Development of Chitosan Scaffold Carrier Device for Absorption and Controlled Release of Iron Chloride Using In Vitro Methods of Characterization

1. Introduction

Chitosan is a polymeric amino polysaccharide derived from the deacetylation of chitin.

Despite its potential, the development of chitosan scaffolds with optimal properties and their applications remain underexplored. Traditionally, crosslinkers are used in chitosan preparations, but these substances can be toxic, leading to cell death and polymerization contraction, which must be mitigated or avoided. Additionally, chitosan scaffold to carry iron chloride effectively. This study proposes a novel protocol that combines the coagulation method with freeze-drying, avoiding cross-linking and toxic agents, for the development of chitosan spheres. The aim is to facilitate the transport and controlled release of iron chloride in a controlled pH environment.

The objective of this work was to develop a novel chitosan sphere scaffold protocol that is potentially biocompatible, low-cost, and feasible as a biomaterial for carrying iron chloride and enabling controlled release at specific pH levels [1]. Following coagulation, freeze-drying, and lyophilization without cross-linking agents, the chitosan spheres were characterized physicochemically. Tests included degradation, cytotoxicity, and controlled release experiments. The release profile and patterns of iron were modeled considering various concentrations and pH levels of the medium in which the spheres were immersed. A theoretical curve was developed to predict diffusion behavior when the carrier is placed in a finite and limited volume solution [2,3], followed by protocol adjustments and complex modifications [4,56]. The modeling was conducted using Fortran (IBM, USA) and Meduza (Informer Technologies, USA) software [6,7].

Chitosan’s versatility allows the preparation of microcapsules and beads of various shapes and sizes. Its applications extend to different parts of the human body due to its biocompatibility, biodegradability, and moldability [5,8,9,10,11,12,13,14]. The spherical shape attributed to the chitosan spheres offers lower energy for the carrier system, making it suitable for various applications, including enzyme immobilization and directed drug diffusion [16,17]. The development of this carrier could address the high costs associated with current scaffold systems, which can exceed $10,000, making them inaccessible to poorer populations. The controlled release allows for predictable and planned therapeutic dosing [18,19]. Administering drugs with controlled release involves managing three basic factors: pH and concentration of the medium, release rate, and gastric transit, which can be simulated from experimental data. The behavior of iron under varying pH and concentration conditions, as well as the release profile of the spheres in a limited volume, was simulated. The material must meet cytotoxicity and biodegradability standards. Chitosan forms complexes with various molecules, and the stability of these complexes depends on factors such as charge density, molecular weight, net charge, solvent properties, ionic strength, pH, and synthesis temperature [20,21,22,23,24]. The release rate of molecules or ions may be linked to the chemical degradation process, which can be measured by indices such as the degree of swelling (GI), degradation index (ID), and loss of water absorption, influencing the rate of drug release [25,26] in the biomedical field [27]. The development and understanding of controlled release and system modeling have been previously explored [28,29]. Some mathematical models, including the Fick equation, describe the controlled release mechanism, particularly in limited volumes [30].

This work focused on developing new chitosan scaffolds, performing appropriate physicochemical characterization using techniques such as scanning electron microscopy (SEM), analysis of the main chemical groups of chitosan by Fourier transform infrared spectroscopy (FTIR) and Raman analysis (RS), analyze the crystallinity by X-Ray Diffraction (XRD), perform the cytotoxicity test in cell culture, promote the appropriate degradation tests and measurement of the indices, model using Fortran software (IBM, United States) the finite volume analysis, and promote the estimation modeling of iron behavior with the aid of Meduza software (Informer Technologies, USA) for iron behavior estimation.

2. Materials and Methods

 Production of Chitosan Spherical Carriers (Scaffold)

Chitosan spheres were produced using an innovative protocol developed from ultrapure chitosan derived from crab shell carapace (Sigma Aldrich^® Merck KGaA, Darmstadt, Germany) with a deacetylation degree (DD) of 87.7%. The synthesis process began by preparing a chitosan solution. An analytical balance was used to weigh 0.125 g of chitosan and was dissolved in 6.25 mL of 2% acetic acid (v/v) solution under constant magnetic stirring until a homogeneous gel, free of residual granules, was obtained. The gel was free of bubbles and chitin residues, contained chitosan at its maximum degree of purity.

