One-Pot Synthesis of Amphiphilic Biopolymers from Oxidized Alginate and Self-Assembly as a Carrier for Sustained Release of Hydrophobic Drugs

In this paper, we developed an organic solvent-free, eco-friendly, simple and efficient one-pot approach for the preparation of amphiphilic conjugates (Ugi-OSAOcT) by grafting octylamine (OCA) to oxidized sodium alginate (OSA). The optimum reaction parameters that were obtained based on the degree of substitution (DS) of Ugi-OSAOcT were a reaction time of 12 h, a reaction temperature of 25 °C and a molar ratio of 1:2.4:3:3.3 (OSA:OCA:HAc:TOSMIC), respectively. The chemical structure and composition were characterized by Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (1H NMR), X-ray diffraction (XRD), thermogravimetry analyser (TGA), gel permeation chromatography (GPC) and elemental analysis (EA). It was found that the Ugi-OSAOcT conjugates with a CMC value in the range of 0.30–0.085 mg/mL could self-assemble into stable and spherical micelles with a particle size of 135.7 ± 2.4–196.5 ± 3.8 nm and negative surface potentials of −32.8 ± 0.4–−38.2 ± 0.8 mV. Furthermore, ibuprofen (IBU), which served as a model poorly water-soluble drug, was successfully incorporated into the Ugi-OSAOcT micelles by dialysis method. The drug loading capacity (%DL) and encapsulation efficiency (%EE) of the IBU-loaded Ugi-OSAOcT micelles (IBU/Ugi-OSAOcT = 3:10) reached as much as 10.9 ± 0.4–14.6 ± 0.3% and 40.8 ± 1.6–57.2 ± 1.3%, respectively. The in vitro release study demonstrated that the IBU-loaded micelles had a sustained and pH-responsive drug release behavior. In addition, the DS of the hydrophobic segment on an OSA backbone was demonstrated to have an important effect on IBU loading and drug release behavior. Finally, the in vitro cytotoxicity assay demonstrated that the Ugi-OSAOcT conjugates exhibited no significant cytotoxicity against RAW 264.7 cells up to 1000 µg/mL. Therefore, the amphiphilic Ugi-OSAOcT conjugates synthesized by the green method exhibited great potential to load hydrophobic drugs, acting as a promising nanocarrier capable of responding to pH for sustained release of hydrophobic drugs.


Introduction
Polymer nanocarriers made from naturally occurring and biodegradable polymers have attracted much attention, especially in various drug delivery systems, since they offer a promising means by which to enhance the therapeutic values of drugs by improving their bioavailability, solubility and retention time, as well as benefitting patients due to physical properties, such as the molecular weight of the copolymer, vary with the source and type of alginate [36]. However, alginates with high molecular weights are difficult to degrade through cleavage of glycosidic linkages under physiological conditions [37]. This mainly restricts its applications in drug delivery. It is now well-established that the oxidation of alginate with sodium periodate on the hydroxyl group (-OH) at the C-2 and C-3 positions of the uronic units can enhance its biodegradability and reactivity, providing an interesting opportunity for broadening the biomedical applications of alginate [38][39][40]. However, there are few reports on OSA-based amphiphilic polymer as a smart carrier for drug delivery, especially as a pH-responsive carrier for oral hydrophobic drugs.
Herein, we report a catalyst-free approach for green and efficient preparation of the innovative OSA-based amphiphilic conjugate (Ugi-OSAOcT) through an Ugi-4CR from octylamine (OCA), oxidized sodium alginate (OSA), acetic acid (HAc) and tosylmethyl isocyanide (TOSMIC) in distilled water at room temperature. The chemical structure and thermal property of the Ugi-OSAOcT conjugate were characterized by FTIR, 1H NMR, XRD, EA, GPC and TGA. The influencing factors, including the reaction time, temperature, and feed molar ratio of the reactant on the degree of substitution (DS) were investigated. The self-assembly behavior, particle size, zeta potential, morphology and stability of the micelles in aqueous solutions were evaluated by FM, DLS and TEM. In addition, the in vitro cytotoxicity of the Ugi-OSAOcT conjugates was evaluated by MTT assay. Ibuprofen (IBU), which served as a model poorly water-soluble drug, was incorporated into the Ugi-OSAOcT micelles by dialysis method. The physicochemical characteristics of the IBU-loaded Ugi-OSAOcT micelles and in vitro drug-release behavior under different pH values were also investigated. The results revealed that the amphiphilic Ugi-OSAOcT conjugates could have potential applications for effective entrapment and oral delivery of hydrophobic bioactive compounds.

Synthesis of Ugi-OSAOcT Conjugates 2.2.1. Oxidation of Sodium Alginate
OSA was prepared according to the protocol previously reported by Gomez et al. with some modifications [39]. In brief, 4.0 g (20.2 mmol) of sodium alginate (SA) was dissolved in 200 mL of distilled water, the required amount of sodium periodate was added and the mixture was stirred for 24 h under dark conditions at room temperature. The molar ratios of sodium periodate to monomeric unit of SA were 1:10, 3:10 and 5:10, respectively. The reaction was quenched by adding equimolar ethylene glycol to sodium periodate and stirring for 0.5 h. Then, 500 mL of ethanol and 1.50 g (0.025 mol) of NaCl was added to the mixture. The precipitate was collected by vacuum filtration, re-dissolved in deionized water and dialyzed against distilled water using a dialysis bag (MWCO 3500 Da) for 3 days and, finally, lyophilized to obtain oxidized sodium alginate (OSA), named as OSA 10 , OSA 30 and OSA 50 , respectively. In this paper, the subscripts in OSA 10 , OSA 30 and OSA 50 mean the theoretical oxidation degree of OSA.
The degree of oxidation of OSA was followed by determining the concentration of unreacted periodate by iodometry, according to the previous method [39,40]. Based on titration results, the degree of oxidation of OSA 10 , OSA 30 and OSA 50 can be calculated as 9.51%, 27.76% and 44.25% (Table 1), respectively.  [41]. Briefly, 2.0 g (10.0 mmol) of OSA (OSA 10 , OSA 30 and OSA 50 ) was first dissolved in 200 mL of deionized water (1.0%, m/v), and 4.0 mL (24.0 mmol) of OCA was added under mechanical stirring at room temperature for 30 min. Then, 1.71 mL (30.0 mmol) HAc and 6.44 g (33.0 mmol) of TOSMIC dissolved in 5.0 mL of DMF was added into the reaction solution and mechanically stirred at room temperature for 12 h. Subsequently, volume of absolute ethanol five times greater was added to the mixture to precipitate product. The precipitate was collected by centrifugation, re-dissolved in deionized water and dialyzed against distilled water using a dialysis bag (MWCO 3500 Da) for 3 days. Subsequently they were lyophilized to obtain the target Ugi-OSAOcT, named as Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT, respectively.

