Investigating the rheological behavior of Poloxamer-chitosan thermogel for in situ drug delivery of doxorubicin in breast cancer treatment: designed by response surface method (RSM)

doi:10.21203/rs.3.rs-4369490/v1

This research investigates rheological behavior of Poloxamer-chitosan thermogel system for the release of doxorubicin, which is a chemotherapy agent. In order to design the experiment, the response surface method (RSM) was used to optimize the formula and investigate the mutual effects of the variables on the rheological properties of the system. In this xperimental design, Poloxamer as a thermogel matrix (15-20%) and chitosan biopolymer as an additive (0.1-0.3%) were used and the pH of the test environment was determined in the range of 2.5-7.5. The results showed that the rheological behavior of Poloxamer-chitosan combination has the best fit according to the Hershal-bulkey model with a correlation coefficient of 100%. Also, adding chitosan to Poloxamer decreased the gelation temperature and gelation time. The results showed that the concentration of Poloxamer and chitosan as well as system temperature have a significant effect on the rheological behavior of thermogel. The optimized formula showed favorable rheological properties including high viscosity and appropriate degradation rate. The study showed the sustained release of the drug in the in-vitro environment of the thermogel system during 144h. Therefore, the design of Poloxamer-chitosan thermogel system has the potential to be used as an in-situ drug delivery system for doxorubicin.

Poloxamer

Chitosan

Drug Delivery

Doxorubicin

Thermogel

Rheology

Cancer occurs when normal cells transform into tumor cells in a multistep process that usually begins with a precancerous lesion [1]. Breast cancer is a malignant and uncontrolled growth of epithelial cells that covers the ducts or lobules of the breast tissue, which are caused by a series of molecular mutations at the cellular level [2]. Despite advances in surgery, radiotherapy, and chemotherapy, breast cancer remains the second leading cause of death among women in the United States [3]. The transfer of the drug in the blood circulation system and delivering it to the tissues, the transfer of the drug from the vessel wall to the location of tumors with long distances and the transfer from the interstitial space in a tumor cause only a part of the prescribed dose to reach the tumor cells, which this issue limits the effectiveness of the treatment and causes toxicity for healthy tissues [4]. Many studies have shown that doxorubicin plays a role in the treatment of various types of cancers such as hepatocellular carcinoma [5], breast cancer [6], prostate cancer [7] and lung cancer [8]. Doxorubicin is one of the effective drugs in cancer treatment that prevents the growth and development of cancer cells in the body [9]. Hydrogels are widely used in the treatment of breast cancer and prevent the growth and development of cancer cells in the body [9]. Hydrogels have a significant advantage in localized drug release, where the drug can be released directly into the infected or damaged part of the body. Hydrogels can release a wide variety of drugs and biomolecules [10]. The release profile of this carrier changes depending on the drug loading method in the hydrogel. A drug can be loaded directly before cross-linking the polymers or after cross-linking. In the first method, the drug is simultaneously added to the polymers and crosslinks, and in the second method, a drug is loaded by distribution in the cross-linked hydrogels. Typically, a large amount of drug can be released during the initial swelling in both methods, which can be controlled by changing the binding density that slows down the release. Encapsulation in a hydrogel membrane is another method used to entrap cells and biomolecules. In these systems, the samples are not kept in the hydrogel matrix like being trapped in a volumetric polymer, but are suspended in small water parts surrounded by a membrane [11]. The membrane prevents the release of cells or encapsulated molecules and is permeable to smaller molecules under the layers and their products [12].

Temperature-sensitive hydrogels are three-dimensional networks that consist of hydrophilic polymers and are able to absorb and maintain a significant amount of water or biological fluids [13]. Among temperature-sensitive polymers, we can refer to poly-N-isopropyl-acrylamide, poly-N,N-diethyl-acrylamide, polyethylene oxide and polypropylene oxide [14–17]. Injectable thermogels are a type of hydrogel that can be transferred to a specific part of the body through injection. They are composed of hydrophilic polymers that form a three-dimensional network and are able to absorb and retain large amounts of water. Injectable thermogels have attracted considerable attention in the field of drug delivery due to their ability to create sustained release of drugs and improve therapeutic results [18]. In some of these polymers, the mentioned temperature is around the temperature of the human body. In fact, the value of this temperature depends on the proportion of hydrophilic and hydrophobic groups in the polymer structure and can be changed [17]. There are two different types of temperature-sensitive hydrogels, which undergo gelation by cooling below the highest critical temperature (UCST) or by heating below the gelation temperature (LCST), respectively [19]. Hydrogels with LCST behavior and sol-gel transition at 37°C have attracted more attention in the medical field as carriers of cells, drugs, and biomolecules and have increased the possibility of encapsulation under mild conditions near 37°C [20, 21]. Network injection system is a type of injection system. Networking in these thermogel systems takes place according to temperature and pH changes and enzymes at the injection site [22]. Thermogel polymers are among the degradable injection systems in the target tissue [18]. Some drugs, such as the drugs used to treat cancer, need to work in certain places and not spread throughout the body. A polymer system in which the drug is loaded can be placed near the target tissue and create a local concentration of the drug in a long period of time [23]. In this case, the side effects related to this system disappear in a high concentration of the drug; because the drug is released gradually. These systems are released in the body either in the form of implantable networks or in the form of drug injection systems [24].

