Results and Discussion

3.1. Synthesis and Characterization of PLGA-Cys, PAH-Cit, and PAH-SS-PLGA Conjugates

The PAH-SS-PLGA co-polymer was synthesized by conjugating the carboxyl group of PAH-Cit with an amino group of PLGA-Cys in the presence of EDC and NHS, as shown in Scheme1. In this study, PAH-Cit was used as the backbone of the carriers, whereas cystamine were used as the GSH-sensitive segments. Furthermore, the hydrophobic segment of PLGA facilitates micelle formation to encapsulate the hydrophobic drugs, TOS and curcumin. The structures of PLGA-Cys, PAH-Cit, and PAH-SS-PLGA were confirmed using¹H-NMR and FTIR. In addition, UV-VIS was used to confirm TOS as well as curcumin loading and release from the PAH-SS-PLGA micelle. As shown in Figure1A, the¹H-NMR peaks of free PAH that were observed at 1.2 ppm correspond to the methylene protons (-CH2-) on the PAH polymer backbone, those at 1.7 ppm correspond to the backbone methine protons, and the 3.20 ppm peak corresponds to the methylene protons adjacent to the amine group. After the conjugation of PAH with citraconic anhydride, additional peaks were observed at the chemical shifts of 1.9, 5.6, and 5.8 ppm, which confirmed PAH-Cit was successfully synthesized. Two isomers of PAH-Cit were obtained, with the methyl group either distal or proximal to the newly generated amide bond. The ratio of proximal to distal isomers was about 1.37. The degree of grafting (dg) of citraconic acid to PAH was approximately 46% by considering the integral area ratio of distal/proximal CH3 of Cit to CH2 group of PAH. Similarly, as shown in Figure1B, PLGA was successfully conjugated with cystamine.

The chemical shifts at 2.98 and 3.01 ppm were observed, which correspond to the different methylene (2H, –CH2–S–; 2H, –CH2–N) groups of the cystamine. Moreover, PAH-SS-PLGA was successfully synthesized by conjugating PAH-Cit and PLGA-Cys.

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backbone methine protons, and the 3.20 ppm peak corresponds to the methylene protons adjacent to the amine group. After the conjugation of PAH with citraconic anhydride, additional peaks were observed at the chemical shifts of 1.9, 5.6, and 5.8 ppm, which confirmed PAH-Cit was successfully synthesized. Two isomers of PAH-Cit were obtained, with the methyl group either distal or proximal to the newly generated amide bond. The ratio of proximal to distal isomers was about 1.37.

The degree of grafting (dg) of citraconic acid to PAH was approximately 46% by considering the integral area ratio of distal/proximal CH3 of Cit to CH2 group of PAH. Similarly, as shown in Figure 1B, PLGA was successfully conjugated with cystamine. The chemical shifts at 2.98 and 3.01 ppm were observed, which correspond to the different methylene (2H, –CH2–S–; 2H, –CH2–N) groups of the cystamine. Moreover, PAH-SS-PLGA was successfully synthesized by conjugating PAH-Cit and PLGA-Cys.

Scheme 1. (A) Scheme of poly(allylamine)-citraconic anhydride-poly(lactic-co-glycolic acid)-cystamine (PAH-SS-PLGA) synthesis and (B) endocytosis-mediated uptake of curcumin/TOS-loaded micelle. TOS: tocopheryl succinate.

Figure 1. Cont.

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Figure 1. ¹H-NMR of (A) PAH and PAH-Cit and (B) PLGA-Cys, PAH-Cit, and PAH-SS-PLGA. Note:

D²O was used as a solvent for PAH and PAH-Cit, whereas DMSO and CDCl3 solvent were used for PLGA-Cys and PAH-SS-PLGA, respectively. PAH: poly (allylamine hydrochloride), PAH-Cit:

poly(allylamine)-citraconic anhydride, PLGA-Cys: poly(lactic-co-glycolic acid)-cystamine.

