Chitosan and (Phenylthio) acetic acid-based ion pair self assembled for temperature and oxidation-responsive drug release

ABSTRACT Chitosan (CTSN), a cationic biopolymer was used to prepare Ion pair self-assembly (IPSAM) with (phenylthio)acetic acid (PTA) as hydrophobic counter ion which is responsive to temperature and oxidation. The IPSAM was formed when the amino to carboxylic group molar ratio was 1/9 ~ 4/6. CTSN/PTA IPSAM was found as sphere-like nanoparticles whose diameter was 30–50 nm on TEM image. The ion pair showed the upper critical solution temperature (UCST) that increased with increasing PTA content and decreased when PTA of the ion pair was oxidized by H2O2. The ion pair exhibited amphiphilic property which was responsible for interface activity which decline upon the oxidation of the PTA. The surface tension was low for the ratio of CTSN/PTA (3/7), which made the 3/7 ratio suitable for further studies. The ionic interaction between CTSN and PTA and the oxidation of PTA were confirmed by FT-IR spectroscopy. The payload’s release (i.e. Nile red) in IPSAM was constrained below the UCST but it was prompted above the phase transition temperature possibly due to the disassembly of the IPSAM and PTA oxidation. The release was found to be higher in tumor environment temperature in the presence of H2O2 indicating the release was dependent on temperature and oxidation.


Introduction
Chitosan is a natural biopolymer, also much attention seeker as a polysaccharide that has gained interest in many research fields.3] The versatility of chitosan relies on the ability of the polycationic charge molecule at physiological pH due to the protonation of the D-glucosamine in its polymer structure. [2]Chitosan's cationic nature confers mucoadhesive characteristics for mucosal medication delivery applications such as ocular and intranasal administration. [4,5][11] Vaccines made with chitosan exhibit high mucosal absorption and macrophage activation due to chitosan's mucoadhesive and adjuvant activities. [12]The pKa value of the chitosan is 6.5 and suitable in an acidic solution the protonated species is capable of complexing a diverse range of anionic biomolecules via self-assembly and can form micro or nanoparticles through polyelectrolytic interaction. [13]In the presence of the hydrophobic moiety, it can form an amphiphilic molecule in an aqueous solution.
Amphiphilic molecules show distinct behavior as they can aggregate themselves in which the hydrophobic moiety orients within the cluster and hydrophilic portions encapsulate the solvent. [13]The hydrophobic portion of the molecule typically consists of nonpolar or partially nonpolar groups, such as hydrocarbon chains, while the hydrophilic region comprises polar or ionic groups, such as carboxylates, sulfates, or quaternary ammonium ions.This structural duality is essential for the unique properties and behaviors exhibited by amphiphiles.The self-assembly behavior of amphiphiles allows for the creation of nanostructures with welldefined architectures which plays a crucial role in drug delivery systems.[23][24] Even if a medicine is successfully transported to a target site by a drug carrier, it must be released promptly for its efficacy to be realized.One way to obtain target site-specific release properties is to imbue drug delivery vehicles with stimuli-responsiveness. [25][26][27][28] Among many stimuli-responsive systems, ion pair self-assembly capable of responding to two or more environmental changes has sparked the interest of researchers for two reasons.Firstly more than one stimulus can evoke the multi-responsive characteristic continuously or sequentially enabling enhanced spatiotemporal control. [29,30]Secondly, because behavioral changes in biological systems are frequently the result of a mixture of environmental variables rather than a single component multi-responsive materials provide an ideal artificial platform for mimicking natural biological processes. [31,32][40] Temperature and oxidationresponsive drug carriers that promote the release of the payload have become a major interest for researchers.The release of the payload aided by oxidation and the oxidation-induced release were more visible at higher temperatures.The oxidation of the oxidation-responsive amphiphilic copolymer led to an increase in the lower critical solution temperature and loss of amphiphilicity leading to di-micellization, accounting for the promoted release. [19,40]However, the manufacture of the dualresponsive polymeric micelles took a long time and effort since copolymers, the micelles' building blocks, had to be produced and purified through a difficult process.
Using Chitosan (CTSN) as a cationic hydrophilic polymer and PTA as a hydrophobic counter ion, a temperature, and oxidation-responsive IPSAM was created in this study (Scheme 1 and 2).Because CTSN is a cationic polymer with a primary amino group and readily soluble in an aqueous solution slightly acidic pH (pH 4), it may establish a stable ion pair with PTA and the ion pair will result in a persistent IPSAM.Furthermore, chitosan is a natural polymer with an amino group that can increase the utilization and added value of bioresources when used to create highly valued materials such as a stimuli-responsive smart medicine carrier.The current study demonstrated a simple, quick, and low-cost approach for creating a stimuli-responsive carrier employing a polysaccharide-based polymer as a material.