Poloxamer 407 (P407), also known as Pluronic® F127 that is a member of the Poloxamer family has been studied more due to its good solubilization capacity, low toxicity, good drug release properties, and compatibility with numerous biomolecules and chemical excipients [25]. Poloxamer 407 is a triblock copolymer with a central hydrophobic chain of polypropylene oxide (PPO) and two identical side hydrophobic chains of polyethylene oxide (PEO). P407 has become one of the most widely used temperature-responsive materials due to its reversible gel property with temperature [26]. The phase transition temperature of P407 mainly depends on its concentration. However, an aqueous solution of P407 with a concentration higher than 20% forms a hydrogel upon warming to ambient temperature. The P407 solution at such a concentration has a lower phase transition temperature of 25°C, so P407 solutions turn into gels above room temperature [16]. Thermogels undergo sol-gel transition under certain temperature conditions and the temperature of the gel shows the temperature of the phenomenon. Therefore, the basic requirement to create a viscous preparation is to determine the sol-gel transition temperature [27]. Chitosan is a hydrolyzed polysaccharide derivative of chitin and a widespread biopolymer in nature, which is non-toxic, biocompatible and biodegradable [28]. This substance has many useful properties for drug delivery systems. Some studies aimed at improving the application of Poloxamer gels in drug delivery systems have focused on chitosan mucoadhesive polymer due to its mechanical and mucoelastic properties. The addition of chitosan has been shown to improve the mechanical properties and mucoadhesiveness of temperature-sensitive gels (Poloxamer) [29–32]. Thermogel-based formulations have been widely developed for sustained release of anticancer drugs [33, 34]. Maintaining the thermogel formulation is essential and challenging to improve clinical performance. This challenge is especially important for the context of cancerous tissues where time-independent formulations can overcome potential issues around acceptable and adaptive [35]. Since the behaviors in the sol-gel phase transition can provide a lot of information, it is very important for the application of Poloxamer-chitosan, so far efforts have been made in this field [36].

Recent studies have highlighted the use of Poloxamer-chitosan in drug delivery such as temperature-sensitive gels [37–39], cobosome [40], ophthalmology [41], release of chemotherapy drugs and gene release [23, 42], breast cancer [43] and vaginal [ 44]. The effective delivery of nanomedicines, especially the delivery of anticancer drugs to tumors, has recently been revealed [45].

The rheological behavior of polymers depends on their molecular structure. The flow behavior of Poloxamer-chitosan can be described by various rheological models (such as power law, Bingham, Hershal-Bulkey and Casson equations) [46]. In the field of drug delivery, rheology plays an important role in the design of drug delivery systems to achieve the optimal release of drugs and improve their therapeutic effects [47]. Hence, the response surface method (RSM) may be considered as an efficient method to deal with the limitations of the conventional method. RSM involves the statistical design of experiments in which all factors are varied simultaneously and can be a useful technique for optimizing, analyzing, and modeling the effects of multiple variables and their responses [48]. Response surface method (RSM) is used to select the variables that are used in the preparation of Poloxamer-chitosan thermogel. In summary, rheology is an important tool in the design and optimization of drug delivery systems. By understanding the rheological behavior of drug delivery systems, it is possible to control the release and distribution of drugs, improve their therapeutic effects, and ensure their stability during storage and transportation.

The relationship between these two polymers, Poloxamer and chitosan, has not been well investigated. The aim of this research is to study the rheology of Poloxamer-chitosan thermogel in different concentration, temperature and pH conditions in order to choose the best conditions and formulations in practical conditions for biomedical uses. Also, the interaction of these variables has been investigated and from the results obtained, it is possible to obtain the best conditions for the in situ drug release system and to design a system that can inject the drug directly into the target tissue without affecting other organs. Also, the effect of adding chitosan to Poloxamer on the degradation and release of the drug at different concentrations has been investigated. The aim of this study was to create a thermogel system with favorable rheological properties for in situ drug delivery of doxorubicin.

Materials

Chitosan (M_W 190,000-310,000 Daltons, 75%) was purchased from Merck, Germany. Poloxamer 407 was purchased from Sigma-Aldrich, Germany. Doxorubicin was obtained from Iran's Nanooxir Company. Citric acid was obtained from Merck with a purity of 99.5%.

Methods

Preparation of Poloxamer-chitosan thermogel systems

In the investigation and study of the Poloxamer-chitosan thermogel system and the effect of the test environment due to the large number of variables and the interaction of each variable on the properties and rheological behavior of the thermogel, it was first used to design the experiment (RSM) with Minitab Pro19 software. Considering that there were three important variables in general and 15 formulas were proposed by the software with the experiment design.

To prepare Poloxamer at different concentrations (15, 17.5 and 20% by weight) a volume of Poloxamer was added to distilled water and placed for 24 h at a temperature of 20 ^oC at a stirring speed of 100 rpm until the solution became completely smooth and transparent. The obtained sample was covered by cellophane so that the moisture of the solution does not escape. In the preparation of chitosan solution at concentrations of 0.1, 0.2 and 0.3% (w/v) of the material with a ratio of 99:1, water and citric acid were added, respectively, and the mixing was continued until chitosan was completely dissolved and a uniform solution was reached after 24 h.

After preparing two solutions at different concentrations, first, according to the formula suggested by Minitab software, each formula was mixed with the specified concentration of Poloxamer-chitosan in a ratio of 90:10 (20 mL) for 2 h. Considering the pH range provided for each formula, it was adjusted to the desired environment using 0.1 M hydrochloric acid and 0.1 M sodium hydroxide solutions, and finally, each of the formulas was kept as a thermogel system in a closed container at a temperature of 4 ^oC until the experiments were performed.

Investigating the rheological properties of systems

Evaluation of the rheological properties of the samples and examination of the time and temperature of the gelation of the samples were done using an oscillating rheometer device model Brookfield DV-II made in the United States. In this study, the effect of concentration on the gelation temperature of thermogel samples was investigated. Poloxamer-chitosan thermogel solutions were placed in the rheometer and covered with a mineral oil to prevent evaporation of the dissolved water during the measurement process. Viscosity changes curves in the temperature range of 4–40 ^oC were considered to measure the gelation temperature with an increase of 1 ^oC for every 1 min.

Gelation temperature of thermogels

In this study, thermogel samples were placed in the material compartment (sample container) of the rheometer device and to further investigate the behavior of the samples, in this test, the sol-gel transition temperature of the formulation was studied for injection into the tissue. To determine the sol-gel transition temperature, a sample container containing thermogel was placed in a water bath at a temperature of 4 ^oC. The sample container was heated to 2°C every minute and continuously stirred at 100 rpm. Its temperature was uniformly heated by a temperature control device in the range of 4–40 ^oC, and the temperature at which the spindle rotation stopped was measured as the gelation temperature.