In addition to ¹H-NMR, the chemical structures of the PAH, PAH-Cit, and PAH-SS-PLGA were further confirmed using FTIR, as shown in Figure 2. All major characteristic peaks of PAH, PAH-Cit, and PAH-SS-PLGA were observed within the range of 4000−620 cm⁻¹. Several major peaks were observed at approximately 3200–3364 cm⁻¹ (N-H stretch), 2883−2915 cm⁻¹ (C−H stretch), 1756 cm⁻¹ (C=O, stretch), and 1320–1410 cm⁻¹ (C−H bending). In addition, there are new peaks at 1619 cm⁻¹ (amide I bond) and 1538 cm⁻¹ (amide II bond), which confirm amide bond formation between PAH and citraconic anhydride and PAH-Cit and PLGA-Cys to form PAH-Cit and PAH-SS-PLGA, respectively. Overall, both ¹H-NMR and FTIR results confirmed the successful synthesis of PAH-Cit and PAH-SS-PLGA.

Figure 2. FTIR peak of (a) PAH, PAH-Cit, and (b) PAH-SS-PLGA.

3.2. Preparation and Characterization of Empty Micelles and Drug-Loaded Micelles

The PAH-SS-PLGA conjugate could self-assemble into micelles in aqueous solution due to its amphiphilic nature and was further used to encapsulate hydrophobic TOS and curcumin drugs via emulsion solvent evaporation methods. The mean particle size of TOS or curcumin-loaded micelles was 172.93 ± 1.1 nm and 194.17 ± 1.7 nm, respectively, with a low polydispersity index, indicating the formation of nanoparticles with a narrow and unimodal distribution, as shown in Table 1, based

Figure 1.¹H-NMR of (A) PAH and PAH-Cit and (B) PLGA-Cys, PAH-Cit, and PAH-SS-PLGA. Note:

D₂O was used as a solvent for PAH and PAH-Cit, whereas DMSO and CDCl3 solvent were used for PLGA-Cys and PAH-SS-PLGA, respectively. PAH: poly (allylamine hydrochloride), PAH-Cit:

poly(allylamine)-citraconic anhydride, PLGA-Cys: poly(lactic-co-glycolic acid)-cystamine.

In addition to¹H-NMR, the chemical structures of the PAH, PAH-Cit, and PAH-SS-PLGA were further confirmed using FTIR, as shown in Figure2. All major characteristic peaks of PAH, PAH-Cit, and PAH-SS-PLGA were observed within the range of 4000−620 cm⁻¹. Several major peaks were observed at approximately 3200–3364 cm⁻¹(N-H stretch), 2883−2915 cm⁻¹(C−H stretch), 1756 cm⁻¹ (C=O, stretch), and 1320–1410 cm⁻¹(C−H bending). In addition, there are new peaks at 1619 cm⁻¹ (amide I bond) and 1538 cm⁻¹ (amide II bond), which confirm amide bond formation between PAH and citraconic anhydride and PAH-Cit and PLGA-Cys to form PAH-Cit and PAH-SS-PLGA, respectively. Overall, both¹H-NMR and FTIR results confirmed the successful synthesis of PAH-Cit and PAH-SS-PLGA.

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Figure 1. ¹H-NMR of (A) PAH and PAH-Cit and (B) PLGA-Cys, PAH-Cit, and PAH-SS-PLGA. Note:

D²O was used as a solvent for PAH and PAH-Cit, whereas DMSO and CDCl3 solvent were used for PLGA-Cys and PAH-SS-PLGA, respectively. PAH: poly (allylamine hydrochloride), PAH-Cit:

poly(allylamine)-citraconic anhydride, PLGA-Cys: poly(lactic-co-glycolic acid)-cystamine.

In addition to ¹H-NMR, the chemical structures of the PAH, PAH-Cit, and PAH-SS-PLGA were further confirmed using FTIR, as shown in Figure 2. All major characteristic peaks of PAH, PAH-Cit, and PAH-SS-PLGA were observed within the range of 4000−620 cm⁻¹. Several major peaks were observed at approximately 3200–3364 cm⁻¹ (N-H stretch), 2883−2915 cm⁻¹ (C−H stretch), 1756 cm⁻¹ (C=O, stretch), and 1320–1410 cm⁻¹ (C−H bending). In addition, there are new peaks at 1619 cm⁻¹ (amide I bond) and 1538 cm⁻¹ (amide II bond), which confirm amide bond formation between PAH and citraconic anhydride and PAH-Cit and PLGA-Cys to form PAH-Cit and PAH-SS-PLGA, respectively. Overall, both ¹H-NMR and FTIR results confirmed the successful synthesis of PAH-Cit and PAH-SS-PLGA.