Preparation of Ion Pair Self-Assembly of CTSN/PTA IPSAM
Chitosan CTSN was dissolved in Trizma buffer with 1% acetic acid (30 mM, pH 4) achieving a concentration of 20 mg/ml.The varied volume of CTSN solution (20 mg/ ml, 0.355¬2.485ml) was added to a 5 ml vial before being diluted to 3 ml with the same buffer.To get the final concentration of CTSN plus PTA 20 mg/ml, a varied amount of PTA (10.3 mg¬52.9mg) was added to the CTSN solution.The molar ratio of the amino group of CTSN to the carboxyl group of PTA was 1:9, 2:8, 3:7, 4:6, 5:5; 6:4, 7:3, 8:2 & 9:1 accordingly.PTA was dissolved in the mixture completely by rolling the vial on a roller mixer for 24 hours at room temperature (20-23°C).The molar ratio of the amino group from CTSN to the carboxyl group of the PTA was a/b respectively, and CTSN/PTA solution was prepared by mixing CTSN and PTA in a buffer solution.The self-assembled Ion pair of CTSN/PTA (x,y) was termed IPSAM (x,y).

Transmission Electron Microscopy(TEM)
The geometric shape of IPSAM (x/y) was investigated using a negative staining method.To prepare the suspension for the IPSAM staining, uranyl acetate solution (1% w/v) was added in a 1:1 volumetric ratio.The suspension was then left at room temperature in the dark for two hours.The deposition of IPSAM onto the grid surface required 5 minutes after a drop of stained IPSAM suspension was placed on a formvar/coppercoated grid (200 mesh, Electron Microscopy Science, Hatfield, PA).The filter paper was used to remove the excess suspension, and the wet grid was allowed to air dry at room temperature in the dark.A transmission electron microscope (LEO912AB OMEGA, LEO, Germany, housed at Korea Basic Science Institute (KBSI)) was used to capture the image of IPSAM

Measurement of Hydrodynamic Diameter and Zeta Potential
Dynamic light scattering equipment (ELSZ-2000.Zeta potential & Particle size analyzer, Otsuka Electronics, ERAETECH, Korea) was used to measure the hydrodynamic diameter and the zeta potential after reducing the light scattering intensity of all IPSAM suspensions to between 50 and 200 Kcps and filtered using 0.45 µm syringe filter.The ratios of CTSN/PTA from 1:9, 2:8, 3:7 & 4:6 respectively were analyzed for the differentiation of the size and charge of the species.

