Multi-sensitive curcumin-loaded nanomicelle based on ABC-CBA block copolymer for sustained drug delivery

Abstract A type of multi-sensitive ABC-CBA block copolymer with thermal, glutathione and pH-responsive bonds was synthesized via ring opening polymerization along with cationic ring opening mechanisms. In continuum, the synthesized copolymer strands self-assembled into nanomicelles. The linear copolymer is comprised poly (methoxy ethylene glycol)-b-poly (2-ethyl-2-oxazoline)-b-poly (ε-caprolactone)-cystamine (i.e. [mPEG-b-PEtOz-PCL]2-Cys) and the curcumin was encapsulated inside the micelles mostly through hydrophobic interaction. The H-NMR, FTIR and GPC analysis were applied to identify the composition structure of the copolymer. The critical micelle concentration (CMC) value was achieved favorably 0.01 mg/mL for the synthesized copolymer. The morphology and particle size of solid nanocarrier were characterized by DLS, Zeta potential, AFM, TEM, and SEM micrographs. The drug loading content for the curcumin was attained 13.3% (w/w), and the entrapment efficacy of the drug in nanocarrier was obtained 79 percent. The in vitro release profile of the drug-loaded micelle was investigated by exposure to different pH, temperature and reduction circumstances, stimulated by tumor microenvironment conditions. The cell viability assay of the drug-loaded nanocarrier demonstrates high cytotoxicity toward HDF cells, while the drug-free nanocarrier has trifling toxicity and good biocompatibility. Therefore, according to the pleasant output of the research, this novel nanomicelle based on ABC-CBA block copolymer can be carried out effectively as an efficient nanocarrier in targeted drug delivery.