FTIR and 1 H NMR Spectroscopy
The structure of the Ugi-OSAOcT conjugates was characterized by FTIR and 1 H NMR spectroscopy. In detail, the FTIR spectra of the sample were recorded on a Nicolet-6700 (Thermo Scientific, Waltham, MA, USA) with KBr pellets in the range of wavenumbers between 4000 and 400 cm −1 for 64 scans with a spectral resolution of 2.0 cm −1 . The 1 H NMR spectra was recorded at 25 • C using an ULTRASHIELD 400 PLUS spectrometer (Bruker, Fällanden, Switzerland) operating at 400 MHz with deuterated water (D 2 O) as a solvent and tetramethylsilane (TMS) as an internal standard; the concentration of the sample was approximately 8.0-10.0 mg/mL.

Thermogravimetric Analysis
Thermal stability was studied by thermogravimetric analysis (TGA). In detail, a 9.0-10.0 mg sample was weighed and placed on a 449F3 thermogravimetric analyzer (TA Instrument, New Castle, DE, USA) with aluminum crucibles as a sample holder. Then, the temperature was increased from 20 • C to 800 • C at a heating rate of 20 • C/min under the protection of high purity nitrogen (100 mL/min, 0.04 MPa). The degree of substitution (DS, %), defined as the number of OCA molecules per 100 sugar residues of OSA, was quantified by elemental analysis according to the method described in the existing literature [41]. The elemental contents of C, H and N (% m/m) of the sample were determined using Elementar's vario EL Cube (Elementar, Germany). The DS was calculated based on the C and N contents (% m/m) according to the following Equation (1): where α, M C and M N erefer to the N/C content ratio, relative atomic mass of C and relative atomic mass of N, respectively.

Gel Permeation Chromatography Analysis
A GPC (Waters e2695, Milford, MA, USA) equipped with an Ultrahydrogel TM 120 (7.8 × 300 mm 2 ) column was applied to measure the weight-average molecular weight (Mw), number-average molecular weight (Mn) and polydispersity index(PDI) of the Ugi-OSAOcT conjugates. The concentration of the sample was 1 mg/mL, and 0.05% sodium azide was used as the mobile phase with a flow rate of 0.6 mL/min at 40 • C.

Measurement of Critical Micelle Concentration (CMC)
The CMC of the Ugi-OSAOcT conjugates in 0.05 mol/L of NaCl aqueous solution was determined using pyrene as a fluorescent probe on a F7000 fluorescence spectrophotometer (Hitachi, Honshu, Japan) [42]. Briefly, a series of concentration ranges of 5.0 × 10 −4 -2.0 mg/mL of the Ugi-OSAOcT solutions were prepared by adding 0.05 mol/L of NaCl solution and 10 µL of pyrene (1.0 × 10 −3 mol/L in methanol) to the aforementioned solution (10.0 mL), respectively. All solutions were sonicated in ultrasonic bath and left to evaporate overnight at 25 • C. Pyrene was excited at 335 nm, and its emission spectra was recorded in the range of 350-600 nm at an integration time of 1 s with a slit width of 2.5 nm. The ratio of fluorescence intensity at 373 and 384 nm (I 1 /I 3 ) was calculated and plotted against the logarithmic concentration of the corresponding samples to determine the CMC value of the conjugates.

Preparation of Blank Ugi-OSAOcT Micelles
The blank Ugi-OSAOcT micelles were prepared by directly dispersing the Ugi-OSAOcT conjugates in deionized water, followed by sonication to facilitate aqueous dispersion and self-assembly. Briefly, the Ugi-OSAOcT conjugates were dissolved in distilled water to make a concentration of 1 mg/mL under gentle stirring at 25 • C for 6 h. The solution was then sonicated for 4 min by a probe sonicator at 120 W and repeated 3 times in order to guarantee optically clear dispersion. The sonication pulse was turned on 2 s with a waiting time of 4 s between pulses. Finally, the micellar solution was further filtered through a 0.45 µm syringe filter and stored at 4 • C.
2.6. Characterization of Polymeric Micelles 2.6.1. Dynamic Light Scattering (DLS) The particle size, polydispersity index (PDI) and zeta potential of the Ugi-OSAOcT micelles in aqueous solution were measured by the DLS experiments using the Zetasizer Nano ZS90 (Malvern, Worcestershire, UK) at 25 • C with an argon laser (He-Ne) light wavelength of 633 nm at a 90 • scattering angle. The solution of conjugates was maintained at a concentration of 1 mg/mL and was passed through a 0.45 µm microfilter before DLS measurement.

Transmission Electron Microscopy (TEM)
The morphologies of the Ugi-OSAOcT micelles were observed on a TEM instrument (JEM2100, JEOL Co., Tokyo, Japan) operated at an acceleration voltage of 200 kV. Prior to obtaining the images, several drops of micelles solution were dropped on a carbon-coated copper grid, stained with 2% (w/v) phosphotungstic acid for 20 s and then left to dry at room temperature for 30 min.