Figure 2. FTIR peak of (a) PAH, PAH-Cit, and (b) PAH-SS-PLGA.

3.2. Preparation and Characterization of Empty Micelles and Drug-Loaded Micelles

The PAH-SS-PLGA conjugate could self-assemble into micelles in aqueous solution due to its amphiphilic nature and was further used to encapsulate hydrophobic TOS and curcumin drugs via emulsion solvent evaporation methods. The mean particle size of TOS or curcumin-loaded micelles was 172.93 ± 1.1 nm and 194.17 ± 1.7 nm, respectively, with a low polydispersity index, indicating the formation of nanoparticles with a narrow and unimodal distribution, as shown in Table 1, based

Figure 2.FTIR peak of (a) PAH, PAH-Cit, and (b) PAH-SS-PLGA.

3.2. Preparation and Characterization of Empty Micelles and Drug-Loaded Micelles

The PAH-SS-PLGA conjugate could self-assemble into micelles in aqueous solution due to its amphiphilic nature and was further used to encapsulate hydrophobic TOS and curcumin drugs via emulsion solvent evaporation methods. The mean particle size of TOS or curcumin-loaded micelles was 172.93 ± 1.1 nm and 194.17 ± 1.7 nm, respectively, with a low polydispersity index, indicating the

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formation of nanoparticles with a narrow and unimodal distribution, as shown in Table1, based on the DLS measurement. The zeta potential results showed that both drug-loaded and empty micelles displayed positive surface charges of approximately 22 mV, which shows that the synthesized carrier is very stable due to charge stabilization (i.e., the electrostatic repulsive forces are high enough to prevent aggregation) [36]. The surface morphology of the synthesized micelle was observed to have a spherical shape, using TEM, as displayed in Figure3a. Moreover, after incubating for 6 h with 5 mM GSH, the synthesized micelle slightly lost its spherical shape, which is due to the cleavage of disulfide bonds, which in turn leads to disassembly of the micelle, as shown in Figure3b.

Table 1. The mean particle size, polydispersity index (PI), and zeta potential of empty, TOS, and/or curcumin-loaded micelles in distilled water (dH₂O) at 25^◦C.

Sample Name Particle Size (nm) Polydispersity Index (PDI) Zeta Potential (mV)

PAH-ss-PLGA micelle 161.73 ± 2.5 0.22 ± 0.02 22.73 ± 1.17

PAH-ss-PLGA-TOS 172.93 ± 1.1 0.13 ± 0.02 21.57 ± 0.84

PAH-ss-PLGA-Curcumin 174.17 ± 1.7 0.16 ± 0. 02 19.77 ± 1.42

PAH-ss-PLGA-Curcumin-TOS 201 ± 2.1 0.21 ± 0.01 17.57 ± 0.54

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micelles displayed positive surface charges of approximately 22 mV, which shows that the synthesized carrier is very stable due to charge stabilization (i.e., the electrostatic repulsive forces are high enough to prevent aggregation) [36]. The surface morphology of the synthesized micelle was observed to have a spherical shape, using TEM, as displayed in Figure 3a. Moreover, after incubating for 6 h with 5 mM GSH, the synthesized micelle slightly lost its spherical shape, which is due to the cleavage of disulfide bonds, which in turn leads to disassembly of the micelle, as shown in Figure 3b.

Table 1. The mean particle size, polydispersity index (PI), and zeta potential of empty, TOS, and/or curcumin-loaded micelles in distilled water (dH²O) at 25 ℃.