The rheological properties of the gels were studied using a TA Instruments’ Rheometer ARG2 (Lukens, New Castle, USA) with plate-plate geometry. Initially, strain amplitude tests were conducted, followed by a frequency scan of the chitosan solution from 6.283 to 62.83 rad/s. A specific sequential structure of adapted syringes was used to ensure correct gel ejection using a controlled drip device into a 5% NaOH (1 mol L−1) solution under constant stirring. The gel beads were then neutralized in a phosphate-buffered saline (PBS) solution (~pH 7) and washed with ultrapure water obtained from a Milli-Q^® device, followed by passage through a sieve sequence.

The samples were lyophilized (Terroni LT-1000, Terroni Equipamentos Científicos, Jardim Jockey Club, São Carlos, Brazil) for 2 consecutive days. After lyophilization, the pellets were stored in amber glass bottles, dried at 50°C for 5 consecutive days, and covered with parafilm. This step was crucial for maintaining the exact size of the spheres in the final process and for avoiding the need for cross-linking the polymer. Besides that, all physicochemical characterizations were conducted before the modeling tests. The protocol was adapted from the works of Benvenutti et al. (2012) and Lima et al. (2016), and further developed by Lima et al. (2013) with modifications to the reagents and procedures for sphere production [31,32,33]. Several modifications were made to refine the protocol and achieve the ideal spherical carrier with long-term stability, as illustrated in the design protocol shown in Figure 1

 Experimental-Physico–Chemical Characterization of Chitosan Carrier Beads (Spherical Scaffold)- before the Controlled Release Test

Scanning Electron Microscopy

Scanning Electron Microscopy (SEM) was utilized to examine the chitosan spheres for surface characteristics, including potential pores, surface roughness, topographic structure, and surface concavities. These features are critical for enhancing cell interaction and biocompatibility by increasing the surface area and, consequently, the contact area with the external environment.

The chitosan samples were metalized on stubs using an Emitech K550X metallizer (Emitech, Dubai, UAE) with a current of 50 mA for 180 seconds. Microstructural analyses were conducted using a Zeiss IVO MA 10 scanning electron microscope (Zeiss, San Diego, CA, USA) with operating parameters set to a working distance of 14 to 15 mm and a voltage range of 20 kV to 21.24 kV. The SEM analyses were performed at the SEM laboratory (SEM-UFF, Volta Redonda, RJ, Brazil).

SEM micrographs were captured at various magnifications, beginning with a wide view of the entire sphere contour to identify areas of interest. Detailed imaging was performed at magnifications of 5000×, 2500×, 1500×, and 500× to capture the fine structural details of the chitosan spheres.

Fourier Transform Infrared Spectroscopy (FTIR)

The standard Fourier Transform Infrared (FTIR) spectroscopy technique is employed to identify the chemical groups present in chitosan structure, providing a fingerprint of the material under investigation. This technique analyzes the vibrational modes of different chemical groups, allowing for the determination of the chemical or structural composition of the material.

For FTIR analysis, chitosan samples were first ground into powders using an agate mortar and pestle. The powders were then stored in 1.5 mL Eppendorf tubes. The samples were prepared as KBr pellets for analysis. FTIR measurements were performed using a Perkin Elmer Spectrum 100 spectrometer (401 Congress Ave, Austin, TX 78701, USA). The analysis involved 32 scans with a resolution of 4 cm−1, and spectra were recorded over a wavenumber range from 4000 to 500 cm⁻¹.

X-ray Diffraction Spectroscopy (XRD)

X-ray Diffraction (XRD) was employed to determine the degree of crystallinity of the chitosan samples. XRD analyses were conducted at room temperature using a Shimadzu XRD-7000 apparatus (Shimadzu Corporation, Kyoto, Japan), with copper Kα radiation (λ = 1.5418 Å), operating at 40 kV and 30 mA. The chitosan samples were analyzed over a 2θ range of 10.0 to 70.0 degrees, with a scanning rate of 2°/min.