Investigating the gelation time of the thermogel system

In order to determine the gelation time of thermogels at 37°C and the effect of different concentrations of Poloxamer-chitosan on it, the inverted tube method was used. In this method, when the test tube containing the thermogel polymer sample is tilted, if the sample flows easily to the end of the tube, it is in the liquid phase and if there is no fluid flow in the tube, it is in the gel phase. The flow of the liquid phase inside the tube is a function of the time and amount of tilting of the tube, the amount of solution and the diameter of the test tube. Therefore, it is necessary to fix these variables before conducting the experiment. For this purpose, test tubes with a diameter of 1 cm containing 1 mL of solution were used. The test tubes were placed in a water bath with the ability to adjust the digital temperature, and they were taken out of the bath in 30 s intervals and tilted at an angle of 45^o to the horizon. In this way, the gelation time of thermogels at different concentrations of Poloxamer-chitosan was determined by repeating three times.

Investigating the effect of pH on the gelation of the Poloxamer system

In order to investigate the effect of pH on the gelation of the system, the temperature of the gel environment was set at 4 ^oC and while stirring the solution at this temperature, by adding 0.1 M hydrochloric acid, the conditions of the gel environments were adjusted to pH 2.5, 5 and 7.5 and then gelation temperature of Poloxamer at different pH values was measured by a Brookfield viscometer.

Investigating the degradation of Poloxamer-chitosan system

In order to study the degradation of hydrogel sensitive to the temperature of Poloxamer-chitosan, the obtained solutions were transferred into a 50 mL beaker and placed in an incubator with a temperature of 37°C to form the gel. After the formation of the gel, a specific amount of phosphate buffer solution was added to the thermogels and the samples were placed at a temperature of 37°C. At specified intervals, the buffer solution was removed and the hydrogels were dried and weighed. After each weighing, the buffer solution was added again on the thermogels. The remaining weight percentage of the samples was obtained with the help of the relationship:

Remaining weight (%) = W_t/W₀×100 (1)

In this relation, W₀ is the initial weight of thermogel, and W_t is the weight of thermogel after time t.

The degradation test was repeated three times for each sample.

Examining drug release from the system

In order to check the release in laboratory conditions, the optimal sample obtained from the results at a concentration of 17.5% Poloxamer and 0.2% (w/v) of chitosan was mixed in distilled water with acetic acid (1%) and kept for 24 h. We gave time for it to be completely resolved. Then, the 90:10 ratio of Poloxamer:Chitosan was mixed together and the obtained sample was added separately to the mixture of 1, 2 and 3% by weight of doxorubicin and Poloxamer-chitosan polymer. One milliliter of the obtained solutions was transferred into a dialysis bag with a cut-off of 3500 and placed at a temperature of 37°C to form a gel. Then 2 mL PBS was added to the dialysis bag and 8 mL of phosphate buffer solution was put into the 50 mL test tube and the dialysis bag with the gel was placed inside the tube. After that, the samples were placed in a shaker incubator under the temperature of 37°C, which was shaken horizontally at 80 rpm.

Statistical analysis

The data obtained from rheological behavior were analyzed by Rheocalc software for Brookfield rheometer. Minitab Pro19 software was used to determine model parameters, model efficiency and compare different rheological models, coefficient of explanation (R²) and standard error square which is shown by root mean square error (RMSE).

Effect of Poloxamer concentration on viscosity

There are many parameters that can affect the thermogel behavior of Poloxamer. It can be clearly said that the effect of concentration can have a very important role on the behavior of this polymer and create a stable role on the drug carrier, but on the other hand, other factors such as additives, pH of the environment, temperature, etc., can affect the behavior of viscosity and rheology.

The effect of different concentrations of Poloxamer on viscosity and rheological behavior was studied. The test samples were prepared at three concentrations of 15, 17.5 and 20% (w/v) of Poloxamer, and the test was performed by a Brookfield rheometer at 25 ℃ and by using an SC4-18 spindle. The results of this test showed that Poloxamer composition with increasing concentration can have a great effect on viscosity and the effect of concentration will be a very important parameter in making the desired thermogel. Figure 1 shows that the effect of concentration on viscosity does not show a continuous behavior and acts independently. Figure 2 shows the effect of different concentrations on the shear rate-shear stress curve of Poloxamer, which shows that as the concentration increases, the shear rate decreases and also the shear stress in the thermogel system increases.

As can be seen from the graph, by increasing the amount of Poloxamer from 15 to 20% (w/v), the viscosity of the Poloxamer solution increases, and with the increase in the shear rate, the shear stress also increases, which indicates the behavior of gelation with shear in Poloxamer solution at different percentages. The behavior of gelation by cutting is one of the important characteristics in the formulation of thermogel polymers. Because by injecting the Poloxamer solution with increasing temperature in the range of body temperature, it causes the formation of a gel and causes the slow release of the drug contained in the Poloxamer thermogel carrier and prevents the presence of high dose drugs in the tissue environment.

The purpose of studying the rheological behavior of Poloxamers made in different ratios is to investigate the relationship between the structure of the material and the carrier intended for drug release. Examining the accuracy of different models in describing the rheological behavior of Poloxamer is important. Also, the changes in the rheological parameters of Hershal-Bulkey, Casson and power law models were investigated by increasing Poloxamer content at 25 ^oC, as shown in Table 1. According to the different models of rheological behavior, it is observed that the Hershal-Bulkey model has the highest correlation coefficient fit (close to 100) compared to other models such as Casson, power law, Hershal, etc. and with the change of different Poloxamer concentrations, this correlation coefficient still follows the Hershal-Bulkey model. In this study, all the prepared thermogels showed a pseudo-plastic flow behavior, which shows an immediate flow after applying stress, and in this way, the rheological parameters specific to this model were calculated with using Eqs. 2, 3 and 4:

τ = τ° +kDⁿ (1)

τ = k₁Dⁿ (2)

√ τ =√ τ° +√(ηD) (3)

In these equations: τ represents shear stress, γ˚ is shear rate (s^− 1), k is the fluid consistency coefficient (dyne/cm) an indication of shear stress, k₁ represents the plastic viscosity (dyne sⁿ/cm²)^0.5, η is the index flow and σ refer to the limit stress (dyne/cm²). Appropriate rheological models were applied to describe the behavior of Poloxamer solution at different concentrations, and in describing the flow behavior of the solution, all three rheological models of

Hershal-Bulkey, power law and Casson were able to analyze the thermogel well in the studied conditions. It should be noted that for all three rheological models, the correlation coefficient is R². All the samples within the range of the studied shear rates showed a good fit with this rheological model and the correlation coefficient (R² > 0.94) was obtained in the studied shear rates in all the samples. The results also showed that among the two rheological models of Casson and power law, the data obtained from the tests showed that the rheological model of Casson has a better fit. Therefore, the rheological models of Hershal-bulkey, Casson and power law are, respectively, the most appropriate equations to describe the rheological behavior of the studied treatments.