Introduction
In the recent century, cancer is the most common reason for human mortality all around the world, so many research concentrations have oriented on achieving a cure-all for this issue [1][2][3]. Different methods for cancer treatment have been applied, which chemotherapy has been used widely for eradicating or soothing the pain of patients in clinics. However, the most significant drawbacks of this treatment branch involve narrow therapeutic window, unfavorable pharmacokinetics, uncontrolled drug release, poor water solubility of the hydrophobic drugs, and low aggregation of drugs at tumor tissues. These factors are complicating the clinical utilization of the drugs which are eventuated to detrimental side-effects as well as high multidrug resistance [4][5][6][7]. To surmount these obstacles, effective nanocarriers for smart drug delivery with a high therapeutic index ought to be developed instantly, which with astronomical development of the nanotechnology, several nanoscale drug delivery systems have been reported including liposome, solid nanoparticles, polymeric micelles, nanogels, and so forth [8][9][10][11][12][13]. Regarding the aforementioned nanocarriers, the polymeric micelles have garnered attention of researcher to be as promising candidates in drug delivery systems with significant benefits such as high drug loading capacity, controlled drug release, excellent biocompatibility, stability in plasma, facile preparation procedure, low cytotoxicity, declined pernicious side effects, and presenting both active and passive targeting mechanisms simultaneously [14,15]. Whereas other types of drug delivery systems have their disadvantages compared to nanomicelles carriers. For instance, dendrimer vectors show toxicity while liposome carriers frequently possess a hydrophobe shell which these factors constrain their application in drug delivery [16,17]. Furthermore, some defined parameters are different in tumor microenvironment (TME) in comparison with normal tissues such as a) the pH value varying from 6.8 to 5.0 in extracellular to subcellular organs namely; b) the higher level of GSH enzyme in solid tumor about 1000 times; and c) the higher temperature about 40 C [18,19]. These parameters have led to the targeted design and synthesis of many stimuli-responsive polymeric micelles such as magnetic field, pH, temperature, biomaterials, redox, light, electrical field, etc. [20][21][22][23][24][25][26].
The scaffold of the nanomicelle is a crucial player to restrain the possible side-effects of the carrier. Hence, selecting the polymeric nanomicelle combined's materials are significantly important. Poly (ethylene glycol), poly (2-ethyl-2-oxazoline) (PEtOz) and polycaprolactone (PCL) are the most applied polymers in the synthesis of the nanomicelle [27,28]. These hydrophilic polymers (PEG and PEtOz) are representing stealth behaviors and minimal interactions with biomaterials, besides excellent biocompatibility with hydrophilic properties which capable them as a good corona for micellization [29,30]. Also, the critical solution temperature (LCST) of PEtOz is 56-60 C in an aqueous solution which can be tuned by changing the length and combining it with other hydrophobic strands. Furthermore, polycaprolactone is miscible with a wide variety of other polymers and owning good biodegradability, additionally it can be a good core due to its hydrophobic properties [31,32]. Moreover, cystamine, which is a disulfide organic compound, has been applied commonly in the structure of the nanomicelles for the sake of its reduction ability by the GSH enzyme which is an ample enzyme in the cancerous tissue [33]. The curcumin (Cur) has been extracted from the rhizome of Curcuma longa as a polyphenolic compound and used in conventional eastern medicine. Current studies have revealed that Cur possessed antineoplastic effects by deactivating nuclear factor kappa B (NF-Kb) [34,35]. Although different nanomicelles combined of the above mentioned materials were synthesized with disparate anticancer drugs, none of them are similar to recent work [36][37][38][39].
In this work, a nanocarrier was designed and will be prepared through a new methodology pathway, inspired by the exclusive characteristics of the defined polymers, cystamine, and curcumin as a potent anti-cancer drug. To efficiently balance the stability and LCST value of the copolymeric nanomicelle, a new kind of linear ABC-CBA block copolymer with environmental-sensitive ability will be prepared by introducing cystamine-conjugated ABC segments. The nanomicelle is going to be sensitive to thermal condition, pH values, and GSH enzyme for the smart drug delivery of Cur. The composition of the copolymer will consist of poly methoxy ethylene glycol (mPEG), poly 2-ethyl-2-oxazoline, and poly e-caprolactone which anchor to cystamine as a core (i.e. [mPEG-b-PEtOz-PCL] 2 -Cys or [ABC] 2 -Cys in short ABC-CBA). The synthesis producer will be included three steps and at the end, the efficiency of the novel synthesized copolymer will be determined. Herein, the questions posed with the current research are that whether the newly designed ABC-CBA copolymer will form stable nanomicelle in an aqueous medium with the proper size and morphology, or will the Cur entrap inside the core of the nanocarrier. Moreover, whether disulfide bonds will cleave and swelling will occur at other specified segments to release its pharmacological agent with desirable output, or will the novel prepared nanomicelles represent favorable CMC value, efficient drug loading, etc. to be a potential drug delivery system. Therefore, various measurement techniques are applied to characterize the copolymer structure and the obtained micelles to answer the defined questions, properly.

-Cys copolymer synthesis
Synthesis of AB block copolymer [mPEG-b-PEtOz-OH] To synthesize the represented multi block copolymer in this essay, the reaction was accomplished in different stages. In the first place, the preparation of mPEG-tosylate was a priority, thus the mPEG (6 g, 3 mmol) and dried TEA (0.1 ml, 0.7 mmol) dissolved in 40 ml of CH 3 Cl were applied to a bi-capped flask. Afterward, tosyl chloride (1.32 g, 6.9 mmol) dissolved in CH 3 Cl was also added dropwise to the reaction balloon under N 2 atmosphere and stirred for 24 h in the ambient temperature. The pure product was isolated by filtration and recrystallized with cold diethyl ether (yield %87). Secondly, 1.8 g of the obtained net crystal solid, 20 ml of dried acetonitrile and 2-ethyl-2-oxazoline (7 ml, 0.069 mmol) were added to balloon and refluxed for two days under 120 C temperature. Thereafter, the solvent in the reaction mixture was evaporated by imposing the reduced pressure condition. The obtained yellowish gel was filtrated by recrystallization methods in methanol and diethyl ether for the following steps (yield not calculated).

Synthesis of ABC copolymer (mPEG-b-PEtOz-b-PCL-CHO)
The creamy solid from the previous step, e-caprolactone (2 ml, 18 mmol) and tin (II) ethyl hexanoate (0.01 ml, 0.03 mmol) were dissolved in dried toluene in a two-spin balloon. The chamber of reaction was stirred at less than 60 C temperature under reduced pressure condition, and then the temperature increased to 110 C by a condenser and stirred for 24 h. The ultimate precipitate (mPEG-b-PEtOz-b-PCL-OH) was purified by recrystallization in CH 3 Cl and diethyl ether. Thereafter, the resultant mixture applied for the rest of reaction. For oxidizing alcohol functional group to aldehyde, the precipitate was dissolved in K 2 CO 3 (1.5 mol)/KI (0.25 mol)/I 2 (1 mol) solution, after 30 min the reaction was completed and purified similar to the previous explained strategies (yield %83).