Storage Stability of Blank Ugi-OSAOcT Micelles
To evaluate the storage stability of the prepared micelles, the blank Ugi-OSAOcT micelles (1 mg/mL) were dispersed in PBS (pH 7.4) and incubated at 25 • C for 0, 1, 2, 4, 6, 8, 10 and 15 days. The mean size, PDI and zeta potential of the micelles were monitored by DLS. All measurements were repeated in triplicate to ensure reproducibility of the results.

Preparation of IBU-Loaded Self-Assembled Nanoparticles
The IBU-loaded Ugi-OSAOcT nanoparticles were prepared by an ultrasound dialysis method [43]. Typically, 10.0 mg of Ugi-OSAOcT conjugates were dissolved in 10 mL distilled water. Different volumes of IBU methanol solution (0.5 mg/mL) were then added dropwise to the solution under gentle stirring for 2 h at room temperature and further sonicated for 10 min by probe-type ultra sonicator. Subsequently, the whole solution was transferred to a dialysis bag (MWCO 3500 Da) and dialyzed against distilled water for 48 h to remove the organic solvents and free IBU. Distilled water was exchanged at 4 h intervals during the dialysis procedure. After dialysis, the final solutions were centrifuged at 8000 rpm for 20 min, and the supernatant was filtered through a 0.45 µm syringe filter to remove the insoluble IBU and then lyophilized for 48 h to obtain micelle powders.
To determine the IBU-loading contents, a known amount of freeze-dried IBU-loaded nanoparticles was dissolved in methanol (1 mL), sonicated for 30 min and filtered through a 0.45 µm syringe filter. The IBU concentration was measured by using a UV-vis spectrophotometer (U-3900, Hitachi, Japan) at 222 nm based on the standard calibration curve obtained from free IBU in methanol. The encapsulation efficiency (%EE) and drug loading (%DL) were calculated according to Equations (2) and (3)

In Vitro Drug Release Studies
To evaluate the suitability of the Ugi-OSAOcT nanoparticle for a variety of applications, the in vitro release behavior of IBU from an IBU-loaded Ugi-OSAOcT nanoparticle was investigated by a common dialysis method in PBS buffer media with different pH values (0.1 M, pH 1.2, 5.0 and 7.4) at 37 • C. The PBS media with pH values of 1.2 and 7.4 were respectively used as the simulated gastric fluid and simulated intestinal fluid. Tween 80 (0.5%, w/v) used as a surfactant was added to the PBS media to maintain the sink conditions [44]. Briefly, 1.0 mL of IBU-loaded Ugi-OSAOcT nanoparticle solution was placed in dialysis bag (MWCO 3500 Da) and then immersed completely in 30 mL of release media. The whole release system was incubated in a thermostat water bath and shaken at 100 rpm at 37 • C. At the predetermined time points (0.5, 1, 3, 5, 7, 9, 12, 15, 18, 24, 36 and 48 h), 3 mL of the release media was withdrawn, and the same volume of fresh release media were then added to maintain a constant volume. The released amounts of IBU were measured by a UV-vis absorption experiment at a wavelength of 222 nm. For comparison, similar release experiments were performed with the same amount of free IBU as found in the IBU-loaded nanoparticle. Before the UV absorption experiments, calibration curves of IBU in the PBS-containing Tween80 (0.5%, w/v) were plotted. All experiments were performed in triplicate. The percentage of cumulative drug release was calculated from Equation (4) as follows: where C i and C n refer to the concentrations of IBU extracted from release media at i and n time, respectively, and n is the number of times the sample solution was withdrawn. W loaded IBU is the weight of IBU previously loaded in the Ugi-OSAOcT nanoparticles.

In Vitro Cytotoxicity Assays
The cytotoxicity of the Ugi-OSAOcT conjugates, OSA and SA (control) against RAW 264.7 cells were evaluated by MTT assay [45,46]. In detail, RAW 264.7 cells were seeded into 96 well plates at a density of 4.0 × 10 3 cells/well in the incubator (37 • C, 5% CO 2 ) for 24 h. The culture medium was replaced with 100 µL of the sample solutions containing a series of concentrations (0, 200, 400, 600, 800 and 1000 µg/mL) in DMEM. Following incubation for 48 h, the culture medium was then replaced with fresh DMEM (100 µL), followed by an addition of 20 µL of MTT solution in PBS (5 mg/mL), and incubated for an additional 4 h at 37 • C in CO 2 . The MTT-containing medium was then removed, and 100 µL of DMSO was added to each well to solubilize the formed formazan crystal with gentle agitation for 10 min. The absorbance of the solution was measured at 570 nm with a Multiskan MK3 microplate reader (Thermo, Waltham, MA, USA). All experiments for this study were performed in triplicate, and the cell viability (%) was calculated from Equation (5) as follows: where Abs 570nm blank refers to the absorbance of DMSO at 570 nm without cells. Abs 570nm sample and Abs 570nm control refer to the absorbance at 570 nm in the presence and in the absence of treatment samples, respectively.

Statistical Analysis
All data are presented as the mean value ± standard deviation. A single factor ANOVA was performed by SPSS software to analyze the variables, and a value of p < 0.05 was considered statistically significant.