Raman Spectroscopy (RS)

Raman spectroscopy was performed using a Witec alpha300 system (Witec, Tennessee, USA). The apparatus was equipped with a plain silicon (Si) sample supplied by Witec and operated with a laser wavelength of 785 nm, providing a maximum laser intensity output. Observations were made using a 100× magnification objective with a numerical aperture (NA) of 0.95. During operation, the pinhole diameter was set to 100 μm, with an integration time of 50 ms and a grating of 600 grooves per millimeter.

The spectral analysis focused on the 950 cm−1 region and used a Raman Edge Filter. The Raman spectra were recorded with a high peak measurement of 2000 counts (CTS) and an integration time of 1 second.

Degradation and Physical–Chemical Characterization of Chitosan (ISO 10993)

The degradation assay was planned from the samples’ insertion into a blood plasma simulator solution (SBF~7pH) that mimicked the physiological conditions at a temperature of 36°C in an isothermal bath and calibrated the parameters’ constancy from a coupled thermostat. The sample was placed in the solution at a ratio of 1 g to 10 ml. In the follow-up, time was established for the measurement of the weights in the analytical balance and the definition of the degradation parameters measured by the swelling index (GI), degradation index, and mass loss, as well as the chemical analysis of the degradation measured by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of the samples before and after the assay [34,37]]. In the present study, the assay was carried out under static conditions using the blood plasma simulator solution (SBF pH~7), having the extreme condition of degradation (pH 5 potassium acetate solution) as control [40]. In this way, two types of tests were performed: the extreme solution (acetate); and the blood plasma simulator solution (SBF). The scaffolds were immersed in solutions and analyzed at 7, 24, 48, 96, and 168 h35. The material was analyzed at 0 for reference and at times 1, 2, 7, 14, and 28 days for analyzing degradation over time (ISO10993-5).

Evaluation of Degradation Parameters

Static Degradation indices were evaluated using the degradation index (ID), swelling index (GI), and mass loss. Additionally, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were performed after each experimental period of the Static Degradation Test to assess whether the material remained intact under physiological conditions compared to extreme conditions (5% potassium acetate).

The swelling index (GI) measures the percentage change in mass of the material due to absorption of the test solution, making it a common parameter for assessing degradation. This index indicates the loss or gain of mass as a result of the material’s interaction with the test solution.

Cytotoxicity Assay of Chitosan Beads in Cell Culture

The scaffold was assayed for cytotoxicity by using the supernatant after contacting 1 g of sample per 10 ml of fetal bovine serum-free DMEM (FBS) solution followed by serial dilutions. The scaffolds were incubated for 24 h in a CO₂ incubator (Thermo Fisher Scientific, Third Avenue, USA). The supernatant was then collected and taken by serial dilution and added onto the subconfluency cells preplated on multi-well plates. Cells from mouse fibroblasts balb-c3T3 obtained from ATCC were used. A total of 24,000 cells were plated in multi-well wells, incubated for 24–48 h, and the extracts were added over the cells when in the subconfluence cells stage. After incubation for 24 h, the cytotoxicity pattern was evaluated by classical parameters of cell viability evaluation: mitochondrial activity by reduction of formazan blue (MTT).

The objective was to develop a modeling design based on experimental analysis, as illustrated in Figure 2. The focus was on understanding the diffusion behavior of a solute contained within a sphere into a solution of limited volume, initially free of solute, and determining the experimental diffusion coefficient from laboratory data. Initially, the adsorption capacity of iron chloride on the surface of chitosan carriers was evaluated, alongside the phenomena of swelling and wetting of the carriers. Preliminary testing indicated that the spheres could adsorb approximately 50% of iron chloride (iron chloride III hexahydrate) (Dinâmica, Metaquímica Produtos, SC, Brazil) when incubated for 24 to 48 hours.

