microscopy. This new designing concept may be promising to help the development of a variety of new Ngs for other chemotherapeutic agents, which may offer favorable and novel prospect in cancer therapy.
3.5. Cell cycle arrest analysis To explore the potential of DOX-Ngs to induce the cell growth inhibition, the effect of Ngs, DOX and DOX-Ngs on cell cycle distribution was tested [34] (Fig. 8). For this 9 purpose, the cell populations of dead cells are quantified by flow cytometry analysis. To determine the distribution in different phases of the cell cycle, the DNA content in the cells was detected by propidium iodide (PI) staining on the cells incubated with DOX, DOX-Ngs and Ngs for 24 h. The results showed that DOX-Ngs induced a significant increase in the percentage of the cells in the sub-G0/G1 phase. Therefore, an increase in the portion of apoptotic cells by accumulation of cells in the sub-G0/G1 region enhanced after
Declaration of competing interest The authors confirm that there are no known conflicts of interest associated with this publication and there has been no financial support for this work that could have influenced its outcome. Acknowledgment The financial support from the Stem Cell Research Center, Tabriz 7
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Fig. 8. Effects of the DOX, Ngs (without drugs) and drug loaded Ngs (DOX-Ngs) on cell cycle distribution of MCF-7 cells (human breast epithelial adenocarcinoma cell line).
University of Medical Sciences was gratefully acknowledged.
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