Synthesis and Characterization of Ugi-OSAOcT Conjugates
The Ugi-OSAOcT conjugates with various DS were straightforwardly synthesized by grafting OCA to the OSA backbone via an Ugi-4CR, and the detailed synthetic scheme of the Ugi-OSAOcT conjugate was presented in Figure 1. Water, a non-toxic and safe solvent, can significantly reduce the impact of the process on the environment of the reaction. Accordingly, we envisioned that the utilization of an Ugi-4CR would provide a rapid and effective synthetic way to constructi novel amphiphilic polymers, due to the advantages of simplicity, atom-economy and good yields. Successful synthesis of the conjugates was confirmed by FTIR and 1 H NMR, as shown in Figure 2A,B, respectively.
The FTIR spectra of SA, OSA, OCA and the Ugi-OSAOcT conjugates were represented in Figure 2A. The strong and broad band at 4000-3000 cm −1 was assigned to the O-H stretching vibration of polysaccharide, and the band at 2925 cm −1 was attributed to the C-H stretching vibration of the polysaccharide structure [47]. The characteristic absorption peak of SA at 1621 cm −1 and 1419 cm −1 belonged to the asymmetric and symmetric stretching vibrations of -COO-, respectively [48]. The band at 1100-890 cm −1 was attributed to ether groups of the polysaccharide skeleton (C-O-C stretching) [49]. However, OSA's characteristic band at 1734 cm −1 was too weak and hard to be detected due to the formation of hemiacetal by the free aldehyde groups [50]. The characteristic band of OCA was also identified at 3300 cm −1 (N-H stretching), at 2925 cm −1 (-CH 2 stretching) and at 2842 cm −1 (-CH 3 stretching). After conjugation of OCA to the OSA backbone, the abovementioned typical bands of OSA appeared in the Ugi-OSAOcT conjugate with various DS spectra. In addition, the characteristic absorption peaks corresponding to OCA (2925 cm −1 and 2842 cm −1 ) were both observed, which confirmed successful introduction of OCA in the OSA skeleton [25]. effective synthetic way to constructi novel amphiphilic polymers, due to the advantages of simplicity, atom-economy and good yields. Successful synthesis of the conjugates was confirmed by FTIR and 1 H NMR, as shown in Figure 2A,B, respectively.  The structures of the Ugi-OSAOcT conjugates were further confirmed by 1 H NMR. From Figure 2B, the typical peaks of SA were found at δ = 3.5-5.0 ppm, assigned to the methylene of uronic acid for SA [48]. In comparison with SA, the new signals of OSA at about δ = 5.35, 5.02 and 4.30 ppm confirmed the successful oxidation of SA with sodium periodate, which were attributed to a hemiacetalic proton generated formaldehyde groups with hydroxyl groups present on the adjacent uronic acid subunits on the OSA [39]. On the Ugi-OSAOcT conjugate spectra, the peaks at δ = 0.8, 1.1-1.5 and 2.0-3.0 ppm were attributed to the methyl group (-CH 2 -CH 3 ), methylene (-(CH 2 ) 6 -CH 2 -NH-) and methylene (-(CH 2 ) 6 -CH 2 -NH-) of OCA, respectively. The characteristic peaks at δ = 2.30 and 7.0-8.0 ppm were attributed to the methyl group (Ph-CH 3 ) and its phenyl protons (Ph-H) of TOSMIC, respectively. Therefore, the presence of the typical peaks of OSA, OCA and TOSMIC in the Ugi-OSAOcT conjugate spectra further confirmed successful synthesis of the Ugi-OSAOcT conjugates via the Ugi-4CR [25].
The DS of the Ugi-OSAOcT conjugates was calculated by the element contents of C and N obtained from EA data. The optimum reaction parameters for coupling of the primary amines to OSA via Ugi-4CR were found through a series of experiments (see Table 2). In all the optimization experiments, the influence of the feed molar ratio of OSA to OCA, temperature and amounts of HAc using the same batch of OSA 10 on DS were investigated. As SA was oxidized by sodium periodate to obtain OSA with a dialdehyde structure, all experiments were carried out on the basis of a molar ratio of OSA to OCA (1:2), in order to obtain a high DS. As shown in Figure 3A  The FTIR spectra of SA, OSA, OCA and the Ugi-OSAOcT conjugates were represented in Figure 2A. The strong and broad band at 4000-3000 cm −1 was assigned to the O-H stretching vibration of polysaccharide, and the band at 2925 cm −1 was attributed to the C-H stretching vibration of the polysaccharide structure [47]. The characteristic absorption peak of SA at 1621 cm −1 and 1419 cm −1 belonged to the asymmetric and symmetric stretching vibrations of -COO-, respectively [48]. The band at 1100-890 cm −1 was attributed to ether groups of the polysaccharide skeleton (C-O-C stretching) [49]. However,  gates increased with an increasing reaction time for all combinations of the reaction pa rameters and reached the plateau DS values at 12 h.  The DS could be increased by increasing the amounts of OCA. As shown in Figure 3A, the feed molar ratio of OSA to OCA increased from 1:2 to 1:2.4, corresponding to an increase in the DS from 3.3% to 4.0%. It then continued to increase to 1:2.8 as the DS slightly decreased to 3.9%. The reason for this may be that an increase in the feed amount of OCA will increase the alkalinity of the solution, making OSA more easily decomposed and degraded, resulting in a decrease in DS. Whereas, increasing the temperature from 25 • C to 37 • C had a definite effect on the DS (Figure 3B), indicating that a temperature beyond 25 • C resulted in the degradation or hydrolysis of isocyanide. Therefore, the most appropriate temperature proved to be around 25 • C. In addition, the DS increased from 4.0% to 4.9% with an increase in the molar ratio of OSA to HAc from 1:2.4 to 1:3.5, suggesting that the DS could be substantially increased by increasing the amount of HAc ( Figure 3C). This may be attributed to the fact that the HAc was conducive to the protonation of imine intermediates and promoted progress in the Ugi-4CR.
Overall, the optimum reaction conditions for synthesizing a high DS of the Ugi-OSAOcT conjugate was obtained, which were a reaction time of 12 h, a reaction temperature of 25 • C and a molar ratio of 1:2.4:3:3.3 (OSA:OCA:HAc:TOSMIC), respectively, and a series of Ugi-OSAOcT conjugates with a high DS were prepared using OSA with different oxidation degrees. As shown in Table 1, the DS of the Ugi-OSAOcT conjugates varied from 4.8% to 24.3% with an increase in the oxidation degree of OSA from 9.51% to 44.25%. In addition, the molecular weight of the Ugi-OSAOcT conjugates decreased with an increase in the oxidation degree of OSA ( Figure 3D). These results are consistent with previous findings in other reports [51].
XRD is the most direct and effective method for analyzing the crystallinity of the polymers, which has great influence on drug incorporation, micelle stability, and drug release. Importantly, the research of Zhang et al. has revealed that an amorphous structure was favorable for drug loading [52]. As shown in Figure 4A, the XRD patterns of SA and OSA showed diffraction peaks at 2θ = 14.8, 22.3 • and 2θ = 15.1 • , 22.4 • , respectively, suggesting that the hydrated crystalline structure resulted from the intramolecular hydrogen bonds of SA and OSA. For the Ugi-OSAOcT conjugates, a typical diffraction peak at 2θ = 20 • was observed, indicating that the Ugi-OSAOcT conjugates became amorphous due to the introduction of hydrophobic OCA units in the OSA block and destruction of inter-and intramolecular hydrogen bonds [53]. These results are consistent with those reported by Prabaharan et al. and Zong et al. [54,55].
The thermal stability of the Ugi-OSAOcT conjugates with different DS was evaluated by TGA and DTG. The TGA curves were shown in Figure 4B. SA and the Ugi-OSAOcT conjugates displayed similar thermal degradation behaviors, revealing two notable weight loss stages. The first one began at 80-120 • C, which was attributed to the evaporation of the physically adsorbed and encapsulated water in the samples [56,57]. The second weight loss stage took place between 200-300 • C, corresponding to the decomposition of the Ugi-OSAOcT conjugates' molecular skeletons [58]. It can be also observed that the initiatial decomposition temperature of the Ugi-OSAOcT conjugates was independent of their DS, which was lower than that of SA (248 • C), indicating that the thermal stability of the Ugi-OSAOcT conjugates decreased. This result could be attributed to the introduction of hydrophobic groups that reduced the carboxyl groups of polymers, thus destroying the intramolecular hydrogen bonds of SA, which was consistent with the XRD analysis.

