Dual-responsive Semi-IPN Hydrogels Based on Poly (N-isopropyl Acrylamide-co-Acrylic acid)/ Glycyrrhizin Cross-linked Chitosan for Controlled Drug Release

doi:10.21203/rs.3.rs-4901998/v1

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Research Article

Dual-responsive Semi-IPN Hydrogels Based on Poly (N-isopropyl Acrylamide-co-Acrylic acid)/ Glycyrrhizin Cross-linked Chitosan for Controlled Drug Release

https://doi.org/10.21203/rs.3.rs-4901998/v1

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The development of novel semi-IPN hydrogels composed of a cross-linked chitosan (CC) network and a thermo-responsive linear copolymer, i.e. poly(N-isopropyl acrylamide-co-acrylic acid) [P(NIPAM-co-AA)], with drug release capability in response to both temperature and pH changes has various potential medical applications. The thermo-responsive free copolymer chains inside the CC network were synthesized via free-radical polymerization to prepare the thermal and pH dual-responsive P(NIPAM-co-AA)/CC hydrogels with a semi-IPN structure. The prepared copolymers and semi-IPN hydrogels were characterized by FTIR, TGA, ¹H and ¹³C NMR apparatus, and the LCST transition was determined using UV/Vis spectroscopy. The stronger C-H stretching of the semi-IPN sample at 2920 cm^− 1 than the CC sample showed that the NIPAM and AA monomers successfully polymerized inside the CC network structure. TGA analysis of the semi-IPN sample exhibited peaks at 249, 379, and 290°C, corresponding to the presence of the thermo-responsive copolymer composition and the chitosan polymer, respectively. The results showed that depending on the temperature below and above the LCST, the semi-IPN hydrogel exhibited a lower (194%) and higher swelling percentage (413%) because the copolymer chain conformation changed form the coil to globule. The drug release results implied that above the LCST, the hydrogen bond between the gallic acid molecules (GA, drug model) and the semi-IPN structure may be broken, causing a change in drug release in the range of 4.5 − 39.1%. The anti-bacterial test and cytotoxicity of the selected semi-IPN sample were carried out. In an MTT assay, the highest cell viability of the semi-IPN sample with 7.5 mg/ml at 37°C was 4% more than the control group. The semi-IPN containing GA exhibited anti-bacterial action against the S aureus bacterial strain significantly. This research describes a method to prepare a smart dual-responsive semi-IPN structure with a potential for transdermal applications.

Drug release

hydrogel swelling

cross-linked chitosan (CC)

P(NIPAM-co-AA)

dual-responsive semi-IPN hydrogel

gallic acid (GA)

Smart or responsive hydrogels are those that possess the capacity to change their physical/ chemical characteristics in response to various environmental factors, including temperature, pH, light, magnetic field, ionic strength, and other relevant variables [1]. This behavior is attributed to the presence of diverse functional groups and constituents within the polymers comprising the hydrogel [2, 3]. The term "thermo-responsive" refers to a class of polymers changing their solubility and, consequently, conformation versus the exposed temperature [4]. When the temperature changes above a certain temperature, i.e. the coil to globule transition temperature (T_cg), the conformational feature of the polymer chain changes from the coil to globule with a compact spherical shape. This conformation change considerably decreases the chain solubility in its media and causes polymer phase separation [5, 6]. This transition occurrence has a wide range of applications in drug delivery [7], tissue engineering [8], and biosensors [9]. Poly(N-isopropyl acrylamide) (PNIPAM) with a lower critical solution temperature (LCST) in the range of 30 − 32°C is a proper thermo-responsive polymer candidate for biomedical applications [10, 11] because this temperature range is close to human body temperature. PNIPAM can be synthesized by free radical polymerization initiated by a redox system in aqueous or non-aqueous solvents [12, 13]. Potassium persulfate (KPS)/ N,N,N'N'-tetramethyl ethylenediamine (TEMED) as an initiator/ accelerator combination has been used for NIPAM polymerization in aqueous media [14]. To improve the efficiency of hydrogels in drug delivery systems, it is more important to prepare hydrogel structures that have a high degree of flexibility in controlling the release mechanism. Due to the complex conditions found in living organisms, the hydrogel system must be responsive to multiple factors at the same time [15, 16]. For this purpose, dual or multi-responsive hydrogels with interpenetrating polymer networks (IPN) and semi-IPN structures would be developed. For instance, the incorporation of thermo-responsive P(NIPAM-co-AA) chains in a pH-sensitive cross-linked hydrogel network would provide thermal and pH dual-responsive semi-IPN hydrogels. This hybrid hydrogel structure permits controlled drug release in response to both temperature and pH changes on/in the body [17, 18]. Additionally, the incorporation of some biopolymers, e.g. chitosan, in these hybrid hydrogels leads to desirable properties such as biodegradability and biocompatibility [19, 20]. Different hydrogel systems based on chitosan with semi-IPN structures with sensitivity to various stimuli such as pH and heat have been worked on recently [21, 22].