Determination of Upper Critical Solution Temperature (UCST)
The temperature-dependent optical density of the CTSN/PTA (x/y) solution was used to calculate the UCST of the CTSN/PTA ion pair. 1 ml of CTSN/PTA (x/y) solution was taken in a cuvette and placed in the cuvette holder of a UV spectrophotometer (6505 UV/ Vis Spectrophotometer, JENWAY, UK) fitted with a temperature controller (CW-05 G, Jeio Tech, South Korea).The CTSN/PTA mixture solution was heated at the rate of 2°C/min in the range of 20°C−60°C at the photometric wavelength of 600 nm to obtain the temperature.The change in PTA's hydrophilicity due to oxidation may have an impact on the UCST of the CTSN/PTA ion pair.For oxidation, a 1:1 volumetric mixture of CTSN/PTA (x/y) solution and H 2 O 2 solution was used.Final H 2 O 2 concentrations were 0, 1, 2.5, and 5 mM, whereas the final CTSN mixed PTA concentration was 20 mg/ml.The mixture solution of CTSN/PTA/H 2 O 2 was spun on a roller mixer at room temperature for 1 hour.The term CTSN/PTA (x/y) solution (z mM H 2 O 2 ) refers to the H 2 O 2 -treated CTSN/PTA (x/y) solution with a z mM H 2 O 2 concentration.The temperature at the cross-section of a tangential line passing through points of data in a rising plateau temperature zone and a declining temperature zone was utilized to determine the CTSN/PTA ion pair's UCST.

FT-IR Spectroscopy
The ionic interaction between CTSN and PTA was discovered using FT-IR spectroscopy.5 ml of CTSN (20 mg/ml), PTA (20 mg/ml), and CTSN/PTA (x/y) (20 mg/ml) solutions were freeze-dried, and KBr was added to each, blended in a mortar with a pestle, pelletized via a press, and evaluated by FT-IR spectroscopy (FT-3000-Excalibur, Varian Inc., CA, USA).FT-IR spectroscopy revealed the oxidation of PTA by H 2 O 2 .2.5 ml of CTSN/PTA (3/7) solution (40 mg/ml) were mixed with the same volume of H 2 O 2 solutions (2 mM, 10 mM, 20 mM in Trizma buffer (30 mM, pH7.0)) in a 10 ml vial, stirred at room temperature for 1 hour with a roller mixer, and freeze-dried.The Fourier transform infrared spectrum of the H 2 O 2treated CTSN/PTA ion pair, as well as the H 2 O 2untreated specimens were collected using the similar method mentioned above.

Turbidimetric Experiment
The ionic interaction between CTSN and PTA was further confirmed by the turbidity experiment.The ion pair self-assembled CTSN/PTA (3/7) solution was prepared in trisma buffer.NaCl solutions at various concentrations were also prepared in trisma buffer.A given volume of the CTSN/PTA (3/7) solution was mixed with different concentrations of the NaCl solution at 25°C.UV spectrophotometer (6505 UV/Vis Spectrophotometer, JENWAY, UK) was used to obtain the optical density or absorbance of the mixture at 600 nm.The turbidity of the solution was evaluated with the interaction of the NaCl to show the electrostatic interaction between CTSN and PTA.

H NMR Spectroscopy
CTSN and PTA were dissolved in 1% acetic acid & D 2 O respectively for the NMR analysis.The IPSAM with the final concentration of CTSN + PTA of 20 mg/ml and the amino group to carboxyl group molar ratio of 1:9 to 2:8 ratio solutions were stirred on a roller mixer at room temperature (20-23°C) overnight and freeze-dried and again dissolved in D 2 O before subjecting them to 1 H NMR spectroscopy on a Bruker Avance 400 MHz spectrometer (Karlsruhe, Germany, located in Kangwon National University's Central Laboratory Cente).

Release Responsive to Temperature, Oxidation
Nile red was incorporated in IPSAM by merging 150 μl of Nile red solution (1 mg/l, in methanol) with 15 ml of CTSN/PTA (3/7) solution (40 mg/l), in Trizma buffer (30 mM, pH7) in a 30 ml vial and mixing the combination overnight at the temperature of the room on a roller mixer.A 3 ml fluorescence cuvette placed in a cuvette holder was filled with 1.3 ml of H 2 O 2 solution (0, 1, 2, 10 mM) in Trizma buffer (30 mM, pH7.0), and the temperature was raised to 26°C, 37°C, and 46°C (room temperature, body temperature, and tumor temperature respectively) using a water circulator.In a cuvette containing an H 2 O 2 solution, a corresponding volume of the Nile red-loaded IPSAM suspension was inserted.The IPSAM suspension was stimulated at 556 nm, and the fluorescence intensity was measured using a fluorescence spectrophotometer (Hitachi F2500, Hitachi, Japan) over time at 613 nm repeating 3 times. [41]ere F t is the fluorescence intensity at a time-lapse, and F o is the fluorescence intensity at time zero.