Self-Assembly Behavior of Ugi-OSAOcT Conjugate
The self-assembly behavior of the amphiphilic conjugate was first evaluated by measuring the CMC with a pyrene fluorescence probe technique. Pyrene displays five characteristic emission peaks in the range of 370-400 nm. The peak intensity is sensitive to the micro-environmental polarity surrounding the pyrene molecules. As illustrated in Figure 5A, the ratio of pyrene fluorescence intensities emitted at 373 and 384 nm (I 1 /I 3 ) remained almost unchanged at low concentrations of the amphiphilic conjugate and decreased sharply once the amphiphilic conjugate concentration reached the CMC, indicating that the micelles were formed, and the pyrene probe molecules were encapsulated, in the hydrophobic interior of the micelles. The intensity ratio of I 1 /I 3 was plotted as a function of the logarithm of conjugate concentration to obtain the CMC value. The CMC values of Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT were found to be 0.30, 0.20 and 0.085 mg/mL, respectively, which was significantly lower than that of the surfactant for sodium dodecyl sulfate (CMC = 2.3 mg/mL) [59]. It can be observed from Figure 5B that the CMC value of the Ugi-OSAOcT conjugates decreased with an increase in the DS. This was possibly ascribed to the introduction of an OCA unit that provided more hydrophobicity to the Ugi-OSAOcT conjugates, thus exhibiting a higher tendency for self-assembly in an aqueous solution, which was similar to the previously reported amphiphilic block copolymers [60]. Moreover, an amphiphilic conjugate with a lower CMC value could show a higher thermodynamic stability and retain intact micellar structures even under a high dilution condition, achieving the aim of a long blood circulation time [61,62].

Self-Assembly Behavior of Ugi-OSAOcT Conjugate
The self-assembly behavior of the amphiphilic conjugate was first evaluated by mea uring the CMC with a pyrene fluorescence probe technique. Pyrene displays five charac teristic emission peaks in the range of 370-400 nm. The peak intensity is sensitive to th micro-environmental polarity surrounding the pyrene molecules. As illustrated in Figur sibly ascribed to the introduction of an OCA unit that provided more hydrophobicity the Ugi-OSAOcT conjugates, thus exhibiting a higher tendency for self-assembly in aqueous solution, which was similar to the previously reported amphiphilic block cop ymers [60]. Moreover, an amphiphilic conjugate with a lower CMC value could show higher thermodynamic stability and retain intact micellar structures even under a hi dilution condition, achieving the aim of a long blood circulation time [61,62].

Preparation and Characterization of Ugi-OSAOcT Micelles
An ultrasonic is a commonly used approach to induce the self-assembly of the a phiphilic Ugi-OSAOcT conjugates in order to achieve the formation of micelles in an aqu ous solution. The particle size and zeta potential, characterized by DLS, are crucial para eters for nanoparticle evaluations. Table 3 summarizes the particle size and zeta potent of Ugi-OSA10OcT, Ugi-OSA30OcT and Ugi-OSA50OcT micelles (1.0 mg/mL). As shown Figure 6A, when the DS of the Ugi-OSAOcT conjugates increased from 4.8 to 24.3%, t particle size of the micelles decreased from 196.5 ± 3.8 to 135.7 ± 2.4 nm, suggesting th an increase in hydrophobic block content enhanced the hydrophobic interaction betwe hydrophobic groups, resulting in a more compact cores and a smaller particle size. Z potential is one of the important factors for maintaining the stability of micelles. In ge eral, micelles with an absolute value of zeta potential greater than 30 mV have good s bility due to electrostatic repulsion [63,64]. Figure 6B showed that the zeta potential of Ugi-OSAOcT micelles (1.0 mg/mL) was around −35 mV, indicating the presence of mu ple ionized carboxyl groups (-COO − ) on the micelles' surfaces, favoring long-term stora stability and long circulation in the body, due to strong electrostatic repulsion betwe micelles.
A TEM image showed the morphologies of the self-assembled Ugi-OSA50OcT m celles (1.0 mg/mL). As shown in Figure 6C, all micelles were almost spherical with a s ranging from 90-110 nm and were well-dispersed with no aggregation. The particle s