In this research work, poly(N-isopropyl acrylamide) (PNIPAM) and its linear copolymers with AA comonomer [P(NIPAM-co-AA)] as thermo-responsive polymers were synthesized. AA can be copolymerized with NIPAM to modify the LCST point of the thermo-responsive polymer exhibiting the pH-responsiveness behavior as PNIPAM shows [23–25]. Thereafter, the thermo-responsive copolymers were synthesized inside the pH-sensitive CC to prepare the final P(NIPAM-co-AA)/CC dual-responsive hydrogels with a semi-IPN structure. Thereafter, the GA as a drug was loaded inside the semi-IPN hydrogels. In addition, the influence of the P(NIPAM-co-AA) proportion on the swelling ratio, and the drug release rate of the resulting semi-IPN hydrogels was evaluated. As a hydrogel, this semi-IPN system have the potential to regulate the release of GA by changing the temperature and P(NIPAM-co-AA) proportion for transdermal applications.

2.1. Materials

All chemical reagents were purchased from Sigma-Aldrich Co. (St. Louis MO, USA) unless otherwise stated. N-iso propyl acrylamide (NIPAM) and acrylic acid (AA) as monomers were vacuum distilled and stored in the refrigerator at 5°C before use. KPS and TEMED as initiator and accelerator were stored at 40–70°C and 15–25°C, respectively. Chitosan (deacetylation level > 75%, M_w=210 kDa), GA, and glycyrrhizin (glycyrrhizic acid ammonium salt from glycyrrhiza root (licorice)) (≥ 70% (HPLC), M_w=839.96) were used to prepare the CC samples. Staphylococcus aureus (S aureus) (ATCC-33591, National Cell Bank of Iran) and Escherichia coli (E Coli) (ATCC-35218, National Cell Bank of Iran) were used for anti-bacterial test.

2.2. Characterization

Fourier Transform Infrared spectroscopy (FTIR) in transmittance mode was used in the wavelength range of 450–4450 cm^− 1 to determine the chemical structure of the prepared PNIPAM, P(NIPAM-co-AA), and semi-IPN hydrogel samples.

The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of the linear thermo-responsive polymers were recorded using a NMR spectrometer (Bruker 400 MHz, Germany) to determine the chemical structure of the thermo-responsive polymer samples. For this purpose, the specimens were dissolved in a deuterium oxide (D₂O) solvent [26].

Thermal gravimetric analysis (TGA, BAHR STA 504, Germany) was used to evaluate the thermal stability of the neat chitosan polymer and CC, P(NIPAM-co-AA), and semi-IPN samples. The experiments were conducted under a nitrogen atmosphere at the temperature range of 100–600°C with a heating rate of 10°C/min.

2.3. CC hydrogel preparation

For preparation of the CC, the carboxylic acid groups of glycyrrhizin (100 mg) react indirectly with the amine groups of the chitosan (50 mg) at pH = 4 in 40 ml deionized water [19]. This reaction has occurred by adding ethanolic solutions (10 ml, 80%) containing the non-toxic activating coupling reagents, such as 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide) (EDC, 46 mg) and N-Hydroxysuccinimide (NHS, 28 mg).

2.4. Thermo-responsive polymer preparation

P(NIPAM) and P(NIPAM-co-AA) were synthesized through free-radical polymerization in an aqueous medium. Initially, a solution of 200 mg (1770 µmol) of NIPAM in 10 ml of DDI was prepared. Then different volumes of AA were added to the solution to prepare P(NIPAM-co-AA) copolymers. The polymerization was carried out by the addition of 25 mg (354 µmol) KPS and 11 µl (71 µmol) TEMED to monomers solution stirred by a magnetic stirring bar at a temperature of 4°C for 24 h [27–30]. In order to synthesize the PNIPAM, the abovementioned procedure was conducted without using any AA comonomer. Table 1 indicates the molar ratio of AA/(AA + NIPAM) to prepare the thermo-responsive copolymers with different compositions. A volume of 20 ml of n-Hexane was added to each polymer solution to remove its impurities. The copolymers are insoluble in the n-Hexane, therefore, it remains in the aqueous phase [31]. Thereafter, n-Hexane separated phase was removed using a separating funnel and the purification process was repeated three times. The aqueous phase was then dried in a vacuum oven at 60°C for 24 h.

Table 1

The recipes for the preparation of thermo-responsive copolymers
Sample code	AA (µmol)	NIPAM (µmol)	Mol ratio* (%)
PNIPAM	0	1770	0
P(NIPAM-co-AA-12.5%)	250	1770	12.5
P(NIPAM-co-AA-25%)	590	1770	25.0
P(NIPAM-co-AA-37.5%)	1060	1770	37.5
P(NIPAM-co-AA-50%)	1770	1770	50.0
* AA/(AA + NIPAM) molar ratio in the initial monomers mixture.

2.5. Semi-IPN hydrogel preparation

To fabricate a semi-IPN structure, the prepared CC (100 mg) hydrogel films were immersed in 10 ml DDI as aqueous media with various AA and NIPAM monomer concenterations (Table 2). According to Table 2, the initiator (KPS) and the accelerator (TEMED) were added to the medium to begin the polymerization reaction inside the CC at 4°C for 24 h. This led to the formation of thermo-responsive copolymers within the pH-sensitive CC structure as a resultant semi-IPN hydrogel. Varrying the total monomers concentration in the reaction media (Table 2) will result in different contents of the free thermo-responsive copolymer chains inside the CC network structure. Finally, the synthesized semi-IPN samples were washed three times using n-Hexane and then immersed in a water bath for 24 h to eliminate the trace of residual monomers and other impurities.