Preparation of Ion Pair Self-Assembly (IPSAM)
The CTSN/PTA (a/b) solutions ratio 1:9, 2:8, 3:7 & 4:6 were homogeneous milky suspensions, as illustrated in Supplementary Figure .S1. PTA and CTSN are most likely ionically coupled, producing an ion pair.Using the Henderson-Hasselbalch equation (Ionization degree = 1/[1 + 10( pKa-pH )] 100), PTA included an ionizable carboxyl group with a pKa of around 3.7 and an ionization degree of 99.94% in the suspension medium (Trizma buffer solution (30 mM, pH7.0). [42,43]As a result, the bulk of the carboxyl groups may be able to interact ionically with the positively charged amino group of CTSN.PTA was completely suspended in the CTSN solution, with little PTA sediment, demonstrating that PTA was bound to the soluble water polymer in the aqueous solution.The hydrophobic contact between the phenyl groups of PTA molecules would cause the CTSN/PTA ion pair to spontaneously assemble once created.The self-assembly of CTSN/PTA appeared to scatter visible light, resulting in the creation of milky opaque suspensions (Supplemental Fig. S1).

Transmission Electron Microscopy (TEM)
The transmission electron diagrams of IPSAM (3/7) and IPSAM (2/8) are shown in Figure 1(a-c, e-g).On the micrographs, all of the IPSAMs were discovered to be virtually spherical nanoparticles having diameters from 30 to 55 nm.IPSAM (3/7) and IPSAM (2/8) had mean diameters of 34.48 nm and 34.5 nm, respectively (Fig. 1(d,  h)).The average number of particles in IPSAM (3/7) and (2/8) was found to be 53 and 47 respectively from the TEM images.The ratio of CTSN to PTA has no discernible effect on IPSAM diameter because CTSN/PTA ion pairs are amphiphilic, they were probably generated in an aqueous solution by an entropy-driven approach based on thermodynamics' second rule.PTA phenyl groups would hydrophobically interact, interconnecting CTSN chains, and CTSN chains would reorient toward the aqueous phase, which led to stable IPSAM nanoparticles.

Hydrodynamic Diameter and Zeta Potential Measurements
IPSAM (3/7) and IPSAM (2/8) had hydrodynamic diameters of 50.6 nm and 65.2 nm, respectively (Supplementary Fig. S2 A,B) which was determined by the intensity distribution curve from the DLS.The number distribution curve from DLS analysis also showed similar hydrodynamic diameters for the IPSAM (3/7), and IPSAM (2/8) as 46.7 nm and 58.3 nm respectively (Supplementary Fig. S2 C,D).The dimensions did not differ much from those estimated from electron micrographs.IPSAM (3/7) and IPSAM (2/8) had zeta potentials of +21.6 & +15.4 mV, respectively.The phenyl groups of PTA would hydrophobically interact with one another, whereas CTSN chains would orient to the water phase, leading to the formation of IPSAM.Thus, the cationic polymer chains were most likely responsible for the zeta potential of IPSAM, explaining why the value was completely positive.As the amount of CTSN increased, so did the absolute value.As a result, the cationic polymer chains would have a significant effect on the surface potential, resulting in a substantial positive zeta potential.