Preparation and Characterization of Ugi-OSAOcT Micelles
An ultrasonic is a commonly used approach to induce the self-assembly of the amphiphilic Ugi-OSAOcT conjugates in order to achieve the formation of micelles in an aqueous solution. The particle size and zeta potential, characterized by DLS, are crucial parameters for nanoparticle evaluations. Table 3 summarizes the particle size and zeta potential of Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT micelles (1.0 mg/mL). As shown in Figure 6A, when the DS of the Ugi-OSAOcT conjugates increased from 4.8 to 24.3%, the particle size of the micelles decreased from 196.5 ± 3.8 to 135.7 ± 2.4 nm, suggesting that an increase in hydrophobic block content enhanced the hydrophobic interaction between hydrophobic groups, resulting in a more compact cores and a smaller particle size. Zeta potential is one of the important factors for maintaining the stability of micelles. In general, micelles with an absolute value of zeta potential greater than 30 mV have good stability due to electrostatic repulsion [63,64]. Figure 6B showed that the zeta potential of the Ugi-OSAOcT micelles (1.0 mg/mL) was around −35 mV, indicating the presence of multiple ionized carboxyl groups (-COO − ) on the micelles' surfaces, favoring long-term storage stability and long circulation in the body, due to strong electrostatic repulsion between micelles. A TEM image showed the morphologies of the self-assembled Ugi-OSA 50 OcT micelles (1.0 mg/mL). As shown in Figure 6C, all micelles were almost spherical with a size ranging from 90-110 nm and were well-dispersed with no aggregation. The particle size obtained from TEM was found to be smaller than that obtained by DLS measurement (135.7 ± 2.4 nm), as shown in Figure 6D. This phenomenon was ascribed to shrinkage of the micelles caused by water evaporation during air drying during the TEM analysis, while the DLS measurements were carried out in an aqueous medium, where the micelles were in swollen form [65].   Figure 7A,B illustrates the influence of pH on the particle size and zeta potential o the Ugi-OSAOcT micelles. When the pH decreased from 5.0 to 3.6, the zeta potential in creased promptly from −27.8-−33.1 mV to −15.2-−19.5 mV. Nevertheless, no significan changes in the particle size of the micelles were observed. This may be attributed to the fact that the beginning of protonation of the carboxylic acid groups reduced electrostatic repulsion among the polymer chains and increased the hydrophobicity of micelles, result ing in a decrease in the swelling capacity of the micelles. When the pH increased from 5.0 to 7.4, the particle size of the Ugi-OSA10OcT, Ugi-OSA30OcT and Ugi-OSA50OcT micelles increased from 145,138 and 108 nm to 196,178 and 135 nm, respectively, suggesting tha an increase in deprotonation of the carboxylic acid groups enhanced electrostatic repul  Figure 7A,B illustrates the influence of pH on the particle size and zeta potential of the Ugi-OSAOcT micelles. When the pH decreased from 5.0 to 3.6, the zeta potential increased promptly from −27.8-−33.1 mV to −15.2-−19.5 mV. Nevertheless, no significant changes in the particle size of the micelles were observed. This may be attributed to the fact that the beginning of protonation of the carboxylic acid groups reduced electrostatic repulsion among the polymer chains and increased the hydrophobicity of micelles, resulting in a decrease in the swelling capacity of the micelles. When the pH increased from 5.0 to 7.4, the particle size of the Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT micelles increased from 145,138 and 108 nm to 196,178 and 135 nm, respectively, suggesting that an increase in deprotonation of the carboxylic acid groups enhanced electrostatic repulsion among the polymer chains and hydrophilicity of the micelles, giving rise to the swelling behavior of the micelles, which could be further supported by the decrease in zeta potential from −27.8-−33.1 mV to −32.8-−38.2 mV. A similar swelling behavior of the polymeric nanoparticles responding to pH changes has been reported elsewhere [66].
tential. As shown in Figure 7C,D, the particle size and zeta potential of all micelles did no significantly change during the first six days. With the extension of storage time to 1 days, the particle size and absolute value of the zeta potential decreased slightly due, to certain extent, the degradation of the polymer micelles, indicating good long-term stabi ity in PBS (pH 7.4), which is in line with the fact that the micelles generally have good stability under neutral conditions [67].

Preparation and Characterization of IBU-Loaded Ugi-OSAOcT Micelles
The IBU-loaded Ugi-OSAOcT micelles were obtained by self-assembly and purifie by dialysis in order to remove the organic solvent. A series of IBU-loaded Ugi-OSAOc micelles with various drug feeding ratios were prepared, and their characteristics wer shown in Table 4. It was found that the sizes of the IBU-loaded micelles increased as th loading content increased, suggesting that the IBU molecules were successfully encapsu lated into the hydrophobic inner cores, and the encapsulated IBU molecules increased th size of the Ugi-OSAOcT micelles. The DL and EE of the IBU-loaded Ugi-OSA50OcT m celles increased with an increase in the drug feeding ratio. However, when the feedin mass ratio of IBU to Ugi-OSA50OcT increased from 3:10 to 5:10, the EE reduced from 57. ± 1.3% to 52.4 ± 1.5%, suggesting that the EE reached a maximum value at the mass rati of 3:10 (IBU:Ugi-OSA50OcT). This upward trend in DL and EE could be explained by th The long-term stability of the Ugi-OSAOcT micelles (1.0 mg/mL) of up to 15 days was also performed in PBS (pH 7.4) at 25 • C by measuring the particle size, PDI and zeta potential. As shown in Figure 7C,D, the particle size and zeta potential of all micelles did not significantly change during the first six days. With the extension of storage time to 15 days, the particle size and absolute value of the zeta potential decreased slightly due, to a certain extent, the degradation of the polymer micelles, indicating good long-term stability in PBS (pH 7.4), which is in line with the fact that the micelles generally have good stability under neutral conditions [67].