Table 2

The formulation for the preparation of semi-IPN hydrogel samples
Sample code*	AA (µmol)	NIPAM (µmol)	KPS (µmol)	TEMED (µmol)	Mole ratio** (%)
Semi-IPN-A	590	1770	354	71	25
Semi-IPN-B	196	590	118	24	25
Semi-IPN-C	65	196	39	8	25

* Different AA/NIPAM initial concentrations for the semi-IPN preparation.

** AA/(AA + NIPAM) mole ratio for the semi-IPN preparation.

2.6. The LCST transition of the polymers

The visible light transmittance of the copolymer samples was measured using a UV/Vis spectrometer apparatus (Camlab SB-10, USA) at the wavelength of 500 nm [32] to determine the LCST transition of the thermo-responsive polymer samples in an aqueous solution (1 wt.%) at various pH values, i.e. 3.5, 4.5, 5.5, and 6.5. These experiments were repeated three times for each sample at pH 5.5.

2.7. The copolymer conversion in the semi-IPNs

Conversion of the thermo-responsive copolymers as the free polymer content (FPC) is defined as the weight ratio percentage of the copolymer to the semi-IPN film weight according to the following equation:

$$\:FPC\left(\%\right)=\frac{{W}_{Semi-IPN}-{W}_{CC}}{{W}_{Semi-IPN}}\times\:100\%$$

where W_Semi−IPN and W_CC indicate the weight of the dried resultant semi-IPN hydrogel and the weight of the CC sample, respectively.

2.8. Temperature dependence of the swelling

The determination of the swelling ratio of the hydrogel samples was performed at different temperatures, below (15°C), close to (30°C), and above the LCST transition (45°C). As the pH value of the transdermal applications is usually in the range of 4.2–5.6 [33], the pH value was adjusted to 5.5 in order to establish an acidic environment for the swelling measurements. The swelling ratio (SR) was determined based on the following equation:

$$\:SR\:\left(\%\right)=\frac{{W}_{S}-{W}_{I}}{{W}_{I}}\times\:100\%$$

where W_I and W_S represent the weight of dry and the swelled hydrogel film, respectively.

2.9. Drug release

To examine the drug release, the Semi-IPN-A and Semi-IPN-C sample were loaded in a 150 µg/ml GA solution for 24 h. In all tests, the weight ratio of the semi-IPN samples to distilled water remained at a constant amount of 2.75 mg/ml. The GA-loaded semi-IPN samples were then immersed in distilled water at various temperatures, 15, 30, and 45°C. The solutions were then sampled and measured using the UV-Vis spectrophotometer at 265 nm. The following equation was used to calculate the drug release efficiency (DR) [34]:

$$\:DR\:\left(\%\right)=\frac{Total\:released\:GA}{Total\:GA\:in\:loaded}\times\:100\%$$

The swelling and drug release experiments were repeated three times for each sample.

The following kinetic models were evaluated to determine the drug release mechanism [34, 35].

Zero-order model	$\:Q=k\bullet\:t+{Q}_{0}$	(4)
First-order model	$\:Q={Q}_{0}{\bullet\:e}^{k\bullet\:t}\:$	(5)
Higuchi model	$\:Q=k{\bullet\:t}^{0.5}$	(6)
Korsmeyer-Peppas model	$\:Q=k\bullet\:{t}^{n}$	(7)

The variable k represents the kinetic constant, while the variable n indicates the release exponent. Q₀ and Q represent the quantities of the drug released at the initial time (t = 0) and at any time (t), respectively.

2.10. Cell viability and anti-bacterial evaluation

The cytotoxicity of the Semi-IPN-A sample was assessed by the MTT assay over a 24 h incubation period. The L929 fibroblast cell line (National Cell Bank of Iran) was seeded in a 96-well plate with different doses of the semi-IPN-A sample, i.e. 0, 2.5, 5, and 7.5 mg/ml, which was dispersed in a culture medium. After a 24-h incubation period, the culture medium was removed; then cells were rinsed with Phosphate-buffered saline (PBS) and treated using the solution to each well at a concentration of 200 µl [36]. The 96-well plate was then incubated for another 4 h at 37°C in a dark environment. After that 300 µl/well of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed during the experiment, and then the MTT solution was removed. Finally, the absorbance of visible light was measured in each well using a microplate reader (BioTek, Elx800 Absorbance Reader) adjusted to 570 nm [36].

By using the agar-well diffusion method, the anti-bacterial properties of the Semi-IPN-A and GA-loaded Semi-IPN-A were determined. This technique involved preparing the culture bacterial medium in Mueller-Hinton agar (MHA) with 10⁸ CFU/mL concentrations of the S aureus and E Coli bacterium strains. The samples were distributed among the four wells of a 150 mm Petri dish with various concentrations, 1, 5, 10, and 15 mg/ml. The inhibitory zone was evaluated following a 24 h incubation period at 37°C [37].

3.1. The copolymers and semi-IPN hydrogels characteristics

3.1.1. FTIR spectroscopy

The FTIR spectra of the AA, NIPAM, PNIPAM, and P(NIPAM-co-AA) samples are depicted in Fig. 1a and the specific transmittance peaks are summarized in Table 3 [32, 38–43]. The stretching peaks of C = C bond of the AA and NIPAM monomers appeared at 984 and 986 cm^− 1, respectively. While the N-H stretching peak of NIPAM at 3300 cm^− 1 disappeared in the PNIPAM and its copolymers with AA [44–46]. Furthermore, the C-H stretching peak shifts from 2970 to 2922 cm^− 1 after NIPAM homo or copolymerization was observed.