Following this, experimental procedures were planned and executed to assess the release of iron chloride from the chitosan carriers at various time points. In the initial phase, a test was performed to determine the degree of iron chloride absorption on the surface of the carriers. Twelve beads were incubated in 10 mL of iron chloride solution for up to 24 and 48 hours. Throughout all experimental steps, monitoring and measurement of the finite volume concentrations were conducted using a UV-Vis spectrometer (Bel Equipamentos Analíticos LTDA, Bel Engineering, Piracicaba, Brazil) with a 420 wave compression. The experimental evaluation and modeling were segmented into steps to allow for more precise modeling. Time intervals of 1 hour were defined for 5 consecutive hours, and measurements were taken daily for 5 days at consistent times each day (5-day interval checks).

 Development

The use of models to predict the release of micro-encapsulated drugs has received much attention from the medical community for providing control of the diffusional kinetics that can be modeled. The diffusion coefficient, D, can be determined according to the theory developed by Crank⁴⁴ for diffusion in a scaffold with a known solute concentration for a limited and solute-free volume solution. Through the Fick Laws for diffusion and admitting nearly spheres geometry and constant diffusion coefficient, we have:

$${\displaystyle \frac{\partial C}{\partial t}}=D\left({\displaystyle \frac{1}{r^2}} {\displaystyle \frac{\partial }{\partial r}}\left(r^2{\displaystyle \frac{\partial C}{\partial r}}\right)\right)$$

(1)

where C is the concentration of the diffusive species in the circular scaffold, r is the scaffold’s radius, and D is the diffusion coefficient.

For the case of diffusion of a scaffold immersed in a solution with a limited volume, the following equations and principles apply:

Initial condition:

t = 0 e 0 < r > a, C = C₀

(2)

Contour conditions

$$\mathrm{t} > 0, \mathrm{r}=0, {\displaystyle \frac{\partial C}{\partial r}}=0, t>0, r=a, C^{*}=C_L$$

(3)

where t is the time, r is the position concerning the center of the circular scaffold, a is the radius of the scaffold, and C_L is the concentration of the diffusive species in the solution.

The resolution of the diffusion problem is based on the analysis of unidirectional linear flow. This can be achieved through methods such as separation of variables or Laplace transforms. According to the approach described by Crank [44], the concentration of the diffusive species in the solution is given [36,37,38,39,94].

$${\displaystyle \frac{C_L}{C_{LE}}}=1-\sum _{n=1}^{\infty }{\displaystyle \frac{6\propto (1+\propto )e^{-D{q}_{n }^{2 }t/a^2}}{9+9\propto +q_{n }^2{\propto }^2}}$$

(4)

where we have to:

a = the radius of the scaffold (cm)

t = the time (hours)

V = the volume of the solution (cm³)

n = number of the scaffold

C_LE = substrate concentration at equilibrium

L_C = concentration

q_n = terms of positive root different from zero, dependent on the values of α, described in Table 1 below, where α = the relation between the volume of solution and total volume of beads defined as

$$\propto ={\displaystyle \frac{V}{n4\pi a^{3 }/3}}$$

(5)

The experimental diffusion coefficient D of the sample can be calculated by separating variables in a sequence of algebraic operations

Being A_n and B_n:

$$A_n={\displaystyle \frac{6\propto (1+\propto )}{9+9\propto +q_{n }^2{\propto }^2}} \mathrm{n}=\mathrm{1,2},\mathrm{3,4},\mathrm{5,6}$$

(6)

$$B_n={\displaystyle \frac{q_{n }^{2 }t}{a^2}} \mathrm{n}=\mathrm{1,2},\mathrm{3,4},\mathrm{5,6}$$

(7)

Substituting Eq. 4 and Eq. 5 into Eq. 2, we have:

$${\displaystyle \frac{CL}{C_{LE}}}=1-{\sum }_{n=1}^6{A}_n{e}^{-D{\mathit{B}}_{\mathit{n}}}$$

(8)

Developing the Eq.8

$$1-{\displaystyle \frac{CL}{C_{LE}}}=A1e^{-DB1}+A2e^{-DB2}+A3e^{-DB3}+A4e^{-DB4}+A5e^{-DB5}+A6e^{-DB6}$$

(9)

$$x=e^{-D}$$

(10)

$$y=1-{\displaystyle \frac{C_l}{CLE}}$$

(11)

Substituting Eq. 10 and Eq. 11 into Eq. 9 we have:

$$\mathrm{y}=A1x^{B1}+A2x^{B2}+A3x^{B3}+A4x^{B4}+A5x^{B5}+A6x^{B6}$$

(12)