Table 1: Flow behavior of Poloxamer formulation at 25 °C measured at concentrations of 15% (A), 17.5% (B) and 20% (C) by a rheometer (Brookfield DV-II) using the rheological models of Hershal-bulkey, Casson and power law

Effect of chitosan on the gelation time of Poloxamer

One of the characteristics evaluated for these materials is the gelation time. This time is necessary for the gel formation of the substance in the sol phase at the physiological temperature of 37°C, and during this time, all the samples (Table 2) should be allowed to be injected in the specified place in the liquid form (sol phase) and then turn into a gel. Meanwhile, the material should not remove from the place of use. Vial tilting method was used to visualize gelation with temperature change by measuring the flowability of the prepared hydrogels. As seen in Fig. 3, increasing the concentration of Poloxamer and chitosan improves the gelation time. In Table 2, Poloxamer control samples of P15, P17.5, P20 with Poloxamer concentrations of 15, 17.5, and 20% (w/v) and chitosan concentrations of 0, 0.2, 0.1, and 0.3%, respectively, are presented. It is expected that increasing the concentration of Poloxamer decreases the gel formation time because there are more triple chains that help to form and pack micelles. According to Fig. 3, the addition of chitosan to the Poloxamer composition has the opposite effect and increases the gelation time, because the chitosan chains are entangled, which reduces the hydrophobic interactions of the Poloxamer molecules.

Table 2

Suggested samples to check the gelation time of thermogel
Number	Sample	%Poloxamer	%Chitosan
1	P15 C0	15	0
2	P15 C0.1	15	0.1
3	P15 C0.3	15	0.2
4	P15 C0.3	15	0.3
5	P17.5 C0	17.5	0
6	P17.5 C0.1	17.5	0.1
7	P17.5 C0.2	17.5	0.2
8	P17.5 C0.3	17.5	0.3
9	P20 C0	20	0
10	P20 C0.1	20	0.1
11	P20 C0.2	20	0.2
12	P20 C0.3	20	0.3

Modeling and test design using the response surface methodology (RSM)

Due to the fact that increasing the number of evaluated factors requires a lot of time and money, the test design method was used, which of course makes it easier to understand the overall results of the tests. Three factors of Poloxamer concentration, chitosan concentration and pH of the environment were taken into consideration and the design procedure was done with the help of Minitan 19 software and the response method. The results obtained are shown in Table 3, and also, selected examples of experimental design for three dependent variables according to the central composite design can be seen in Table 4. In this test design, the results of 3 response surfaces with 3 central points and 15 suggested samples are presented.

Table 3

Factors and surfaces measured by response surface method
			Encoded surfaces
X3: pH	X2: Chitosan(%)	X1:Poloxamer(%)	Encoded surfaces
7.50	0.20	20.00	+α (1/682)
7.50	0.30	17.50	1
5.00	0.20	17.50	0
2.50	0.10	17.50	-1
5.00	0.10	15.00	-α (-1/682)

Table 4

Selected samples designed for three dependent variables according to the central composite design
X3: pH	X2: Chitosan (%)	X1: Poloxamer (%)	Samples
7.500	0.200	20.000	1
5.000	0.200	17.500	2
7.500	0.100	17.500	3
5.000	0.300	20.000	4
5.000	0.200	17.500	5
2.500	0.200	20.000	6
7.500	0.200	15.000	7
7.500	0.300	17.500	8
5.000	0.100	15.000	9
5.000	0.100	20.000	10
2.500	0.200	15.000	11
5.000	0.300	15.000	12
2.500	0.300	17.500	13
5/000	0/200	17/500	14
2.500	0.100	17.500	15

Effect of addition of chitosan to Poloxamer on gelation temperature

Poloxamer is the main component of the material affecting the thermogel behavior and this behavior can have a very important effect on the properties of the carrier. The effect of Poloxamer concentration is one of the effective factors that can affect other conditions of Poloxamer such as degradation rate, thermogel swelling, drug release rate, gelation temperature, etc. Rheology is the study of the relationship between the mechanical phenomena produced by a material under external force and the viscosity of the polymer during flow. The role and application of Poloxamer as a drug delivery system in the treatment of diseases has been described. In the study, in situ Poloxamer gels, injection capacity and gelation time are among the key factors. Gelation time is defined as the time required to change from liquid to gel during injection at a constant temperature (37.5 °Ϲ).

It is usually delivered using a 20-gauge needle to stabilize the volume and inject the gel at the target tissue site. Below the injection or gelation threshold, the solution can easily pass through the syringe, while above the injection or gelation threshold it is difficult for the solution to pass through the syringe and therefore to administer it.

The temperature of human body tissue is 37.5°C, so if the gelation temperature is below 25°C, the gel may form at room temperature, leading to difficulties in manufacturing, handling and administration. If the gelation temperature is above 37 °Ϲ, a liquid dosage form is still present at body tissue temperature, resulting in early tissue release of the formulation. In studying the behavior of the proposed samples, the design of the experiment was used according to its manufacturing method.

Table 5

Gelation temperature and viscosity at different ratios of Poloxamer to chitosan at 37°C
Sample	% Poloxamer	% Chitosan	T.G	Viscosity(cp)
1	20.000	0.200	16.530	7686
2	17.500	0.200	21.680	7400
3	17.500	0.100	23.000	7700
4	20.000	0.300	24/200	6797
5	17.500	0.200	27.440	7595
6	20.000	0.200	20.380	7462
7	15.000	0.200	38.000	5341
8	17.500	0.300	32.700	7700
9	15.000	0.100	36.000	2520
10	20.000	0.100	18.930	7644
11	15.000	0.200	32.600	7070
12	15.000	0.300	39.000	5105
13	17.500	0.300	36.700	4102
14	17.500	0.200	27.440	7650
15	17.500	0.100	23.030	7154

Prediction of experimental parameters by RSM

A graph of actual values versus model predicted values is given. This graph in Table 6 shows the predicted response values versus actual values to help identify values or groups of values that are not predicted by the model.