Synthesis of ABC-CBA copolymer (mPEG-b-PEtOz-PCL) 2 -Cys
In the final steps of preparation block polymer, cystamine dihydrocholoride (0.013 g, 0.8 mmol) in dried methanol was added dropwise to mPEG-b-PEtOz-b-PCL-CHO (17 g, 1.6 mmol)/choloform (50 ml) under 50 C temperature while the reaction chamber was covered with foil to fabricate a dark medium. The reaction was accomplished after 8 h and then the reactor was cooled to room temperature. The final ABC-CBA copolymer (yellowish brown) was collected by precipitation in cooled diethyl ether, filtrated, and dried overnight at 40 C in vacuum (yield %85).

The LCST of synthesized ABC-CBA copolymer
The thermo-sensitivity properties of the obtained ABC-CBA copolymers were monitored by optical transmittance measurements. The transmittance of the prepared copolymer in PBS (2 mg/mL) was measured at various temperatures using an UV-Vis spectrophotometer (at 500 nm) with a temperature controller (Jasco ETC-505 T). The temperature was gradually increased from 34 to 45 C.
The LCST value of the copolymer was determined as the temperature escalated.

Micellization of copolymer and drugs entrapment
The polymeric micelles were prepared by the solvent displacement method via utilizing a water-permeable membrane dialysis bag with 8-10 kDa molecular mass cut off. Both of drug-free micelles and drug-loaded micelles were prepared by the same route. Generally, 5 mg of the copolymer dissolved in 2 ml DMF and then the solution was added dropwise in a dialysis bag and dialyzed against 1000 ml pure water for 24 h. As the time elapsed, DMF was replaced with distilled water and the drug-free nanomicelles were gently formed inside the bag. Subsequently, the formation of drug-loaded nanomicelles were followed the same way but only with a negligible difference which it was addition of 1 mg of drug in conjunction with the copolymer (5 mg) in 2 ml DMF. Finally, freeze-drying strategy was used to obtain solid nanocarriers from prepared micelles and complementary analyzes were performed on the dried nanocarriers [40].

CMC measurement
In continuum, the efforts were devoted to measure critical micelle concentration (CMC) of the prepared micelles. A fluorescent-probe methodology using pyrene was applied by a UV-Vis spectrophotometer. The copolymer solutions (10 ml) with various concentrations (5 Â 10 À3 to 0.5 mg/ml) were added to pyrene-coated vials and then permitted to equilibrate for 24 h. The pyrene-loaded vials' spectra with various amount of the copolymers were measured at the detection emission wavelength of k em ¼350 nm. The evaluation of CMC was accounted from intensive ratio of two wavelengths (I 337 /I 334 ) according to copolymer concentration logarithm and finding intersection point of the diagram.

Drug loading and release experiments
The powder of drug-loaded nanocarriers (1 mg) was dissolved in 10 ml dimethyl formamide (DMF), and 50 equiv. of DTT was added and the mixture stirred for 2 h at the room temperature. The amount of curcumin concentration of the sample was measured by using a standard curve of pure Cur/DMF solution. The drug loading content (DLC) and entrapment efficacy (EE) were determined and calculated by absorption reader and the following equations: %DLC ¼ (weight of loaded drug)/(weight of drugloaded micelles) Â100; %EE ¼ (weight of loaded drug)/(weight of drug in feed)Â100.
In vitro profile release of curcumin from the nanocarriers in phosphate buffered saline (PBS) was obtained in four various circumstances as shown in Table 1.