The shift to a lower wave number is owing to the dehydration of the isopropyl groups and chain segments of the resultant polymers [47]. The peak at 2922 cm^− 1 would be attributed to C-H formation bonds within the polymer chain structure. The stretching peaks of carboxyl (C = O) and COO⁻ groups respectively appeared at 1706 and 1426 cm^− 1 belong to AA units in the prepared thermo-responsive copolymers.

Table 3

The remarkable FTIR peaks of AA and NIPAM monomers, and thermo-responsive polymers
Sample	COO⁻ Sym. Stret.	C-H Sym. Stret.	C = O Stret. (Amide I)	N-H Stret.	O-H Stret.	COOH	Vinyl Stret.
AA	1426	-	1636	-	-	1720	984
NIPAM	-	2970	1656	3300	-	-	986
PNIPAM	-	2922	1650	Disappear	3430	-	Disappear
P(NIPAM-co-AA-12.5%)	1426	2922	1650	Disappear	3430	1706	Disappear

Figure 1b indicates the FTIR spectra of the CC, semi-IPN hydrogels, GA-loaded semi-IPN, and GA samples. The notable peaks are summarized in Table 4 [19, 48–50]. The copolymerization of the NIPAM and AA within the CC network led to the resultant thermo-responsive copolymer and production of a semi-IPN hydrogel. The C-H stretching peak appeared at 2920 cm^− 1 in the semi-IPN sample is sharper than that of the CC sample due to the C-H bond of the thermo-responsive copolymer formed in the semi-IPN structure. In addition, the peaks related to N-H stretching bond of the chitosan and C-O-C bond of the chitosan glucosamine units inside the semi-IPN were observed at 3445, and 1025 cm^− 1, respectively [51].

The lowering intensity of the carboxyl group (COOH) peak at 1706 cm^− 1 in the semi-IPN samples in comparison with the thermo-responsive copolymers can be attributed to the ionic interaction between the amine groups (NH₂) of CC and the copolymer' carboxyl group (COOH) [19]. It seems that the presence of the GA as a drug into the Semi-IPN-A structure increases the intensity of the carboxyl groups at 1706 cm^− 1 when compared to the Semi-IPN-A sample without any drug. Also, it appears that the GA carboxylic acid groups would form an ionic reaction (COO⁻) in the acidic medium at 1540 cm^− 1 corresponding to the asymmetrical vibration with the unreacted amine groups present in the semi-IPN samples [35, 52].

Table 4

The FTIR peaks of the GA, the copolymer, GA- unloaded Semi-IPN and GA-loaded Semi-IPN
Sample	COO⁻ Sym.Stret.	C-H Stret.	C = O Stret.(Amide I)	N-H Stret.	O-H Stret.	COOH	C-O-C glucosamine
CC	-	-	1650	3445	3445	1736	1025
Semi-IPN-A	-	2920	1650	3445	3445	Disappear	1025
GA	1426	-	-	-	3496,3282	1708	1025
GA-loaded Semi-IPN-A	-	2920	1650	3445	3445	1706	1025

3.1.2. ¹H and ¹³C NMR

To analyze the NMR spectra, each point of the copolymer segments is given a name, i.e. a, b, c, etc., (Fig. 2). The main ¹H NMR peaks of the thermo-responsive copolymers and their corresponding moieties are indicated (Fig. 2a and Table 5). As shown in Fig. 2a, the proton adjacent to the electronegative atoms (f moiety) corresponding to a chemical shift value in the range of 3.36 − 3.87 ppm is connected to methylene group (-CH₂-) of the AA unit in the copolymers. In fact, increasing the amount of AA in recipes for the copolymerization with NIPAM led to chemical shifting values from 3.36 to 3.87 ppm [11, 32, 53]. According to ¹³C NMR analysis of the thermo-responsive copolymers, the significant chemical shift values of carbon atoms at 21.47, 33.31, 41.94, 42.12, 43.37, and 175.33 ppm are connected to e, a, b, f, d, and c moieties, respectively (Fig. 2b and Table 5) [54]. Also, an apparent peak was observed in all the samples in the range of 50.71 to 51.3 ppm indicating the existence of the C-NR₂ moiety in TEMED and the sulfate one in KPS [55–57].

The proton of the carboxyl group (-COOH) of the AA or the amine group (-C-CO-NH-CHC₂H₆) in the NIPAM units frequently appears as a broad singlet at 4.79 ppm of ¹H NMR due to hydrogen bonding. The addition of D₂O causes the signal to disappear as a result of hydrogen-deuterium exchange [54, 58]. As a consequence, the carboxyl group in AA and the amine group in NIPAM were changed to CO-OD and CO-ND-CHC₂H₆, respectively (Fig. 1S).