Table 6

Actual and predicted values of experimental parameters of temperature glass (̊C) and torque
Torque		Temperature glass (̊C)
Predicted	Measured	Predicted	Measured
126.275	100.000	15.610	16.530	1
93.333	100.000	25.520	21.680	2
82.288	100.000	26.247	22.700	3
83.287	100.000	26.248	24.200	4
93.333	100.000	25.520	27.440	5
99.000	100.000	21.880	20.380	6
77.000	76.000	37.000	38.500	7
90.438	100.000	31.572	32.700	8
80.412	63.700	33.953	36.000	9
91.437	100.000	16.303	18.930	10
73.725	100.000	33.520	32.600	11
19.762	11.200	41.628	39.000	12
32.612	14.900	36.453	40.000	13
93.333	80.000	25.520	27.440	14
109.562	100.000	24.158	23.030	15

According to the proposed design of the experiment and the results obtained from the gelation point and torque of different formulations, it has been shown that the temperatures obtained from the simulation model are relatively close to the temperatures measured in the laboratory and this close numerical agreement of the predicted gelation temperature with the temperatures recorded by the device can show the accuracy of the simulation, but there is a greater numerical difference in the data obtained from the torque, and its estimation and overestimation is unacceptable.

Chitosan is a natural copolymer that has good biocompatibility, antibacterial activity and biodegradability. Chitosan improves the mucoadhesive properties and gel strength of Poloxamer because it has positively charged amine residues that facilitate interaction with the gel solution and provide good mechanical and mucoadhesive properties for the drug, and has antifungal properties, as well as the ability to enhance penetration and healing. Chitosan temperature-sensitive hydrogels are safe and have good pharmaceutical bioavailability. Gel formation is the result of non-covalent interactions such as electrostatic, hydrophobic or hydrogen bonding. The results showed that chitosan-based thermogels have a great advantage in anticancer drug release. The chitosan temperature-sensitive gel microspheres containing the drug prevented the sudden release of the drug at the initial stage and facilitated the slow release of the drug later.

In the development of in situ application of drugs, we must first consider the local physiological conditions, in addition to the capacity to retain and release the drugs. Therefore, P407 is used not only in medicines but also in various additives for safety. To adjust the sol-gel transition temperature, P407 and chitosan were used. According to Fig. 4, the

three-dimensional response surface and the two-dimensional contour versus the concentration of PLX and chitosan show that the higher the proportion of non-ionic polyoxyethylene surfactant parts, the higher the lipophilic-hydrophilic balance value. Therefore, P407 has fat-soluble and water-soluble properties, and it can be dissolved in the aqueous phase and uniformly distributed in the carrier material in the form of molecules, thereby creating a uniform pore structure on the surface of the carrier material. In addition, the addition of chitosan can improve the drug release from the gel in order to improve the effective concentration of the drug in cancer tissues. According to the data, the mixture of P407 (17% by weight) and chitosan (0.2% by weight) is the best choice to obtain the appropriate gelation temperature.

Statistical techniques have been used in the modeling of the surface response procedure using the RSM method. To analyze the results of the desired answers, several effective variables are examined. One of the important actions in RSM design is to find the real dependencies between Y and independent variables. For this purpose, low-order polynomials are generally used. If there is curvature in the system, second- or third-order polynomials are used. To estimate these parameters, the method of least squares in polynomials is investigated.

In the explanation of these models, it can be mentioned that the model used in the response surface method is generally a complete quadratic model equation in the form of the following relationship:

\(Y=\mathop \beta \nolimits_{0} +\sum\limits_{{i=1}}^{k} {\mathop \beta \nolimits_{i} } \mathop x\nolimits_{i} +\sum\limits_{{i=1}}^{k} {\mathop \beta \nolimits_{{ii}} } \mathop {\mathop x\nolimits_{i} }\nolimits^{2} +\sum\limits_{{i=j=1}}^{k} {\mathop \beta \nolimits_{{ij}} } \mathop x\nolimits_{i} \mathop x\nolimits_{j}\)

In this relationship, β₀, β_i, β_ii, and β_ij are constant coefficient, linear regression coefficient, quadratic regression coefficient, and regression coefficient of interaction between two variables, respectively, and the expansion of the above equation is written in simple language as follows:

By examining the main and reciprocal factors on the variables, the response of the surface procedure model was fitted to the data obtained with the Minitab software. The design with three independent variables will be in the form of the following general equation: in this equation:

X1 = Poloxamer concentration, X2 = chitosan concentration, and X3 = pH of the environment.

Y= (\(\mathop \beta \nolimits_{0}\)) +(\(\mathop \beta \nolimits_{1}\)) X1+ (\(\mathop \beta \nolimits_{2}\)) X₂+ (\(\mathop \beta \nolimits_{3}\))X₃ + (\(\mathop \beta \nolimits_{{11}}\)) X₁X₁+ (\(\mathop \beta \nolimits_{{22}}\)) X₂X₂+ (\(\mathop \beta \nolimits_{{33}}\)) X₃X₃ + (\(\mathop \beta \nolimits_{{12}}\)) X₁X₂ + (\(\mathop \beta \nolimits_{{13}}\))X₁X₃ + (\(\mathop \beta \nolimits_{{23}}\))X₂X₃

For the TG (temperature glass (^oC) parameter, the formula will be:

TG = 93/591- 5/748 X₁ – 93/175 X₂ + 6/694 X₃ + 0/113 X₁X₁ + 330/875 X₂X₂ + 0/125 X₃X₃ + 2/270 X₁X₂ – 0/390 X₁X₃ − 6/970 X₂X₃

For the torque parameter, the formula will be:

Torque = 47/75 + 18/29 X₁ – 718/58 X₂ – 39/30 X₃ – 0/75 X₁X₁ – 1994/17 X₂X₂ + 0/85 X₃X₃ + 52/20 X₁X₂ + 0/96 X₁X₃ + 85/10 X₂X₃