Cell viability assay
The MTS assay was implemented to investigate effects of the nanocarriers (empty and drug-loaded) on viability and cell proliferation against human dermal fibroblast. The cells were plated into a 24-well plate (300 Â 1000 cell/cm 2 ) at DMEM/High glucose medium along with 10% fetal bovine serum (FBS), and other complementary materials, and then the cultured cells were allowed to grow for 24 h. The cells were cultured at two different incubators with 37 C and 40 C temperature under 5% of CO 2 atmosphere for 1 day. To the following, cultured cells without presence of the nanocarriers were used for control. The MTS solution was added to the cultured cells mixed with the empty nanocarriers (sham sample) and drug-loaded nanocarriers (to the ratio of 1:5), followed by incubation for 3 h in a dark medium with 5% of CO 2 atmosphere, under 37 C and 40 C temperature. The nanocarriers chamber was exposed to UV irradiation with a wavelength of k em ¼254 nm for 30 min. A microplate reader was utilized to measure the absorbance at a wavelength of 490 nm. The wells with more living cells show higher optical density (OD). Finally, cell viability (%) was calculated by the equation (Mean OD of sample)/(Mean OD of control) Â 100.

Instrumentations
The NMR spectra of all intermediates and final polymer products were recorded on a Varian Mercury Plus 300 spectrometer in CDCl 3 at 25 C and chemical shifts are given in ppm rel. to Me 4 Si. Fourier transform infrared (FT-IR) spectra were recorded on a FT-IR spectrometer (Magna IR 550. USA). The size, size distribution and morphology of the nanocarriers were studied by dynamic light scattering (DLS, ZetaSizerNano ZEN3600, Malvern Instruments, USA), transmission electron microscopy (TEM, FEI Tecnai G2 F30 TWIN 300 KV, E.A. Fischione Instruments, Inc. USA), atomic force microscopy (AFM), and scanning electron microscopy were recorded with a (SEM, KYKY-EM3200 apparatus). The concentration of drug was measured by a UV-Vis spectrophotometer (UV-1800, HIMADU) based on a standard calibration curve of the drug at 490 nm. The Zeta potential measurement was carried out with Malvern Zeta sizer Nano ZS instrument Ltd, Worcestershire, UK. Molecular weights of the polymers were determined by gel permeation chromatography (GPC) measurement which performed using a GPC instrument (Agilant 1100, USA) consisting of a 10 mm PLGel column. Polystyrenes of known molecular weight were used as calibration standards and reagent grade tetrahydrofuran (THF) was used as a mobile phase eluting at a flow rate of 1.0 ml/min (Detector RI and Temp. 30 C).
To show statistical significance for validation of the results, all statistical analysis were presented with n ¼ 3 using SSPS software and p < 0.05. The outputs are expressed as mean ± standard deviation.