Table 5

¹H NMR and ¹³C NMR chemical shift values (ppm) of the thermo-responsive polymers
	Codes	a (-CH₂-)	b (-CH-)	c (-C-)	d (-CH-)	e (-CH₃)	f (-CH-)	D₂O
¹H NMR	P(NIPAM)	1.56	1.99	-	3.87	1.13	-	4.79
	P(NIPAM-co-AA-12.5%)	1.55	1.99	-	3.87	1.11	3.36	4.79
	P(NIPAM-co-AA-25%)	1.55	1.98	-	3.87	1.11	3.34	4.79
	P(NIPAM-co-AA-37.5%)	1.54	1.98	-	3.87	1.10	3.51	4.79
	P(NIPAM-co-AA-50%)	1.56	1.99	-	3.87	1.12	3.87	4.79
¹³C NMR	P(NIPAM)	33.31	41.94	175.33	43.37	21.47	-	-
	P(NIPAM-co-AA-12.5%)	33.30	41.98	175.36	43.47	21.52	43.15	-
	P(NIPAM-co-AA-25%)	31.35	41.83	175.46	43.37	21.52	43.37	-
	P(NIPAM-co-AA-37.5%)	31.36	42.02	175.43	43.38	21.60	43.33	-
	P(NIPAM-co-AA-50%)	31.70	41.84	175.45	43.35	21.48	43.35	-

3.1.3. LCST evaluation

As shown in Fig. 3, PNIPAM is a thermo-responsive polymer exhibiting a lower critical solution temperature (LCST) close to 32°C [59]. LCST of the PNIPAM is primarily determined by intermolecular hydrogen bonding caused by the interaction between water molecules and thermo-responsive polymer with functional units. These units mainly consisted of N-H and C = O bonds comprising polar moieties, and exhibiting hydrogen-bond acceptors. Below the LCST the polymer polar groups are surrounded by water and interactions between the nonpolar-nonpolar groups are much weaker than the hydrogen bonds between polar groups with water molecules. As a result, PNIPAM chains become hydrated and take an expanded conformation. When the temperature exceeds the LCST, the nonpolar interactions within the polymer structure overcome the hydrogen bonding, and the chains tend to a globule or collapsed conformation, which is commonly attributed to the dehydration phenomenon [54, 59, 60].

Figure 4 shows the UV/Vis absorbance spectra of the thermo-responsive polymers at various temperatures and pH values, 3.5, 4.5, 5.5, and 6.5, (These experiments were repeated three times for each sample), when the AA groups are in the undissociated form. The absorbance values were significantly changed with LCST-phase transition. Figure 2S illustrates the absorbance derivative versus temperature curves to determine the LCST transition point [61]. As seen in Fig. 4, the LCST of the PNIPAM was almost insensitive to pH variation [23], while the intensity of the absorbance value was varied. As shown in Fig. 4 and Table 6, further addition of the AA units decreased the cloud point temperature (T_CP) of the LCST-type copolymers at a pH value of 3.5. This phenomenon is consistent with the LCST behavior observed elsewhere [62, 63]. This weakening the hydrophobic character and higher chain solubility with increasing AA content can be attributed to the lowering of intra-chain hydrogen bonds between the two complementary groups in AA and NIPAM units [62, 63]. In contrast, the decrease in UV/Vis intensity value of absorbance and LCST disappearance of the P(NIPAM) and P(NIPAM-co-AA) at higher pH values, i.e. 4.5, 5.5, 6.5, can be connected to the further intra-molecular electrostatic repulsion, which prevents the chains globulization.

The LCST point vanishes when the AA units have sufficient solubility in an aqueous media to oppose the NIPAM units association [62, 64]. In contrast to the thermo-responsive polymers, the prepared semi-IPN hydrogels are insoluble in aqueous media. Therefore, it is not possible to measure their LCST transition points using UV/Vis absorbance spectroscopy.

Table 6

The LCST point of the thermo-responsive polymer (°C) at various pH values
Sample	pH = 3.5	pH = 4.5	pH = 5.5	pH = 6.5
P(NIPAM)	31.5	31.5	31.2	31.8
P(NIPAM-co-AA-12.5%)	30.7	NV	NV	NV
P(NIPAM-co-AA-25%)	30.3	NV	NV	NV
P(NIPAM-co-AA-37.5%)	30.8	NV	NV	NV
P(NIPAM-co-AA-50%)	27.8	NV	NV	NV

3.1.4. TGA evaluation

TGA evaluation was used to distinguish the semi-IPN sample (Semi-IPN-A) structure from chitosan polymer, CC, and the thermo-responsive polymer (P(NIPAM-co-AA-25%)) by thermal degradation (Fig. 5a). Furthermore, Fig. 5b illustrates the TGA derivation diagram (DTGA) to identify the temperature associated with the highest decomposition rate (T_max). The weight loss observed in the temperature range of 20–120°C is related to evaporation of the absorbed water in all the samples. However, the chitosan degradation was started around 250°C. A single DTGA peak with a maximum decomposition rate was observed at 295°C. The residual mass percentage at 600°C was 34% [65–68]. Typically, the degradation begins with the random destruction of β-1,4-glycosidic bonds followed by the degradation of N-acetyl bonds [68]. Although the CC sample exhibited a similar decomposition behavior to the chitosan, its weight loss below 250°C was 4 wt.% higher than that of the chitosan, 9 wt.%. This is due to the glycyrrhizin additional decomposition of the CC sample below 200°C [36, 69]. Therefore, the weight ratio of the cross-linker can be around 4 wt.% based on CC total weight. The CC structure begins to decompose close to 250°C, and the T_max of the sample shifts to a lower temperature of 290°C. The residual mass percentage of the CC was 35.8 wt.% at 600°C.