Preparation and Characterization of IBU-Loaded Ugi-OSAOcT Micelles
The IBU-loaded Ugi-OSAOcT micelles were obtained by self-assembly and purified by dialysis in order to remove the organic solvent. A series of IBU-loaded Ugi-OSAOcT micelles with various drug feeding ratios were prepared, and their characteristics were shown in Table 4. It was found that the sizes of the IBU-loaded micelles increased as the loading content increased, suggesting that the IBU molecules were successfully encapsulated into the hydrophobic inner cores, and the encapsulated IBU molecules increased the size of the Ugi-OSAOcT micelles. The DL and EE of the IBU-loaded Ugi-OSA 50 OcT micelles increased with an increase in the drug feeding ratio. However, when the feeding mass ratio of IBU to Ugi-OSA 50 OcT increased from 3:10 to 5:10, the EE reduced from 57.2 ± 1.3% to 52.4 ± 1.5%, suggesting that the EE reached a maximum value at the mass ratio of 3:10 (IBU:Ugi-OSA 50 OcT). This upward trend in DL and EE could be explained by the enhanced hydrophobic interaction between hydrophobic groups and IBU with the increase in hydrophobic IBU feeding ratio. When the drug content in the micelles reached saturation, severe precipitation occurred with a further increase in the IBU feeding ratio, leading to the decrease in EE. In addition, the size of IBU-loaded Ugi-OSA 50 OcT micelles remained less than 200 nm, which was very crucial for avoiding reticuloendothelial system (RES), as shown in Figure 8A [68,69]. As can be seen in Figure 8B, the TEM image of the IBU-loaded Ugi-OSA 50 OcT micelles also indicated a spherical morphology. 14.6 ± 0.3%, respectively, corresponding to EE values of 40.8 ± 1.6%, 50.6 ± 1.8% and 57 ± 1.3%, respectively. Obviously, the DS influenced both the DL and EE of the IBU-load Ugi-OSAOcT micelles. A possible reason is that more hydrophobic OCA groups graft on the hydrophilic mainchain enhanced the hydrophobic association between the hydr phobic chain, which improved the hydrophobic affinity with the hydrophobic IBU mo cules, thereby providing more available sites for hydrophobic interaction with IBU mo cules in the inner cores, and thus resulting in an increase in EE and DL. This is in accor ance with the results from study by Yokoyama et al. [70].   When the feeding mass ratio of the IBU to Ugi-OSAOcT was maintained at 3:10, the size of the IBU-loaded Ugi-OSAOcT micelles displayed a slight decrease with the rise in DS from 210.8 ± 5.2 nm (Ugi-OSA 10 OcT) to 198.6 ± 4.8 nm (Ugi-OSA 30 OcT) and further to 160.3 ± 5.7 nm (Ugi-OSA 50 OcT). This tendency of the particle size was in agreement with that of the blank Ugi-OSAOcT micelles in terms of DS. In addition, the DL of the Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT micelles were 10.9 ± 0.4%, 13.2 ± 0.5% and 14.6 ± 0.3%, respectively, corresponding to EE values of 40.8 ± 1.6%, 50.6 ± 1.8% and 57.2 ± 1.3%, respectively. Obviously, the DS influenced both the DL and EE of the IBUloaded Ugi-OSAOcT micelles. A possible reason is that more hydrophobic OCA groups grafted on the hydrophilic mainchain enhanced the hydrophobic association between the hydrophobic chain, which improved the hydrophobic affinity with the hydrophobic IBU molecules, thereby providing more available sites for hydrophobic interaction with IBU molecules in the inner cores, and thus resulting in an increase in EE and DL. This is in accordance with the results from study by Yokoyama et al. [70].

In Vitro Release of IBU from Ugi-OSAOcT Micelles
To investigate the potential utilization of the Ugi-OSAOcT micelles as oral drug carriers, in vitro drug release of IBU from micelles was explored in PBS (pH 7.4 and 5.0) at 37 • C. As shown in Figure 9A, IBU release from micelles occurred in a sustained release pattern compared with that of free IBU, which was found to release 93% within 7 h, confirming that IBU molecules were well encapsulated in the inner core of micelles. The cumulative release amount of IBU from Ugi-OSA 10 OcT, Ugi-OSA 30 OcT and Ugi-OSA 50 OcT was 73.2%, 67.2% and 63.1% within 48 h, respectively, displaying a slight decrease with an increasing DS. A possible reason is that the Ugi-OSAOcT micelles with a high DS could form a more compact hydrophobic core and exhibit a higher affinity with IBU molecules, which reduced the rate of drug diffusion from the micellar core. Therefore, it can be considered as a potential strategy to fine-tune the DS of conjugates for better control of the drug release, thereby retaining more drugs in polymeric micelles during their in vivo transport before reaching target tissue/cells, which provides a higher therapeutic efficacy [71]. Figure 9B, about 39.8% and 54.1% of IBU were released, respective from the Ugi-OSA50OcT micelles in PBS (pH = 1.2) and PBS (pH = 5.0), while 63.1% of IB was released in PBS (pH = 7.4). The results showed slower release behavior at a lower p In acidic conditions (pH = 1.2 and 5.0), the weak acid groups (-COOH) in the U OSA50OcT backbone were protonated, and the surface charge of the micelles gradua decreased, which could enhance the hydrophobicity of the micelles and reduce the el trostatic repulsion, thus forming a tighter core-shell structure to make the IBU relea more slowly. In actual drug therapy, the prolongation of release time and ideal cont performance is beneficial for improving the drug efficacy and drug utilization. In a sh period of time after taking the drug, the blood concentration of the drug in the mat could reach the effective concentration relatively quickly, and then, the concentrati could be maintained for a longer period of time, thereby achieving the purpose of su tained release. Moreover, the protonated Ugi-OSA50OcT backbone at a lower pH (sim lated gastric fluid) was conducive for protecting the drug from digestion. Consequent the Ugi-OSA50OcT micelles were suitable for oral drug administration [72]. Converse the release rates of the IBU in PBS (pH = 7.4) showed a faster and greater release behavi which made it a potential carrier for liposoluble nutraceuticals, such as IBU with co trolled release in gastrointestinal fluid.  The IBU loaded micelles also showed a sustained and pH-dependent drug release manner. As shown in Figure 9B, about 39.8% and 54.1% of IBU were released, respectively, from the Ugi-OSA 50 OcT micelles in PBS (pH = 1.2) and PBS (pH = 5.0), while 63.1% of IBU was released in PBS (pH = 7.4). The results showed slower release behavior at a lower pH. In acidic conditions (pH = 1.2 and 5.0), the weak acid groups (-COOH) in the Ugi-OSA 50 OcT backbone were protonated, and the surface charge of the micelles gradually decreased, which could enhance the hydrophobicity of the micelles and reduce the electrostatic repulsion, thus forming a tighter core-shell structure to make the IBU release more slowly. In actual drug therapy, the prolongation of release time and ideal control performance is beneficial for improving the drug efficacy and drug utilization. In a short period of time after taking the drug, the blood concentration of the drug in the matrix could reach the effective concentration relatively quickly, and then, the concentration could be maintained for a longer period of time, thereby achieving the purpose of sustained release. Moreover, the protonated Ugi-OSA50OcT backbone at a lower pH (simulated gastric fluid) was conducive for protecting the drug from digestion. Consequently, the Ugi-OSA 50 OcT micelles were suitable for oral drug administration [72]. Conversely, the release rates of the IBU in PBS (pH = 7.4) showed a faster and greater release behavior, which made it a potential carrier for liposoluble nutraceuticals, such as IBU with controlled release in gastrointestinal fluid.