Result and discussion
Synthesis and characterization of ABC-CBA copolymers The synthetic route of all reactions performed is shown in Scheme 1. This new methodology demonstrates high yield synthesize of ABC-CBA block copolymers with minimum possible stages. The mPEG-b-PEtOz-OH was synthesized through polymerization of activated oxazoline with mPEG-tosylate. In the following, a catalytic amount of Sn(oct) 2 along with caprolactone was added to the reaction chamber for fabrication of ABC block polymer (mPEGb-PEtOz-b-PCL-OH) [41]. Afterwards, to functionalize the hydroxyl group into the aldehyde, K 2 CO 3 /KI/I 2 was added and finally amphiphilic copolymer strands with ABC-CBA structure were obtained by applying cystamine hydrochloride. The dialysis method was applied for the preparation of the final polymeric micelles loaded with drug. The 1 H NMR analysis was performed to determine the structure of all synthesized copolymers (Supplementary Figures S1-S3). In Figure 1(A), the spectrum exhibits major resonances related to two blocks of the AB copolymer (mPEG-b-PEtOz-OH). The resonance at 3.7 ppm that appeared in the spectrum is belonged to the CH 2 -CH 2 protons (peak 'a') of mPEG and other resonances in 3.4 ppm are assigned to -NCH 2 -CH 2groups of PEtOz and O-CH 3 groups of mPEG (peak 'c þ b') which overlapped. The doublet peak at 2.4 and the singlet peak at 1.1 ppm are attributed to -CH 2 C¼O and -CH 3 (peaks 'd and f') of the oxazoline groups, respectively. The specified peak at 7.2 ppm belongs to the solvent (CHCl 3 ) in all given spectra. These denoted peaks prove the successful synthesis of both two blocks in backbone of the copolymer chain.
As shown in Figure 1(B), the prepared mPEG-b-PEtOz-b-PCL-CHO in this way displayed the 1 HNMR signals of mPEG (a and b at 3.7, 3.4 ppm), PEtOz (c at 3.4, d at 2.4, f at 1.1 ppm) and PCL (e at 4.1, i at 2.4, g at 2.0, and h at 1.6 ppm) blocks. Some of the area of the peaks overlapped with each other. The 1 H NMR spectrum of the ABC-CBA copolymer (mPEG-b-PEtOz-PCL) 2 -Cys is also illustrated in Figure 1(C). The signals at 1.4, 2.7, and 4.2 ppm were specified for the proton groups of cystamine in the copolymer chain as well as all signals for three blocks on the 1 H NMR spectrum were clearly observable.
These results were further confirmed by FT-IR spectra which supports the successful polymerization. The composition structure of (mPEG-b-PEtOz-PCL) 2 -Cys was identified by FT-IR spectroscopy in Figure 2. At the FT-IR spectrum, the absorption bands at 2980, 1427 and 1375 cm À1 are referred to C-H in methyl and methylene bonds for all blocks of copolymer. The characteristic peak at 1109 cm À1 is assigned to C-O-C ether groups of the mPEG block. The representing peaks at 1198 and 1640 cm À1 are assigned to C-N bonds and C¼O amide bonds of the PEtOz block, and also the imine (C ¼ N) bonds in other spectra which overlapped. The peaks at 1731, 1173 cm À1 are attributed to C¼O ester bonds and C-O bonds of the PCL block. The absorption bands at 724 and 508 cm À1 are assigned to C-S and disulfide bonds of cystamine.
Gel permeation chromatography (GPC) is an analytical technique for measurement of unknown molecular weight of polymers which separates molecules of material by size and provides molecular weight distribution of a sample. Molecules with higher molecular weight pass through the column first, thereafter materials with lower molecular weight pass the GPC column [42]. Herein, the molecular weight of the prepared block copolymer was analyzed by GPC with a UV detector at wavelength of 245 nm, to check progressing of polymerization reaction. The GPC elution curve of ABC-CBA copolymer (mPEG-b-PEtOz-b-PCL)2-Cys reveals a monomodal peak with Mn of 15.78 Â 10 þ3 and a dispersity data (D) of 1.33. It turned out that prepared macromolecular chains have an appropriate range of weight average molecular weight (M W ) for the micellization and drug entrapment which is 2.1 Â 10 3 g/mol. Hence, this obtained value for GPC confirm the linear structure of synthesized copolymer that is in good agreement with 1 H-NMR spectrum results of the novel copolymer (Supplementary Figure S4).
Typically, thermo-sensitive copolymer with a LCST in the range of 34 and 41 C is pivotal for the controlled release of drug in cancerous tissues. The LCST value of PEtOz is almost between 56 and 60 C which has limited its usages for thermal drug targeting. According to literature, the conjugation of PEtOz with hydrophobic polymers leads to decrease in the LCST value [43]. So, here the copolymerization of PEtOz with PCL was performed to diminish the LCST of the final copolymer up to the cancerous tissues' level.
The cloud point experiments of the (mPEG-b-PEtOz-b-PCL) 2 -Cys is shown in Figure 3. The phase transition of the copolymer occurs at specific temperature, whenever the clear solution changes into a cloudy one. The respective LCST value of the copolymers was obtained just 40.1 C, and the observation is one of the outstanding points of the synthesized copolymer.