In addition, the T_max peaks of the PAA (Poly(AA)) and PNIPAM were observed at 231 and 397°C, respectively [70]. Thus, the T_max peaks that appeared at 249 and 379°C can be related to the degradation of the AA and NIPAM units in the P(NIPAM-co-AA) structure, respectively. Unlike the other samples, the P(NIPAM-co-AA) sample exhibits a residual mass of 14.6 wt.%, which can be attributed to the absence of chitosan in its structure. As shown in Fig. 5b, the Semi-IPN-A sample exhibited three peaks of the PAA, chitosan, and PNIPAM at temperatures 249, 290, and 379°C, respectively. The incorporation of the thermo-responsive copolymer in the semi-IPN sample decreased the thermal stability of the CC sample to some extent.

3.2. The copolymer fraction inside the semi-IPN

The weight percentage of free P(NIPAM-co-AA-25%) in the semi-IPN hydrogel films (FPC) is summarized in Table 7. As seen, the copolymer content in the resultant semi-IPN substantially decreased by lowering the monomers concenteration of the polymerization medium.

Table 7

The weight percentage of the free copolymer content (FPC) and CC content ratio in the semi-IPN hydrogels.
	Semi-IPN-A	Semi-IPN-B
NIPAM (µmol)	1770	590	196
AA (µmol)	590	196	65
FPC (%)	27.8	22.5	1.3
CC content ratio (%)	72.2	77.5	98.7

3.3. Swelling behaviour of semi-IPN hydrogels

The swelling ratio (SR) of the semi-IPN hydrogels is illustrated in Fig. 6. The results indicate that the semi-IPN hydrogel samples quickly reach a constant amount of SR. The hydrogel swelling rate at the lowest temperature, 15°C, is slower than that of the higher temperatures, i.e. 30 and 45°C, because of the lower interaction between the water molecules and the hydrogel segments and, subsequently, lower water diffusion into the hydrogels. Figure 6 shows that the hydrogel swelling ratio is directly proportional to the CC content ratio (Table 7). The Semi-IPN-A and Semi-IPN-C samples exhibited the equilibrium SR of 195 and 326% at 15°C, respectively. The weight ratio of CC to semi-IPN samples was 72.2 and 98.7 wt.%. Therefore, the Semi-IPN-C sample with a higher CC content (lower amount of P(NIPAM-co-AA)) exhibits more swelling behavior than the Semi-IPN-A sample at pH = 5.5. It seems that a higher content of the free copolymer chains inside the semi-IPN sample would increase the interaction area and entanglement level of the copolymer chains with the CC network structure to prevent further swelling at the same temperature. These behaviors can be attributed to the further interaction area between the unreacted amine group of the CC segments and the carboxylic acid groups of the AA units of the thermo-responsive copolymer chains, and between the hydroxyl group of CC and N-H and C = O groups of the copolymer segments [71–73].

Moreover, the swelling ratio of each sample after exposure to different temperatures below the LCST, i.e. 15 and 30°C, is nearly identical, whereas the swelling ratio of all the samples increases significantly after exposure to the temperatures above the LCST. According to Fig. 6, the average swelling ratio of the semi-IPN hydrogels at 15 and 30°C is 195 and 194% for Semi-IPN-A, 273 and 264% for Semi-IPN-B, and 326 and 322% for semi-IPN-C. While each semi-IPN sample above the LCST point (T > 30°C) at T = 45°C exhibited a swelling ratio close to 221, 326, and 413% for Semi-IPN-A, Semi-IPN-B, and Semi-IPN-C, respectively.

When the thermo-responsive copolymer passes the LCST transition point, it changes from a random coil conformation to a compact/ globular conformation which causes a phase separation phenomenon. It seems that this issue decreases the interfacial interactions and surface between the CC network and the copolymer and, subsequently, increases the free volume and swelling of the hydrogel. Additionally, at T > 30°C, swelling of the CC hydrogel is also related to the temperature directly [73, 74]. Therefore, the CC swelling above 30°C is affected by both pH and temperature variable. The results indicated taht the swelling ratio of the semi-IPN further increased at the higher temperatures than the lower temperatures.

3.4. Drug release of the semi-IPN hydrogels

The standard curve, light absorbance versus GA solution concentration, was generated to figure out the solute concentration in unknown samples [19]. Figure 7 illustrates the drug release (DR) values versus time for the Semi-IPN-A and Semi-IPN-C at 15, 30, and 45°C. According to Fig. 7, the rate of DR values increases with increasing the temperature. Raising the temperature above the LCST point of the thermo-sensitive copolymer, enhanced the mesh size of the CC network, and provided more space to transfer further drug into/ out of the hydrogel matrix. This behavior can be ascribed to thermal swelling of the CC hydrogel and changing the conformation of the copolymer chains from coil to globule state [73–76]. This issue causes more drug molecules to diffuse out of the semi-IPN hydrogel during drug release.