Sample Name Particle Size 3.3. Drug Loading and Drug Release Study from PAH-SS-PLGA Micelle

The encapsulation efficiency (EE%) of the PAH-SS-PLGA micelle was assessed both for curcumin and TOS. The calculated EE% of the micelles was 85% and 95.5% for TOS and curcumin, respectively. Furthermore, to validate that GSH induced TOS/curcumin release from the micelle, the drug-release behavior was determined by a dialysis method in PBS buffer (pH 7.4) in the presence and absence of GSH (5 mM). The amount of TOS and curcumin released was measured by UV−VIS spectrophotometer at λmax of 286 nm and 420 nm, respectively. In vitro drug release study revealed that excess TOS and or curcumin release was observed in the presence of GSH (5 mM) at the physiological pH value, as shown in Figure 3c,d, showing that the synthesized PAH-SS-PLGA micelle is GSH sensitive.

Figure 3. TEM image of PAH-SS-PLGA-micelle in the absence (a) and presence of 5 mM GSH (b), in vitro curcumin (c) and TOS (d) release from PAH-SS-PLGA micelle in the presence and absence of GSH (5 mM). Data are expressed as mean ± SD values (n = 3).

Figure 3. TEM image of PAH-SS-PLGA-micelle in the absence (a) and presence of 5 mM GSH (b), in vitro curcumin (c) and TOS (d) release from PAH-SS-PLGA micelle in the presence and absence of GSH (5 mM). Data are expressed as mean ± SD values (n= 3).

3.3. Drug Loading and Drug Release Study from PAH-SS-PLGA Micelle

The encapsulation efficiency (EE%) of the PAH-SS-PLGA micelle was assessed both for curcumin and TOS. The calculated EE% of the micelles was 85% and 95.5% for TOS and curcumin, respectively.

Furthermore, to validate that GSH induced TOS/curcumin release from the micelle, the drug-release behavior was determined by a dialysis method in PBS buffer (pH 7.4) in the presence and absence of GSH (5 mM). The amount of TOS and curcumin released was measured by UV−VIS spectrophotometer at λmax of 286 nm and 420 nm, respectively. In vitro drug release study revealed that excess TOS and or curcumin release was observed in the presence of GSH (5 mM) at the physiological pH value, as shown in Figure3c,d, showing that the synthesized PAH-SS-PLGA micelle is GSH sensitive.

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3.4. In Vitro Cytotoxicity Studies

Dependent on the drug release mechanism or their direct interaction with the cell of interest, nanoformulated drugs show different anti-cancer activity in the in vitro cytotoxicity study in comparison to free drugs. Hence, in order to investigate the in vitro cytotoxicity effect of free TOS, free curcumin, TOS + curcumin, or TOS/curcumin-loaded micelles, cell viability was evaluated by the MTT assay using PAN02 pancreatic cancer cells. As shown in Figure 4a,b, free TOS, free curcumin, and TOS/curcumin-loaded micelles could inhibit the growth of PAN02 cancer cells in a concentration-dependent manner. Most interestingly, nanoformulated drugs showed higher cytotoxicity than the free drugs in Figure4c, which is maybe due to the greater accumulation of nanoformulated drugs via endocytosis and slow drug release mechanism, which will prevent the drugs’ efflux. Similarly, the cytotoxicity of the materials (PAH, PAH-Cit, and PAH-SS-PLGA) was also investigated, as shown in Figure4d. The result revealed that PAH shows higher cytotoxicity at the tested higher concentration in comparison to its derivative (PAH-Cit and PAH-SS-PLGA). This is due to the cationic nature of PAH, which will disrupts the integrity of the cellular membranes. It was reported that substitution of the amino groups of PAH (such as guanidinylation, glycolylation, or imidazolyl) significantly reduces its cytotoxicity [22,23]. Hence, it is expected that surface modification of the PAH polymer with citraconic anhydride and PLGA-Cys could also minimize the PAH-related cytotoxicity.