Self-assembly and micellar properties of copolymer
The thermodynamic stability of the copolymer micelles (that influence the initial release of the drug) were determined by the CMC  measurement. Herein, dialysis method was applied for preparation of the micelles, and pyrene as a florescence-probe was used for measurement of the CMC values. As shown in Figure 4(A), the CMC evaluation was obtained in two intensive wavelength (I 337 / I 334 ) and the CMC point was estimated 0.01 mg/ml from finding intersection point of the copolymer concentration logarithm diagram (Figure 4(B)). To achieve favorable CMC amount, the volume of hydrophobic (PCL) and hydrophilic blocks (PEG, and PEtOz) of copolymers should be balanced precisely to decline the CMC value and consequently increase the nanomicelles retention time in blood strain. On the one hand, the large amount of the PEtOz is not applicable and its chain length should be restricted to be able to play its thermo-responsiveness role. On the other hand, reaching a desirable CMC value is an important paramount, thus the PEG as a hydrophilic segment was utilized to pay the way, besides having its significant merits [44]. Moreover, the synthesized Cur-loaded nanocarriers are combined of a balanced amount of hydrophilic copolymers in their outer surface and hydrophobic moieties in their inner side that can easily self-assemble in aqueous solution and fabricate hydrophilic nanomicelles at lower concentrations of CMC which demonstrate the tendency of (mPEG-b-PEtOz-b-PCL) 2 -Cys toward micellization as an important factor for drug delivery.

The stability measurement of the nanomicelle
The amount of electric charge at the surface of the micelles (zeta potential) indicated the stability of micelle solutions [45]. As can be seen in Figure 5 the zeta potential value for the copolymer micelle solutions [(mPEG-b-PEtOz-b-PCL) 2 -Cys] was gained À10.5 mV (Figure 6(A)) and did not change with a function of time ( Figure 5(B)). As can be observed, the zeta potential trends were measured at pH¼ 5.0 and 7.4 within 6 h. At the initial part of the diagrams the zeta potential value is À10.5 mV while this trend at pH 7.4 approximately stayed stagnant, indicating prevention of the nanomicelle agglomeration due to repulsive electrostatic forces. Whereas at pH¼ 5.0 a significant change is observable from À10.5 mV to þ8 mV which is possibly because of the alterations in the outer shell of the nanomicelle. In fact, the mPEG has a negative charge on its surface that in the acidic condition is prone to absorb protons of the milieu which is leading to the nanomicelles aggregation and deformation. Furthermore, according to the principle of the organic chemistry and many reported literatures, the imine groups of the synthesized copolymer in the acidic circumstances also absorb the protons of the environment and thus micelle dissociation is inevitable [18][19][20]23]. Therefore, it is supposed that the presence of mPEG as well as PEtOz together in the hydrophilic segments can efficiently balance the stability of polymer micelles as well as good LCST value.

Characterization of drugs loaded nanocarriers
The morphology, size, and size distribution of drug-loaded nanocarriers are a crucial player in the rational design of smart drug delivery systems due to the direct effects of the nanoparticle size into the stability, drug loading and drug release of nanocarriers [43]. Using distinguishing techniques are noteworthy owning to the different advantages representing everyone for exact measurement outputs. Thus, various measurements were investigated to characterize the obtained copolymer micelles. Figures 6 and 7 summarized the results of evaluation concerning various aspects of the prepared nanocarriers.

TEM and SEM analysis
The TEM micrographs of the nanocarriers exhibited the copolymers had self-assembled in the form of spherical nanostructures  in aqueous solution and have nano-sized range around 60-100 nm ( Figure 6(A,B)). According to the TEM images, the inner morphology of the nanocarriers was not uniform because of the presence of drug and different polymeric blocks (Supplementary Figure S5).
The SEM image in Figure 7(A), has confirmed 40-100 nm scales for the nanocarriers which is in accordance with the TEM results. Prior to observation, the micellar powder was dusted on gold sputter-coated to fabricate electrically conductive particles. As can be observed, micelles dispersed as nanoparticles with regularly spherical shapes proving the well micellization without any aggregation.

Size measurement by DLS technique
The DLS size distribution profile analysis in Figure 7(B) has exhibited negligible differences in the scales from 15 to 80 nm, referring to the basic differences in the measurement route of these techniques, since in the TEM and SEM measurements, the field of view has been very small and the properties of the analyzed area fail to be attributed to the whole sample [46].

AFM analysis
The surface morphology of the synthesized nanocarrier was achieved by AFM technique. The three-dimensional image ( Figure  7(C)) and the two dimensional image of the nanomicelles ( Figure  7(D)) were obtained by the AFM technique which confirm the nano-sized structure of the nanocarrier. As it can be observed, the roughness of the surface is not high.