Table 7

Regression coefficients for the estimated model temperature glass (̊C)
P	T	Standard deviation coefficients	Coefficients	Symbol
0/432	0/855 ^ns	109/493	93/591	constant (\(\mathop \beta \nolimits_{0}\))
0/646	-0/489 ^ns	11/755	-5/748	X₁ (\(\mathop \beta \nolimits_{1}\))
0/600	-0/560 ^ns	166/437	-93/175	X₂ (\(\mathop \beta \nolimits_{2}\))
0/361	1/005 ^ns	6/657	6/694	(\(\mathop \beta \nolimits_{3}\)) X₃
0/746	0/342 ^ns	0/329	0/113	(\(\mathop \beta \nolimits_{{11}}\)) X₁× X₁
0/169	1/608 ^ns	205/831	330/875	(\(\mathop \beta \nolimits_{{22}}\)) X₂ × X₂
0/721	0/378 ^ns	0/329	0/125	(\(\mathop \beta \nolimits_{{33}}\)) X₃ × X₃
0/786	0/287 ^ns	7/910	2/270	(\(\mathop \beta \nolimits_{{12}}\)) X₁ × X₂
0/273	-1/233 ^ns	0/316	-0/390	(\(\mathop \beta \nolimits_{{13}}\)) X₁ × X₃
0/419	-0/881 ^ns	7/910	-6/970	(\(\mathop \beta \nolimits_{{23}}\)) X₂ × X₃
*Significant at p ≥ 0.05
**Significant at p ≥ 0.01
***Significant at p ≥ 0.001
^ns No significant correlation

Table 8

Variance analysis of temperature glass model (̊C)
T	F	Corrected mean square	Corrected sum of squares	Sequential sum of squares	Degree of freedom	Ref.
0.037	5.57*	87.099	783.890	783.890	9	regression
0.654	0.58 ^ns	9.040	27.121	704.615	3	linear
0.646	0.24 ^ns	3.740	3.740	545.490	1	X1
0.600	0.31 ^ns	4.903	4.903	155.232	1	X2
0.361	1.01 ^ns	15.815	15.815	3.892	1	X3
0.504	0.90 ^ns	14.026	42.077	42.077	3	Second order
0.746	0.12 ^ns	1.829	1.829	0.633	1	X1×X1
0.169	2.58 ^ns	40.423	40.423	39.204	1	X2×X2
0.721	0.14 ^ns	2.239	2.239	2.239	1	X3×X3
0.548	0.79 ^ns	12.400	37.199	37.199	3	multiplication
0.786	0.08 ^ns	1.288	1.288	1.288	1	X1×X2
0.273	1.52 ^ns	23.766	23.766	23.766	1	X1×X3
0.419	0.78 ^ns	12.145	12.145	12.145	1	X2×X3
		15.643	78.215	78.215	5	residual error
0.393	1.69 ^ns	18.699	56.097	56.097	3	Mismatch error
		11.059	22.118	22.118	2	Pure error
				862.106	14	Total error
R² = 90/93%, R² (pred) = 0/00%, R² (adj) = 74/60%
*Significant at p ≥ 0.05
**Significant at p ≥ 0.01
***Significant at p ≥ 0.001
^ns No significant correlation

According to the results obtained from Table 8, in the mentioned model, about 74% of the changes in the response variable can be justified with predictive results. The results of R² and adjusted R-squared (R²adj) calculations are related to the optimal fit of the data.

Investigating the effect of pH on the gelation properties of Poloxamer PLX

Among the other factors mentioned earlier about the effect of thermogel, we can mention the application environment or the pH of the thermogel environment. Considering that the intended drug carrier is used in the environment of cancer cells and according to previous studies, cancer cells decrease the pH of the environment and the presence of Poloxamer thermogel in this environment can affect its release. According to the results obtained from Fig. 5, the three-dimensional and two-dimensional diagrams of the effect of pH of the environment and its reaction on Poloxamer concentration, it seems that the increase of Poloxamer from 15–20% decreases the gelation temperature from 39 ℃ to 16℃ in the environment with pH 7.5, but with the change of the environment towards acidity, the temperature becomes 34 − 20 ℃ in the environment with pH 2.5, and this data shows that the pH factor is an independent factor and necessarily different concentrations do not lead to a decrease in gelation with the acidification of the environment, and as it is clear from the three-dimensional images, increasing the concentration in the range of 20% in an environment with pH 2.5 increases the gelation temperature, and on the other hand, at the concentration of 15% and in a neutral environment at pH 7.5, it reduces the gelation temperature.

Investigating the effect of pH on gelation properties by adding chitosan

According to Fig. 6 of the three-dimensional response surface diagram and two-dimensional contour, the role of chitosan as an improver of the properties of Poloxamer is of great interest and also this polymer can affect the thermogel behavior of Poloxamer, on the other hand, other parameters such as pH can also affect the properties On the other hand, other parameters such as pH can also affect the properties of this compound and change the structure of the drug carrier.

In examining the three-dimensional form of this effect, it was observed that by increasing very small concentrations of chitosan, it can increase the gelation temperature, and on the other hand, by changing the environmental conditions from neutral to acidic at a concentration of 0.1% chitosan, the gelation temperature decreases. In addition, in the concentration of 0.3% chitosan, these environmental conditions increase the gelation point in the response level. According to the previous study of the effect of pH on the thermogel behavior of Poloxamer, it was also observed that the results of different concentrations follow a non-dependent spectrum, and in the investigation of the effect of adding chitosan to Poloxamer, it was also observed that this

non-dependent effect is also evident.

In the review of the data obtained from the results of the experimental design by RSM according to Table 8, in the conditions of the lowest and the highest amount of Poloxamer- chitosan in different environments, it showed that in the combination of 20% Poloxamer, 0.2% chitosan and pH 7.5, it has the lowest gelation temperature in the range of 16.5 ^oC, and in combination with 15% Poloxamer, 0.3% chitosan and pH 2.5, it has the highest temperature of 39 ^oC. In the analysis of the data from the results of torque, it can be stated that in combination with 17.5% Poloxamer, 0.2% chitosan and pH 5, torque has maximum state and also in combination with 15% Poloxamer, 0.3% chitosan and pH 2.5 it has shown the lowest torque limit.