On the other hand, the GA has several hydrogen bonding sites. The acidic hydrogen atom of the carboxyl group (O-H) of the GA is a hydrogen bond donor, donating its hydrogen to the N-H functional groups of the CC structure, C = O and N-H functional groups of the NIPAM, and the C = O functional groups of the AA repeating units [63, 77, 78]. Also, the carbonyl bond (C = O) of the GA strongly accepts the hydrogen atom of the CC OH groups in the semi-IPN structure [63, 77, 78]. It seems that passing up the LCST point weakens the hydrogen bonds and makes the water molecules stick together [79, 80], and consequently, increases the DR values. This issue seems to decrease the contact surface area between the copolymer/CC segments and GA molecules to enhance the DR values.

Although the Semi-IPN-A involves more copolymer content than the Semi-IPN-C, the DR values at 15°C do not approximately change with increasing the FPC value. However, the degree of swelling decreased. It seems that the ionic or hydrogen bonds created between the GA functional groups and the semi-IPN functional groups at 15°C remain stable, and the GA release is solely influenced by the presence of the unbound GA molecules within the semi-IPN hydrogel network [78].

For T ≥ 30°C, when the temperature passes the LCST transition of the copolymer, the chain conformation is changed from coil to globule, which leads to the further breaking of hydrogen bonds between the copolymer segments and the drug molecules. This phenomenon causes more semi-IPN swelling and drug release.

Above the LCST of the copolymer, increasing the FPC value increased the DR values, while decreased the swelling ratio. The release of the GA appears to be a result of an ionic bond formed between the carboxylate ions ($\:{\text{R}\text{C}\text{O}\text{O}}^{-}$) of the GA and teh ammonium ion ($\:{\text{N}\text{H}}_{3}^{+}$) of the semi-IPN structure at pH = 5.5 [54]. Also, it seems that a more content of the thermo-responsive copolymer in the semi-IPN-A caused more drug loading and, therefore, furthere expected drug release above LCST point when compared with the Semi-IPN-C sample.

3.4. Drug release kinetics and mechanism

Drug release from a polymer network in vitro process can be controlled by drug diffusion and polymer network swelling. Therefore, the drug release kinetics and transport mechanism are key parameters. Table 8 summarizes the mathematical kinetic model fitted based on quantitative data obtained from release rates. The Higuchi model most accurately characterizes the release kinetics of water-soluble and encapsulated compounds [81–83]. As a result, the Higuchi model illustrated a satisfactory level of agreement with the release data (Table 8).

Table 8

Kinetics model parameters based on drug release results for semi-IPN samples
			Kinetic models
			Zero-order			First-order			Higuchi		Korsmeyer-Peppas
Sample	T(°C)			$\:k$	$\:{R}^{2}$		$\:k$	$\:{R}^{2}$	$\:k$	$\:{R}^{2}$	$\:k$	$\:n$	$\:{R}^{2}$
Semi-IPN-A		15		0.024	0.57		0.007	0.40	0.63	0.89	1.34	0.352	0.87
Semi-IPN-A		30		0.107	0.93		0.011	0.57	1.7	0.99	1.75	0.507	0.92
Semi-IPN-A		45		0.299	0.65		0.015	0.40	5.3	0.94	1.81	0.792	0.86
Semi-IPN-B		15		0.052	0.64		0.008	0.34	1.34	0.91	2.19	0.418	0.79
Semi-IPN-B		30		0.113	0.48		0.009	0.24	3.03	0.88	3.60	0.507	0.69
Semi-IPN-B		45		0.386	0.83		0.015	0.44	6.03	0.98	2.19	0.772	0.90
Semi-IPN-C		15		0.029	0.49		0.007	0.41	0.74	0.89	1.36	0.382	0.89
Semi-IPN-C		30		0.083	0.93		0.011	0.61	1.33	0.99	1.58	0.461	0.87
Semi-IPN-C		45		0.205	0.62		0.015	0.41	3.63	0.93	1.32	0.774	0.89

3.6. Cell survival

As shown in Fig. 8, the semi-IPN sample significantly improved cell survival when compared to the control group (at zero concentration) and the CC sample (at 5 mg/ml). Chitosan hydrogels usually exhibit sufficient biocompatibility (more than 80%) for biological research [84]. However, the incorporation of chitosan in a semi-IPN and IPN structure and formation of hybrid hydrogels can lead to enhanced biocompatibility [85, 86]. The cell survival rates for Semi-IPN-A samples at concentrations of 2.5, 5, and 7.5 mg/ml were 101, 102, and 104%, respectively. The semi-IPN samples showed a 10% increase in cell survival compared to the CC one at 5 mg/ml.

3.7. Anti-bacterial activity

Chitosan polymer had Minimum Inhibitory Concentration (MIC) values in the range of 0.03-2 (degree of acetylation = 16–48%, Mw = 3–224 kDa) and 0.8–10 mg/ml (Mw = 28–1106 kDa) against E Coli and S aureus bacteria, respectively [87]. As seen in Fig. 9, the incorporation of 15 mg/ml Semi-IPN-A did not affect strains of both bacterial species. If the polycationic feature of the chitosan is removed or reversed, the anti-bacterial capacity can be decreased or abolished [19]. Protonation of the amino groups and attaching the proton to the C-2 on the chitosan backbones plays a crucial role in electrostatic interactions. A large number of amino groups can boost anti-bacterial activity [73]. However, chitosan chains in the CC structure are cross-linked by amidation reaction in which the amino groups are transformed into the amide bonds. Meanwhile, the GA-loaded Semi-IPN-A exhibited a significant anti-bacterial activity against S aureus compared to E Coli (Fig. 9). The GA-loaded Semi-IPN-A on the S aureus bacterial strain increases the inhibitory zone around the samples. This observation suggests that the In vitro anti-bacterial properties of the semi-IPN are related to the GA loaded inside the semi-IPN structure [88, 89].