UCST Determination
Figure 2(a) depicts the temperature-dependent optical density curve of the CTSN/PTA (x/y) solution.CTSN/ PTA (3/7) solution has a tremendous optical density in the 20-46°C temperature range before rapidly falling in the higher temperature range.In an aqueous solution, the amphiphilic CTSN/PTA ion pair would tend to spontaneously assemble, and the self-assembling phenomena can potentially be ascribed to the hydrophobic interaction that exists between the phenyl groups of PTA molecules self-assembly (i.e.IPSAM) was a type of nanosphere on the TEM image (Fig. 1), scattering visible light, so the IPSAM solution was whitish and very turbid, which gave it a high optical density.As the temperature rises, the phenyl group becomes hydrated, and the ion pair loses its amphiphilic function, resulting in the depletion of IPSAM.This could explain why optical density reduces fast at higher temperatures.When the phenyl group is thermally hydrated, it is improbable that an entropy-driven process for self-assembling the ion pair will occur, and the ion pair will stay non-aggregated.The UCST of CTSN/ PTA (3/7) was calculated using the intersection of two tangential lines.The optical density, which is dependent on temperature, of the other CTSN/PTA solutions was somewhat the same in shape and lesser than CTSN/PTA (3/7).Using the same strategy, the UCSTs of CTSN/ PTA (4/6) and CTSN/PTA (2/8) were discovered to be around 27°C and 29°C, respectively.The CTSN/PTA (1/ 9) showed turbidity in temperature lower than 20°C and other ratios of CTSN/PTA (5:5), (6:4), (7:3) (8:2) do not show the milky white suspensions (Supplemental Fig. S1) and no UCST was observed from the UV spectrum analysis (Fig. 2(a)).According to the results provided in Figure 2(a), the UCST rose as the PTA concentration increased.As the PTA content increased, the hydrophobic interaction intensity for the self-assembling of the CTSN/PTA ion pair increased, requiring more thermal energy for the disassembly of IPSAM, resulting in a higher phase transition temperature (i.e.UCST) [19] The temperature-dependent optical density behavior of the CTSN/PTA (3/7) solution (z mM, H 2 O 2 ) is shown in Figure 2  measured to be 30°C for the CTSN/PTA (3/7) solution (1 mM, H 2 O 2 ) and CTSN/PTA (3/7) solution (2.5 mM, H 2 O 2 ) had a similar optical density as the later with a decrease in temperature to 28°C respectively.The CTSN/PTA (3/7) solution (5 mM, H 2 O 2 ) and the CTSN/PTA (3/7) solution (10 mM, H 2 O 2 ) showed clear solution without any UCST values.After H 2 O 2 treatment, the UCST of the CTSN/PTA ion pair was reduced (Fig. 2(b)).PTA is a sulfide chemical, and it was assumed that the oxidizing agent would oxidize it to sulfoxide and/or sulfone.PTA becomes polar and hydrophilic when oxidized, the hydrophobic interaction that exists between the phenyl groups of PTA molecules declines, and the thermal energy requirement to disassemble the IPSAM reduces, giving rise to a decrease in UCST.CTSN/PTA (3/7) solution (10 mM, H 2 O 2 ), on the other hand, was transparent across the temperature range tested and showed no UCST in the temperature window.Because the PTA oxidation degree is proportionate to oxidizing agent concentration, the PTA of CTSN/PTA (3/7) solution (10 mM, H 2 O 2 ) was anticipated to be more polar than CTSN/PTA (3/7) solution (1 mM, H 2 O 2 ) and CTSN/ PTA (3/7) solution (2.5 mM, H 2 O 2 ).As a result, the UCST of the CTSN/PTA (3/7) ion pair in the higher H 2 O 2 concentration solution would be lower than that of the ion pair in the lower H 2 O 2 concentration solution.Therefore, the ion pair of CTSN/PTA (3/7) solution (10 mM, H 2 O 2 ) showed UCST to be lower than the temperature window examined.