The colony assay is the other alternative assay to monitor a cancer cell’s ability to produce a viable colony after treatment. The colony-forming assay gives information about a long-term proliferative potential of cells that cannot be determined by short-term assays. Hence, combining the colony-forming assay with assays that measure cell death or cell viability in short time periods is likely to offer more information about the fate of cells in a population. Hence, in this study, colony formation assay was done to investigate the long-term anti-cancer potential of free TOS, curcumin, or nanoformulated TOS and curcumin using PAN02 pancreatic cancer cell lines. As shown in Figure4e–g, the colony formation results revealed an improved therapeutic efficacy of nanoformulated TOS and or curcumin than free TOS and or curcumin at a lower dosage. The enhanced anti-cancer therapeutic efficacy of nanoformulated TOS and/or curcumin compared to free TOS or curcumin is due to the slow release of TOS or curcumin from internalized nanocarriers in PAN02 pancreatic cancer cells. Together with MTT assay, this result indicates that nanoformulated drugs have better anti-proliferative actions against PAN02 pancreatic cancer cells.

The combination effects of the TOS and curcumin were also investigated for PAN02 pancreatic cancer cells, as shown in Figure4c. As shown above, a combination of TOS and curcumin as well as dual-loaded micelle shows better therapeutic efficacy than the free drugs alone. In addition, the dose-dependent effects of the drugs when used alone and in combination were analyzed using CompuSyn, which is a computer program that analyzes dose–response data according to the Chou and Talalay median effect principle [34]. The program generates combination index (CI) values from the dose–response curves and provides an indication as to whether the interaction between the two drugs results in synergistic (CI< 1), additive (CI = 1), or antagonistic (CI > 1) effects. As shown in Table2, at all tested concentrations, the calculated CIs value were less than one, indicating synergistic effects for all nanoformulated-drugs (PAH-SS-PLGA-TOS-Curcumin), whereas at lower concentrations, the combination of free TOS+ free curcumin elicited an antagonistic effect on the cellular proliferation inhibition of PAN02 pancreatic cancer cells.

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Figure 4. MTT in vitro cytotoxcity results using PAN02 pancreatic cancer cells (a) free curcumin and curcumin-loaded micelles, (b) free TOS and TOS-loaded micelles, (c) a combination of TOS and curcumin, (d) PAH, PAH-Cit, PLGA-Cys and PAH-SS-PLGA, and (e–g) representative images of the crystal violet-stained colonies using PAN02 pancreatic cancer cells at different doses of free TOS and PAH-SS-PLGA-TOS, free curcumin and PAH-SS-PLGA-curcumin, and a combination of free TOS Figure 4. MTT in vitro cytotoxcity results using PAN02 pancreatic cancer cells (a) free curcumin and curcumin-loaded micelles, (b) free TOS and TOS-loaded micelles, (c) a combination of TOS and curcumin, (d) PAH, PAH-Cit, PLGA-Cys and PAH-SS-PLGA, and (e–g) representative images of the crystal violet-stained colonies using PAN02 pancreatic cancer cells at different doses of free TOS and PAH-SS-PLGA-TOS, free curcumin and PAH-SS-PLGA-curcumin, and a combination of free TOS and curcumin and nanoformulated TOS/curcumin, respectively. Data are expressed as mean ± SD values

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Table 2. Effects of combined TOS and curcumin treatment on PAN02 pancreatic cancer cells.

CI: combination index.

Drugs Concentration of Drugs

(µg/mL) % Cell Viability CI Value Effects

Free TOS+ Free Curcumin 50+ 25 7.23 ± 0.053 0.40125 synergism

25+ 12.5 17.81 ± 0.028 0.53959 synergism

12.5+ 6.25 31.34 ± 0.049 0.56649 synergism 6.25+ 3.125 50.85 ± 0.039 0.65387 synergism 3.125+ 1.5625 81.12 ± 0.115 1.48333 antagonistic 1.5625+ 0.78125 87.63 ± 0.087 1.28316 antagonistic PAH-ss-PLGA-TOS+

PAH-ss-PLGA-Curcumin 50+ 25 9.95 ± 0.016 0.68485 synergism

25+ 12.5 15.68 ± 0.028 0.61559 synergism

12.5+ 6.25 32.35 ± 0.020 0.89538 synergism 6.25+ 3.125 50.17 ± 0.044 0.97939 synergism 3.125+ 1.5625 58.42 ± 0.039 0.76110 synergism 1.5625+ 0.78125 64.89 ± 0.069 0.52037 synergism

3.5. Apoptosis and Cell Cycle Analysis

Apoptosis is the other key factor that accompanies cancer cell death in chemotherapy. The induction of apoptosis can be recognized by several molecular mechanisms (including the activation of caspases and cleaved poly (ADP-ribose) polymerases (cPARP) or a change of cellular morphology [37].