This study aims to generate a novel semi-IPN hydrogel structure, i.e. P(NIPAM-co-AA)/CC semi-IPN hydrogel. For possible drug release uses in transdermal applications, the prepared hydrogels regulate the GA release through the thermo-responsive behavior of the free P(NIPAM-co-AA) chain below or above its LCST transition inside the semi-IPN hydrogel. This study investigates the influence of temperature changes on the semi-IPN hydrogel behavior in acidic medium. The results showed that the semi-IPN samples expanded further at 45°C than 15 and 30°C. Above the LCST transition point, the nonpolar-nonpolar interactions of the thermo-responsive copolymer overcome the hydrogen bonding between the copolymers and CC segments in the semi-IPN hydrogel. The thermo-responsive polymer changes from a random coil conformation to a compact/ globular conformation. It seems that this issue decreases the interfacial interactions and surface between the CC network and the thermo-responsive polymer and, subsequently, increases the free volume and swelling of the hydrogel. Additionally, at T > 30°C, swelling of the CC hydrogel is also related to the temperature directly and the swelling ratio of semi-IPN increases more than the lower temperatures. Furtheremore, the drug release results showed that the GA (drug model) would form several hydrogen and ionic bonds with the functional groups of the semi-IPN samples. Temperature enhancement above the LCST point would substantially break the bonds between the GA and semi-IPN segments, leading to increasing the drug release. Increasing the concentration of the thermo-responsive copolymer inside the semi-IPN provided a higher drug loading and release at the temperatures above LCST ytansition point.

The present work demonstrates the potential use of the novel semi-IPN for drug release applications with high biocompatibility and enough anti-bacterial properties, specifically in the area of transdermal drug delivery. Additionally, it highlights the feasibility of developing thermo-responsive chitosan-based semi-IPN hydrogels.However, it is conceivable in future studies to achieve the LCST point of the semi-IPN structure near the human body temperature, i.e, 37°C, and tune the drug release through employing an external energy agent remotely, such as loading magnetic nanoparticles in hydrogels structure to use the magnetic hyperthermia process.

Conflict of interest:

The authors declare no conflict of interest.

Data availability:

Data will be made available on request.

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You are reading this latest preprint version

Dual-responsive Semi-IPN Hydrogels Based on Poly (N-isopropyl Acrylamide-co-Acrylic acid)/ Glycyrrhizin Cross-linked Chitosan for Controlled Drug Release

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Characterization

2.3. CC hydrogel preparation

2.4. Thermo-responsive polymer preparation

2.5. Semi-IPN hydrogel preparation

2.6. The LCST transition of the polymers

2.7. The copolymer conversion in the semi-IPNs

2.8. Temperature dependence of the swelling

2.9. Drug release

2.10. Cell viability and anti-bacterial evaluation

3. Results and discussion

3.1. The copolymers and semi-IPN hydrogels characteristics

3.1.1. FTIR spectroscopy

3.1.2. ¹H and ¹³C NMR

3.1.3. LCST evaluation

3.1.4. TGA evaluation

3.2. The copolymer fraction inside the semi-IPN

3.3. Swelling behaviour of semi-IPN hydrogels

3.4. Drug release of the semi-IPN hydrogels

3.4. Drug release kinetics and mechanism

3.6. Cell survival

3.7. Anti-bacterial activity

4. Conclusion

Declarations

Conflict of interest:

Data availability:

References

Supplementary Files

Status:

Version 1

Zero-order model	\(\:Q=k\bullet\:t+{Q}_{0}\)	(4)
First-order model	\(\:Q={Q}_{0}{\bullet\:e}^{k\bullet\:t}\:\)	(5)
Higuchi model	\(\:Q=k{\bullet\:t}^{0.5}\)	(6)
Korsmeyer-Peppas model	\(\:Q=k\bullet\:{t}^{n}\)	(7)

Dual-responsive Semi-IPN Hydrogels Based on Poly (N-isopropyl Acrylamide-co-Acrylic acid)/ Glycyrrhizin Cross-linked Chitosan for Controlled Drug Release

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Characterization

2.3. CC hydrogel preparation

2.4. Thermo-responsive polymer preparation

2.5. Semi-IPN hydrogel preparation

2.6. The LCST transition of the polymers

2.7. The copolymer conversion in the semi-IPNs

2.8. Temperature dependence of the swelling

2.9. Drug release

2.10. Cell viability and anti-bacterial evaluation

3. Results and discussion

3.1. The copolymers and semi-IPN hydrogels characteristics

3.1.1. FTIR spectroscopy

3.1.2. 1H and 13C NMR

3.1.3. LCST evaluation

3.1.4. TGA evaluation

3.2. The copolymer fraction inside the semi-IPN

3.3. Swelling behaviour of semi-IPN hydrogels

3.4. Drug release of the semi-IPN hydrogels

3.4. Drug release kinetics and mechanism

3.6. Cell survival

3.7. Anti-bacterial activity

4. Conclusion

Declarations

Conflict of interest:

Data availability:

References

Supplementary Files

Status:

Version 1

3.1.2. ¹H and ¹³C NMR