Interfacial Tension Measurements Between Air and Water
The air/water interfacial tension distribution of the CTSN/PTA (x/y) solution is depicted in Figure 3(a).CTSN/PTA (3/7) solution showed a quick decrease in interfacial tension from 71.8 to 51.72 dyne/cm when the percentage of PTA content was increased to 35.5% and a steady decline to 47.4 dyne/cm in the enduring concentration range of PTA content.The amount of PTA attached to the chitosan in the formation of selfassembled ion pair CTSN/PTA was determined by UV spectrum (Supplemental Fig. S3 and supplementary Table T1).This is because when the percentage of PTA content increases, interfacial tension is thrown along the path of a saturation curve, as is characteristic of surface-active agent interfacial tension behavior. [13,44]s previously stated, PTA might be connected as an anchor to the CTSN chain via an ionic contact to produce an ion pair.The CTSN/PTA ion pair was amphiphilic and potentially interface-active because the anchor's phenyl group (i.e.PTA) is hydrophobic and the cationic polysaccharide biopolymer derivative (i.The oxidizing agent could oxidize the sulfide molecule (i.e.PTA) to a sulfoxide/sulfone, causing it to become polar, and CTSN/PTA ion couples to lose their amphiphilic ability, resulting in a decrease in interfacial activity.
The interface surface tension was decreased at all x/y ratios across the entire CTSN/PTA concentration range when H 2 O 2 concentration increased.This was achieved because increased H 2 O 2 concentrations allowed for more effective oxidation of PTA, causing the amphiphilic property of the ion pair to decrease more quickly.Because PTA is not a surface-active agent, its interfacial tension was unlikely to alter with H 2 O 2 concentration.Because the CTSN solution included no oxidizable groups, H 2 O 2 concentration did not affect its interfacial tension.Because the CTSN/PTA ion combination was interface-active and oxidizable, the oxidizing agent concentration might have a significant effect on the solution's interfacial tension.

Thermofluorescence Infrared Spectroscopy
The FT-IR spectroscopy of CTSN, PTA, and CTSN/ PTA (x/y) ion pairs is shown in Figure 4(a,b).CTSN's spectra revealed C-N at 1020 cm −1 , C-H at 2940 cm −1 , and O-H at roughly 3350 cm −1 .The phenyl group was discovered in the spectrum of PTA at 750-600 cm −1 , carboxylic C-O at 1197 and 1310 cm −1 , and carboxylic C=O at 1700 cm −1 .The phenyl group of PTA was identified in the spectra of the CTSN/PTA (3/7) ion pair at 750-600 cm −1 , the C-N of CTSN at 1020 cm −1 , the carboxylic C-O at 1197 and 1310 cm −1 , the carboxylate (COO-) of PTA at 1552 and 1630 cm −1 , the C=O of PTA at 1700 cm −1 , and the O-H of CTSN at 3350 cm −1 .The presence of the carboxylate (COO-) in the spectrum of the CTSN/PTA (3/7) at 1310 cm −1 ion pair suggests that PTA was conjugated to CTSN via an ionic connection Figure 4(b).The distinctive peaks of CTSN and PTA, as well as the carboxylate signal, were identified in the spectra of the other CTSN/PTA (x/y) ion pairs at the same position as the signals of the CTSN/PTA (3/7) ion pair.
Figure 4(c) depicts the FT-IR spectra of PTA and the CTSN/PTA (3/7) ion pair before and after H 2 O 2 treatment.The typical peaks of PTA were detected in the spectra of H 2 O 2 -treated IPSAM, coupled with the signals of sulfoxide and sulfone at 1024 and 1296 cm −1 , respectively.This revealed that H 2 O 2 treatment might oxidize PTA sulfide to sulfoxide and sulfone.The previously indicated ion pair's distinctive peaks were seen in the spectra of the H 2 O 2 -untreated CTSN/PTA (3/7) ion pair, but no signal of the oxides was observed.The characteristic peaks of the ion pair were found alongside the signals of sulfoxide and sulfone in the spectrum of the H 2 O 2 -treated CTSN/PTA (3/7) ion pair, indicating that the PTA of the ion pair could easily be oxidized by H 2 O 2 treatment.
The turbidity experiment was done in this work to verify the electrostatic interaction between the CTSN and PTA molecule.The change in the turbidity of the ion pair self-assembled CTSN/PTA (3/7) was evaluated at different salt concentrations: 0, 1, 2, 3, and 5 mM NaCl (Supplemental Fig. S4).The result exhibited that turbidity can be used as a sensitive measure for the electrostatic interaction of the self-assembled ion pair formation.The different ratios of the CTSN/PTA (x/y) ion pair were prepared as mentioned above named as IPSAM (x/y).Among them, IPSAM (1/9), IPSAM (2/8), IPSAM (3/7), and IPSAM (4/6) exhibited turbidity indicating the formation of the insoluble complexes in solution.The presence of turbidity shows higher absorbance which decreases depending on the ionic strength of the salt added. [45]An increase in the ionic strength of a solution in which complexes preexisted due to electrostatic interaction may lead to the solubilization of the complexes, disappearing the turbidity partially or completely. [46,47]In the initial stage, when the NaCl concentration was 0 mM, the turbidity was maximum found in CTSN/PTA (3/7) ratio followed by CTSN/PTA (2/8), CTSN/PTA (4/6) and CTSN/PTA (1/9).As the NaCl concentration was increased gradually the turbidity seemed to disappear thereby reducing the optical density.When the concentration of NaCl was increased from 3 to 5 mM the turbidity of the ion pair disappeared completely giving a clear solution indicating the solubilization of the self assembled ion pair formed.The electrostatic interactions, which have been identified as the primary molecular interactions in the creation of polyelectrostatic complexes, operate at a longer distance than hydrogen bonds and Van der Walls interactions. [48]At increased salt concentrations, the ions protect the charge of CTSN in solution, reducing the impact of electrostatic interactions, and the importance of non-electrostatic forces on complexation increases.In high concentration of Na+/Cl-the turbidity disappears due to the electrostatic screening effect, which reduces the attraction between CTSN and PTA in forming the ion pair.