In Vitro Cytotoxicity of Ugi-OSAOcT Conjugates
The in vitro cytotoxicity of the Ugi-OSAOcT conjugates, OSA and SA (control) against RAW 264.7 cells were evaluated by MTT assay. In general, a percentage of cell viability greater than 80% is considered to be a low cytotoxic in MTT assay analysis [73,74]. As shown in Figure 10, the Ugi-OSAOcT conjugates displayed a similar cell viability to SA; the cell viability of the RAW 264.7 cells remained above 90% after incubation for 48 h with a concentration of Ugi-OSAOcT conjugates ranging from 0 µg/mL to 1000 µg/mL. This indicated that an introduction of alkyl chains into OSA would not cause significant cytotoxicity against RAW 264.7 cells, even though the Ugi-OSAOcT concentration reached 1000 µg/mL. More importantly, the biocompatibility was independent of the concentration and DS of the Ugi-OSAOcT conjugates. These results were in good agreement with other studies on the development of amphiphilic OSA derivatives [75,76].

In Vitro Cytotoxicity of Ugi-OSAOcT Conjugates
The in vitro cytotoxicity of the Ugi-OSAOcT conjugates, OSA and against RAW 264.7 cells were evaluated by MTT assay. In general, a perce viability greater than 80% is considered to be a low cytotoxic in MTT assay ana As shown in Figure 10, the Ugi-OSAOcT conjugates displayed a similar cel SA; the cell viability of the RAW 264.7 cells remained above 90% after incuba with a concentration of Ugi-OSAOcT conjugates ranging from 0 μg/mL to This indicated that an introduction of alkyl chains into OSA would not caus cytotoxicity against RAW 264.7 cells, even though the Ugi-OSAOcT concentra 1000 μg/mL. More importantly, the biocompatibility was independent of th tion and DS of the Ugi-OSAOcT conjugates. These results were in good agr other studies on the development of amphiphilic OSA derivatives [75,76].

Conclusions
In the present study, amphiphilic Ugi-OSAOcT conjugates with differ successfully synthesized in distilled water at room temperature using OCA and TOSMIC via the Ugi-4CR for sustained release of IBU. The optimum rea eters were a reaction time of 12 h, a reaction temperature of 25 °C and a m 1:2.4:3:3.3 (OSA:OCA:HAc:TOSMIC), respectively. The Ugi-OSAOcT conju self-assemble into stable micelles in aqueous solutions with low CMC values of 0.40-0.085 mg/mL, a small size in the range of 135.7 ± 2.4-196.5 ± 3.8 nm a zeta potential in the range of −32.8 ± 1.4-−38.2 ± 1.8 mV. The DL and EE of the Ugi-OSAOcT micelles (IBU/Ugi-OSAOcT = 3:10, w/w) reached as much as 10 ± 0.3% and 40.8 ± 1.6-57.2 ± 1.3%, respectively. The in vitro release study d that the IBU-loaded micelles exhibited sustained and pH-responsive drug re ior, indicating that the micelles are suitable for oral drug delivery. In additio

Conclusions
In the present study, amphiphilic Ugi-OSAOcT conjugates with different DS were successfully synthesized in distilled water at room temperature using OCA, OSA, HAc and TOSMIC via the Ugi-4CR for sustained release of IBU. The optimum reaction parameters were a reaction time of 12 h, a reaction temperature of 25 • C and a molar ratio of 1:2.4:3:3.3 (OSA:OCA:HAc:TOSMIC), respectively. The Ugi-OSAOcT conjugates could self-assemble into stable micelles in aqueous solutions with low CMC values in the range of 0.40-0.085 mg/mL, a small size in the range of 135.7 ± 2.4-196.5 ± 3.8 nm and negative zeta potential in the range of −32.8 ± 1.4-−38.2 ± 1.8 mV. The DL and EE of the IBU-loaded Ugi-OSAOcT micelles (IBU/Ugi-OSAOcT = 3:10, w/w) reached as much as 10.9 ± 0.4-14.6 ± 0.3% and 40.8 ± 1.6-57.2 ± 1.3%, respectively. The in vitro release study demonstrated that the IBU-loaded micelles exhibited sustained and pH-responsive drug release behavior, indicating that the micelles are suitable for oral drug delivery. In addition, the DS of the hydrophobic segment on the OSA backbone had an important effect on the IBU-loading and drug release behavior. Additionally, the Ugi-OSAOcT conjugates exhibited a lower cytotoxicity against RAW 264.7 cells, which indicates that the Ugi-OSAOcT micelles could be considered a safe carrier for biomedical applications. Therefore, it could be an ideal candidate for hydrophobic drug delivery in the biomedical field.