The apoptotic effect of free TOS, curcumin, and drug-loaded micelles against PAN02 cancer cells was investigated using an annexin V and PI assay. As shown in Figure5a, an increase in apoptosis of PAN02 cancer cells was observed after treatment with free TOS, curcumin, and nanoformulated drugs for 48 h. The percentages of necrosis, early, and late apoptotic cells among those treated with free TOS (30 µg/mL) were 12.7%, 24.3%, and 24.7% respectively, whereas the figures were 0.2%, 0%, and 0% in untreated cells, respectively. The percentages of necrosis, early, and late apoptotic cells among those treated with nanoformulated TOS (at a TOS concentration of 30 µg/mL) were 36.2%, 20.9%, and 3.5%, respectively. Similarly, the percentages of necrosis, early, and late apoptotic cells among those treated with free curcumin (15 µg/mL) were 33.0%, 27.6%, and 7.9% respectively, whereas the figures were 22.8%, 30.2%, and 11.6% for nanoformulated curcumin, respectively. Most interestingly, the percentages of early and late apoptotic cells among those treated with combination drugs or free curcumin and free TOS were 60.4% and 9.4%, respectively, while those of nanoformulated curcumin–TOS were 49.5%

and 7.8% (at curcumin and TOS concentration of 15 µg/mL and 30 µg/mL, respectively). Similarly, several researchers reported that curcumin can induce apoptosis and subsequently contribute to cancer cell death [38,39]. In addition, proapoptotic activities of TOS are also reported in cancer cells via mitochondrial inhibition, the most effective mitocans in inducing the apoptosis, and the production of superoxide radicals [40–42]. Collectively, these results suggested that a co-treatment of curcumin and TOS would enhance the apoptotic cell death of PAN02 pancreatic cancer cells.

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while those of nanoformulated curcumin–TOS were 49.5% and 7.8% (at curcumin and TOS concentration of 15 μg/mL and 30 μg/mL, respectively). Similarly, several researchers reported that curcumin can induce apoptosis and subsequently contribute to cancer cell death [38,39]. In addition, proapoptotic activities of TOS are also reported in cancer cells via mitochondrial inhibition, the most effective mitocans in inducing the apoptosis, and the production of superoxide radicals [40–42].

Collectively, these results suggested that a co-treatment of curcumin and TOS would enhance the apoptotic cell death of PAN02 pancreatic cancer cells.

Furthermore, investigation of cell cycle distribution and cell proliferation is essential for studying cell growth differentiation, apoptosis, and senescence. During cell cycle progression, there are variations in the genetic content, and proliferating cells consecutively undergo different stages, such as G0G1 (preparation for DNA synthesis), S (DNA synthesis), and G2M phases (preparation of cell division and mitosis), which can be observed by fluorescent dye, such as propidium iodide (a fluorescent DNA-intercalating dye), using flow cytometry. In this study, the cell cycle analysis was carried out for PAN02 pancreatic cancer cells after treating with free TOS, curcumin, and TOS/curcumin-loaded micelles for 48 h. As shown in Figure 5b, PAN02 cells treated with free TOS and TOS + free curcumin showed a higher accumulation of cells in the G0G1, while TOS and or curcumin-loaded micelles showed cell inhibition at the G2M phase compared to that of the untreated PAN02 pancreatic cancer cells. A similar observation was observed that showed curcumin-mediated cell cycle arrest at G0G1 and G2M checkpoints [43].

Figure 5. (a) Apoptosis assay of free TOS, free curcumin, free TOS + free curcumin, and TOS/curcumin-loaded micelles and (b) cell cycle analysis of free TOS, free curcumin, and

Figure 5. (a) Apoptosis assay of free TOS, free curcumin, free TOS + free curcumin, and TOS/curcumin-loaded micelles and (b) cell cycle analysis of free TOS, free curcumin, and