Temperature and Oxidation Affect Release
The release diagram of Nile red enclosed in CTSN/PTA IPSAM (3/7) at 26°C with H 2 O 2 concentrations of 0, 0.5, 1, and 2.5 mM is shown in Figure 6(a).When the oxidizing agent was removed from the release medium, the maximum release degree (i.e. the release degree in 180 minutes) was 38%.The release medium temperature (26°C) was significantly lower than the UCST of CTSN/PTA IPSAM (3/7) (ca.32°C, Fig. 3), implying that IPSAM (3/7) would be stable at 26°C without considerable release.The release was slightly increased when the H 2 O 2 concentration was 0.5 mM compared to the release without the oxidizing agent, with a maximum release degree of about 12.2%.Higher H 2 O 2 concentrations accelerated the release much more.The maximal release degree at 1 and 2.5 mM H 2 O 2  concentrations was 53.4% and 54.6%, respectively, substantially greater than at 0 and 0.5 mM H 2 O 2 .The PTA sulfide might be converted to sulfoxide and the sulfone, CTSN/PTA ion pair's amphiphilic property would reduce, leading the IPSAM to be weakened and the release hastened.The UCST of ion pair (3/7) treated with 1 mM H 2 O 2 solution was approximately 29°C (Fig. 3), which was near to the release medium temperature (i.e.26°C), implying that a few molecules of IPSAM might be dissolved at 26°C, resulting in a promoted release.The ion pair (3/7) treated with 2.5 mM H 2 O 2 solution, in particular, did exhibit UCST behavior around much closer to the medium temperature so the release was almost similar to the 1 mM H 2 O 2 concentration.After the addition of the H 2 O 2, the solution becomes clear eventually indicating the destructure and promoted release.
The dye release profile in CTSN/PTA IPSAM (3/7) at 37°C, when H 2 O 2 concentrations were 0, 0.5, 1, and 2.5 mM, is shown in Figure 6(b).When no H 2 O 2 was present in the medium, the degree of release grew slowly along the trajectory of a saturation curve, reaching a maximum of 42.4%, a little higher than the 4.2% found at 26°C.The UCST of IPSAM (3/7) was estimated to be at 32°C, which was a little less than the release medium temperature (37°C), implying that IPSAM would be disintegrating much faster, boosting the release.Aside from the thermally induced instability of IPSAM, the increased mobility of the ion pair would also contribute to the greater release.Because the ion pair's mobility increases with temperature, IPSAM would be fluidized following heating, resulting in a promoted release.When H 2 O 2 was present in the release medium, the maximum release degree was nearly 58% at all concentrations tested (54.8 at 0.5 mM, 58% at 1 mM, and 56.7% at 2.5 mM).Following H 2 O 2 treatment, the UCST dropped, implying that IPSAM was likely to be entirely disintegrated at that temperature, resulting in a nearly higher payload release.
The dye release profile in CTSN/PTA IPSAM (3/7) at 46°C, when H 2 O 2 concentrations were 0, 0.5, 1, and 2.5 mM, is shown in Figure 6(c).When no H 2 O 2 was present in the release medium, the release degree grew significantly in a saturation manner, and the maximum release degree was 53.3%, which was significantly greater than the 42.4% reported at 37°C.Because the release medium temperature (i.e.46°C) was higher than IPSAM (3/7)'s UCST (32°C), IPSAM would be disintegrated, resulting in an extensive release.When the oxidizing agent was incorporated into the release medium, the release degree was nearly 72%.When the CTSN/PTA ion pair (3/7) was exposed to H 2 O 2 , the UCST dropped substantially below the release medium temperature (46°C) (Fig. 3).Maximum release of 72.5% could be ascribed to the complete disintegration of IPSAM (3/7).