Curcumin loading and release study
The loading of drug molecules into the core of the empty micelles depend on the way how the micelles are prepared [47]. Herein, curcumin as the most frequently used anticancer drug with poor solubility in water, was loaded into the assembled micelles via using the dialysis method. The entrapment efficacy (EE) and the drug loading content (DLC) were measured by UV-Visible  spectrometer. The DLC for the curcumin was attained 13.3% (w/ w), and the EE of the drug into the nanocarriers was obtained 79 percent, which are desirable percentages and this is likely because of multi-block structure of the synthesized nanocarrier which entraps the drug efficiently and attributes to controlled release of the drug by imposing their influence in the copolymer biodegradation, and diffusion mechanisms [48,49]. It was speculated the ABC-CBA scaffold design of the synthesized micelle likely accelerated the micelle dissociation.
In this study, the vast majority of drug release occurs through the biodegradation of nanocarriers. Hence, the Cur was implemented as chemotherapeutic model agent to investigate the releasing and loading properties of the nanocarriers under presence of glutathione, various pH, and thermal conditions. The in vitro study of the drug release was conducted at pH 5.0 and 7.4, in the ambient or 40 C temperature with or without 10 mM DTT along with 1 mg.ml À1 polymeric concentration. As shown in Figure 8(A), the release rate of curcumin in pH 7.4 without presence of the DTT was restricted efficiently (about 35%). Whereas, the Cur-loading release with the existence of the reductive agent (pH 7.4 þDTT) was escalated dramatically (just over 65%) because of disulfide bonds cleavage in the Cys parts and also DTT likely can act as a competing H-bond donor and a reducing agent, and thereby facilitates drug release. Fast drug release of curcumin was accelerated greatly (approximately 80%) when the temperature increased up to 40 C or the pH factor diminished to 5.0 due to the imine-bonds' dissociation of the micelle in conjunction with the disruption of the hydrogen-bonds presented between the drug and the polymeric carrier or the swelling of the PEtOz block in the imposed conditions.

Cytotoxicity assays
The in vitro studies of the nanocarriers were examined against HDF cells by utilizing the MTS assay to recognize the number of viable cells due to its unique optical properties [49]. As shown in Figure 8(B), the results were investigated in two disparate temperatures for the cultured medium as control, the cultured cells with the blank nanocarriers as negative control, and the Cur-loaded nanocarriers. Inferring from the statistics, the cell viability for the blank cultured medium was negligible in the both given temperatures showing the viability potential of the HDF cells. Whereas, the results represented considerably high cell viability by about 99%, and 93% for the empty micelles at 37 C and 40 C, respectively. These values are demonstrating low cytotoxicity and excellent biocompatibility for the synthesized nanocarriers against HDF cells. In contrast, the Cur-loaded nanocarriers showed 66% cell viability after 24 h incubation under 37 C temperature. Whereas with increasing the temperature up to 40 C, the cell viability trend decreased to 44% for the HDF cells, meaning the delivery of the Cur-loaded nanocarriers delineated the highest cytotoxicity assay or more than of 50% HDF cell's death.

Conclusion
To recapitulate, a novel ABC-CBA block copolymer combining m-PEG-b-PEtOz-PCL-cystamine-based were successfully synthesized by self-assembly into nanomicelle with thermal, glutathione (GSH), and pH-sensitive bonds. The balance between the stability ABC-CBA copolymer micelles was evaluated. The LCST obtained 40.1 C, and the CMC value with approximately 0.01 mg/ml delineates the easiness of the nanocarriers' self-assembly and their high thermodynamic stability. The particle size distribution was examined which have depicted around 40-100 nm scales and the zeta potential measurement also revealed excellent stability for the micelles. Furthermore, Cur as the most effective drug in the cancer therapy was entrapped inside the core of the nanocarriers readily. The multi-sensitivity of nanocarrier was investigated by in vitro assay. The cytotoxicity assay against HDF cells has represented negligible toxicity for the blank nanocarriers and the maximum cytotoxicity for the Cur-loaded nanocarriers. Therefore, the answers to the questions which were posed with this research have gained meticulously and propitiously desirable, so the nanocarriers can be applied effectively as a potential candidate in drug delivery systems for improving cancer therapy.