Table 8

The minimum and maximum range of gelation temperature and torque
		Poloxamer (%)	Chitosan (%)	pH
Temperature glass (̊C)	Max = 39	15	0.3	2.5
Temperature glass (̊C)	Min = 16.5	20	0.2	7.5
Torque	Max = 100	17.5	0.2	5.0
Torque	Min = 21.84	15	0.3	2.5

Characterization

Analysis and identification of thermogel using FTIR

Infrared Fourier transform (FTIR) spectroscopy is one of the methods to identify the molecular structure and functional groups of compounds. In this study, when the radiation frequency and the molecular bond frequencies are in the same area, the energy of the radiation beam occurs in the absorption bond area and causes a change in the intensity of the vibrations. The spectrum obtained from the analysis shows the intensity of the passage in terms of wavelength. Each peak represents the amount of radiation energy absorption corresponding to the wavelength of that peak. Figure 7 shows the infrared spectrum of the produced Poloxamer-chitosan composite. In order to analyze the peaks in the obtained spectrum, the functional groups in the structural formula of the aforementioned Poloxamer and chitosan samples and the location of the infrared absorption frequencies of each group have been collected according to spectroscopic books. By comparing the frequency of the peaks in the graph related to the produced Poloxamer-chitosan and the values related to the frequency of each functional group, the following results are obtained:

In Fig. 7 spectrum A, the frequencies related to Poloxamer composition can be obtained. In this spectrum, it has the main absorption peaks in the 2870 cm^− 1 region related to the stretching bond (C-H) in the aliphatic structure, and at 1350 cm^− 1 associated with the bending bond (O-H) as well as in the 1108 cm^− 1 region related to the bond (C-O). The spectrum related to Poloxamer has an index peak in the area of 1251 cm^− 1 related to C-O stretching vibrations. In addition, the peak created in the region of 3518 cm^− 1 belongs to the vibrations of hydroxyl nodes (H-O). The peak in the 1108 cm^− 1 region is related to strong bonds (C = H) and also the strong peak in the 2870 cm^− 1 region corresponds to (C-H) bonds. In Fig. 7 related to spectrum B, related to chitosan composition, the stretching vibration of the hydroxyl group (O-H) in a bifunctional carboxylic acid has a broad absorption peak with medium intensity in the range of 2400 to 3400 cm^− 1, which is completely obvious. The bending vibration of this group also has an average absorption peak in the range of 1395 to 1440 cm^− 1, which is also present in the obtained spectrum in the region of 11419 cm^− 1. The symmetric and asymmetric stretching vibrations of the N-H functional group in a first-type amide, like the N-H group in acrylamide, have two broad peaks with relatively strong intensities at 3180 and 13350 cm^− 1, respectively. Also, the stretching vibration of this group in an amide is of the second type. It should be noted that all 3 mentioned peaks interfere with the broad peak resulting from the O-H group.

By studying the spectra obtained in Fig. 8, it can be seen that the bending vibration of the N-H group in both the first and second type amides has a relatively strong peak in the range of 1550 to 1680 cm^− 1, which is in the mentioned spectrum with the absorption peak of the C = O group as separate peaks are in the same range. The stretching vibration of the carbonyl group (C = O) in a bifunctional carboxylic acid and an amide have strong absorption peaks in the ranges of 1700 to 1730 cm^− 1, respectively. These peaks in the obtained absorption spectrum are in the region of 1714 cm⁻¹.

Degradability of Poloxamer-chitosan thermogel

Degradation rate in laboratory conditions of Poloxamer-chitosan thermogel was used to investigate its effect on drug release. The results in Fig. 9 show that Poloxamer-chitosan thermogel has lost 51% of its initial weight in phosphate buffered saline (PBS = 7.4) after 5 days and 76% of its original weight after 10 days. In addition, the rate of degradation in phosphate buffered saline (PBS = 5.5) was significantly degraded under acidic conditions and after 10 days, 100% of the thermogel was degraded. According to the obtained results, it can be stated that this thermogel can be used for the long-term release of DOX during the treatment. Hydrogel degradation should be adjusted to completely remove the thermogel after drug release. Also, the rheology behavior can help in designing drug release systems to withstand different biological environments and maintain their stability during storage and transportation.

Investigating the effect of adding chitosan to Poloxamer in the release of doxorubicin

The tests performed on the role of Poloxamer 17.5% (w/v) on the release behavior of doxorubicin drug showed that samples of Poloxamer combination with 1% by weight of doxorubicin can be used as a carrier in many release systems. According to the data obtained in this carrier, it can be concluded that the high release rate of this drug in environments with pH 5.5 and 7.4 caused toxicity in the target tissue and also caused the destruction of healthy tissues next to the tumor. In further investigation of the release conditions of the doxorubicin drug directly in the Poloxamer-chitosan thermogel carrier, the drug was added freely in the thermogel structure and considering that the best thermogel conditions close to body temperature were obtained in the conditions of 17.5% Poloxamer and 0.2% chitosan, so these conditions were used as the basis for the continuation of the work. To investigate these conditions, doxorubicin was added to the Poloxamer-chitosan combination in amounts of 1, 2, and 3% by weight separately, and the release conditions of the drug were carried out in two PBS mediums with pH 7.4 and pH 5.5 in a shaker incubator.

From the release of DOX from Poloxamer-chitosan thermogel with 1% drug in PBS with pH 7.4, explosive release of DOX drug was observed during 9 h (Fig. 10). Sustained release of DOX was then observed with 49% release of the drug in 24 h. In this research, the initial rapid release of DOX from the Poloxamer-chitosan thermogel is expected because the thermogel with large pores and high water content provides a path for the rapid release of compounds with low molecular weight and, as a result, the rapid release of DOX. From the results obtained from the two PLX-CS-DOX and PLX-DOX carriers, it can be pointed out that the presence of chitosan can change the release rate from 48 to 140 h in PBS environment with pH 5.5. This is in the case that in PBS medium with pH 7.4, the release rate in 48 h in the PLX-DOX carrier was about 96%, and in comparison with the same conditions, the release rate in the PLX-CS-DOX carrier was about 55% of the total drug. These results can clearly show that the role of adding chitosan to Poloxamer in drug release and its use improve other mechanical and rheological properties of injectable drug carriers.

Effect of adding different concentrations of DOX on its release

In further studies of DOX release from Poloxamer-chitosan thermogel in PBS at pH 5.5, more explosive release of DOX was observed than from Poloxamer-chitosan thermogel in PBS at pH 7.4, and the results showed that this drug carrier in pH 5.5 environment within 120 h can release all the medicine in it.