Conclusions
CTSN/PTA IPSAM nanoparticles were created as temperature and oxidation-responsive nanoparticles where the amino group of CTSN to the carboxyl group of PTA with a molar ratio of 3/7, showed good IPSAM properties.On the TEM image, the IPSAM took the form of sphere-like nano-sized particles.PTA content and PTA oxidation may alter the UCST of the CTSN/PTA ion pair (i.e., PTA treatment with H 2 O 2 ).The ion pair showed increased air/water interface activity with increasing PTA content and decreased with growing PTA oxidation and it rose significantly when the ion pair was oxidized with H 2 O 2 .FT-IR spectroscopy displayed that PTA molecules were connected to the polymeric chain of CTSN via an ionic connection, and it confirmed that the oxidizing agent treatment oxidized the PTA of the ion pair to sulfoxide and sulfone.The cargo (i.e.nile red) loaded in IPSAM (3/7) was restricted when the release medium temperature was 26°C, below the UCST, but triggered when the temperature was 37°C and 46°C over the phase transition temperature.The triggered release could be attributed to the thermally induced disintegration of the IPSAM.The assembly was dissolved when the IPSAM was exposed to an oxidizing situation, presumably because the UCST was reduced below the release medium temperature by oxidation.The CTSN/PTA IPSAM created in this study would be used as a drug carrier that is temperature and oxidation-sensitive.
(b).The CTSN/PTA (3/7) solution was selected for the experiment in which the influence of PTA oxidation on the UCST was studied because the CTSN/PTA (3/7) ion pair exhibited the closest to body temperature CTSN/PTA (3/7) solution (0 mM, H 2 O 2 ) had a very high optical density in the temperature range of 20-38°C and was found to be 32°C.The temperature of the phase transition was

1 H
NMR SpectroscopyFigure5(a) depicts the 1 H NMR spectra of IPSAM (1/9), IPSAM (2/8), IPSAM (3/7) and IPSAM (4/6).The methyl protons of the CTSN molecule were determined to be 2.1 ppm and the methylene proton to be 3.73 and 3.863 ppm.The protons of the phenyl group from PTA were discovered at 7.228-7.429ppm.Chemical interactions involving an ionic connection can result in the chemical shift of proton signals.The protons were downfield shifted from 7.407-7.429to 7.401-7.410ppm, possibly due to the ionic interaction of CTSN and PTA (Fig.5(g)).The peak of CTSN shifted from 3.733-3.863to 3.681-3.753potentially as a result of ionic contact between CTSN and PTA.FT-IR spectroscopy, also demonstrated that PTA interacted with CTSN via an ionic interaction.