Applying Ln in Eq. 10 we have:

$$D=-Ln\left(x\right)$$

(13)

Calculation of Sample Experimental Diffusion Coefficient

Eq. 6 to Eq. 13 calculate the diffusion coefficient from the experimental data. The root of the polynomial of Eq. 12 was obtained through the Principle of the Bisection method described in Eq. 14. The interval of application of the method was defined by analyzing the signals of Eq. 13 so that the value of the diffusion coefficient is D > 0, Eq. 15. The algorithm was implemented in the Fortran language (Annex 2), with an error stopping criterion less than 1 × 10⁻⁶.

$$f\left(a\right)-f\left(b\right)<0 \to x_0={\displaystyle \frac{a+b}{2}}$$

(14)

where a and b range and x0 the root

$$S=\left\{x \in R/0<x<1\right\}$$

(15)

With the data in Table 2 below, the experimental diffusion coefficient was calculated. A coefficient of 8.583106 × 10⁻⁶ cm²/h was obtained.

The rate of drug release across a membrane can be expressed by the ratio dQ/dt, where D is the diffusion coefficient, S is the contact surface area between the solution and the membrane, C is the concentration difference of the drug across the membrane, and, l is the thickness of the membrane [40,60]. According to Fick’s law, this equation can be utilized to analyze the initial 60% of drug release [40].

The beneficial mathematical model was developed by Lopes et al. (2015) to achieve a specific release rate by calculating the size and shape of hydrophilic matrices. The Monte Carlo method, which utilizes a random number generator, was implemented in the C++ programming language [42]. Siepmann later expanded upon this model in 2008. Currently, various mathematical models are applied to control drug delivery systems based on matrix-controlled mechanisms. Many of these models are rooted in the diffusion concept developed by Crank and are used to simulate experimental trials [43,44,45]. So, by understanding the conditions of the medium—such as predetermined volume, pH, and the total volume of the solution before and after immersion—it is possible to predict the surface adsorption behavior and the release mechanism through diffusion. This can be achieved by calculating the controlled release coefficient using Fortran software, which is commonly employed for simulating diffusion assays [45,46].

Key parameters used in the modeling include:

A: the diameter of the sphere (1 mm)

α =3V/n4πa³: the volume of the solution producing the beads, using 6.25 mL

N: the total number of beads, which is 42.

This modeling approach, based on the diffusion coefficient obtained from experimental data and Crank’s model, allows for the prediction of the release behavior of iron chloride under different pH conditions. The behavior of iron and iron chloride in various pH environments was specifically analyzed using the MEDUZA software [47].

The experimental and theoretical modeling of the controlled release from the spheres was conducted, considering the factors involved in both practical experiments and different diffusion mechanisms. And, in summary, considering the finite element approximation we can represent the same system by the geometric form below:

4. Conclusions

This work made possible the development of the synthesis of a new protocol chitosan carrier, maintaining all the characteristics and properties inherent to chitosan as initial raw material, being confirmed by the physico–chemical analysis and analysis of the parameters: favorable topography by cell spreading (SEM, semi-quantitative analysis), identification of functionally chemical groups (FTIR and Raman), and degree of crystallinity and semi-crystalline polymer (XRD). It was possible to predict the controlled degradation of the material by the classical tests of degradation and analysis of the degree of swelling, ID, and loss of mass. The spherical chitosan carrier biomaterial is noncytotoxic from the cytotoxicity assay’s cell death parameter that evaluates mitochondrial activity (MTT assay). Analysis of iron release in the form of iron chloride and adjustments of specific parameters, it was possible to make the interrelationship of experimental data with classic simulations adsorption and release on the surface of iron chloride obtaining a response between the experimental part delineated and interrelated with the simulation models used from the Fortran and Meduza software and with that try to predict the release response from an unpublished trial and simulation leading to reagent savings, time, and cost of spherical chitosan no reticulated. Experimental predictability helps plan other similar assays by modifying fixed model parameters and thereby obtaining a standard-type profile for these types of assays, including spherical chitosan scaffold.