In the study, drug release was investigated at different concentrations of doxorubicin. The results showed that Poloxamer-chitosan thermogel is combined with 2% of doxorubicin due to the change in the nature of the molecular bond of the structure and the distance between the chains of the thermogel, which causes a decrease in the connection between the bonds and more penetration of water molecules into the structure and the release of the drug to the outside. The reason for the speed of drug release in this thermogel composition will change. According to Fig. 11, it is possible to see the release rate of doxorubicin in different environments. According to the results, it can be said that in the environment with pH 5.5, due to acidification, the structure breaks down faster and releases more, and in the first 24 h, 70% of the drug is released, and the comparison of the same compound in the environment with pH 7.4, only nearly 42% of the drug was released for 24 h, and during 144 h, all the drugs were completely released in two environments.

Considering that the drug dosage factor can be very important in the prescription of drugs for treatment, and also due to long-term use of the drug, it can cause drug resistance in the patient and change the behavior of the drug effect on the patient, so the disease requires an increase in the drug dose or changing the type of medicine. For this reason, the concentration of the drug in the Poloxamer-chitosan carrier was investigated. As a result of this study shown in Fig. 12, increasing the concentration of the drug doxorubicin in the range of 3% caused a lot of explosive release behavior in the environment of the buffer solution and the duration of 24 h in the environment with pH 5.5 caused the release of more than 82% of the drug in the medium that was compared to the release rate of the drug at the same time at pH 7.4, in which 73% of the drug was able to release doxorubicin. From the obtained results, it can be concluded that the environment of buffer solution can increase the drug release and by increasing the drug concentration, it can play an important role in the structure of the thermogel and its bonding, as well as the loaded drug could be released in a short period of time.

Placing a bioactive compound in a thermogel system is a new approach for in situ administration due to the easy injection of the formulation in the target tissue and the controlled release of the remaining drug. In this article, for the first time, it was possible to use Poloxamer-chitosan thermogel carrier for in situ administration of doxorubicin. Drug entrapment in Poloxamer-chitosan-based hydrogels is expected to have many advantages. In these conditions, the liquid behavior at low temperature allows for easy preparation and manageability, making them suitable injectable formulations. A thermogel that forms in the body can prolong their shelf life at the site of administration, allowing for controlled drug release and reducing side effects from the number of necessary intravenous injections. These features can increase patient compliance and reduce treatment costs. The ability of Poloxamer-chitosan to promote the solubility of poorly soluble molecules in water is one of the well-known features of this material. Due to its specific chemical nature, chitosan can effectively interact with the copolymer as a result of hydrophobic as well as electrostatic interactions. Thanks to their versatility, these formulations can be used for various biomedical applications and administration routes such as subcutaneous or used inside the tissue. Furthermore, the anticancer properties of doxorubicin in the Poloxamer-chitosan thermogel may be enhanced, as is the case with other drugs administered as Poloxamer-based formulations. RSM was used to optimize the rheological behavior of Poloxamer-chitosan thermogel and three-dimensional dimensions, as well as to identify the effects of temperature, polymer ratio, pH, and polymer concentration on the rheological parameters of Poloxamer-chitosan thermogel. Flow behavior by Hershal model was well described and the Poloxamer-chitosan thermogel behaved as a pseudo-plastic fluid under yield stress. Also, the presence of the drug doxorubicin in the combination of Poloxamer-chitosan carrier caused the slow release of this drug in the tissue site, and the drug released the largest amount of the drug in a period of 14 days according to the environment and created a safe environment for the tissue and its efficiency and effectiveness and increased tolerance and also reduced the drug dose to the minimum required amount, and according to the obtained results, this property can reduce the recovery time and minimize the involvement of other body tissues.

Rheological characteristics of drug delivery systems play an important role in determining their effectiveness and safety. In the field of drug delivery, rheology plays an important role in the design of drug delivery systems to achieve the optimal release of drugs and improve their therapeutic effects. Rheological properties of drug delivery systems, such as viscosity, elasticity, and surface tension, are important factors that affect drug release and distribution. A high-viscosity drug delivery system can slow drug release and prolong its release time, while a low-viscosity system can lead to faster drug release and a shorter therapeutic effect.

It is very important to evaluate the properties of different thermogels. Therefore, rheology is a simple method to investigate the evolution of gelation time and the effect of polymer

cross-linking, polymer concentration, ionic strength, pH and solvent effect to regulate the mechanical strength of thermogels. Gels designed for this purpose must show acceptable mechanical and rheological properties to ensure ease of application and maintenance. Carriers exhibit a wide range of mechanical (hardness, compressibility) and rheological (flow and dynamics) properties. For enhanced clinical performance in particular, the highly elastic structure of the Poloxamer-chitosan formulation, its acceptable flow and controlled release characteristics, may offer significant clinical promise. By controlling the rheological properties of drug delivery systems, drug solubility, bioavailability and targeting efficiency can be improved. Overall, injectable hydrogels are a promising drug delivery system that can provide sustained drug release, improve therapeutic outcomes, and reduce the need for invasive surgical procedures. Their ability to form gels in situ and tunable rheological properties make them ideal for a wide variety of drug delivery applications.

Furthermore, advances in rheological characterization techniques and computational modeling have opened new avenues for optimizing drug delivery systems based on their rheological properties. As such, further research in this area will continue to provide valuable insights into the design and optimization of drug delivery systems with improved safety and efficacy profiles, as well as understanding the relationship between rheology and drug release to design effective delivery systems that can achieve bioavailability. It is essential to improve medication, reduce toxicity and increase patient compliance. Based on the in vitro characteristics of this study, we concluded that breast cancer tissue injectable thermogel with Poloxamer 407 and chitosan may be a promising and innovative treatment.

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contribution

Mehdi Mehrazin, conceptualization, methodology, investigation, resources, visualization, software, writing—original draft.Azadeh Asefnejad: methodology, investigation, resources, visualization, software, writingFarid Naeimi: investigation, resources, and reviewing and editingHossein Ali Khonakdar: investigation, resources, and writing—reviewing and editing.

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No competing interests reported.

Investigating the rheological behavior of Poloxamer-chitosan thermogel for in situ drug delivery of doxorubicin in breast cancer treatment: designed by response surface method (RSM)

Status:

Journal Publication

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Abstract

Figures

Introduction

Materials and methods