1. Introduction
Polymeric microparticles can accommodate drugs/bioactive agents within their structures and withhold their release, thus, having a more prolonged release mechanism compared to oral or systemic administration. There is also the possibility to accommodate cells within their structure, hence, they can also be a useful system for tissue engineering [
1]. Gellan gum (GG) and alginate (Alg) are biocompatible, non-toxic and biodegradable polymers very used for biomedical purposes. The production of Drug Delivery Systems (DDS) from the mixture of these two polymers is an easily accessible alternative. The mixture of these two polymers has been used to produce microparticles for different biomedical purposes [
2,
3] such as bacteria encapsulation [
4,
5] and drug delivery [
6]. However, the optimization of microparticle production with a sustainable, simple, and non-expensive method, is meet the required standards before clinical application.
A simple microparticle production method is the coaxial airflow technique [
7]. It is a replicable, and inexpensive method that can produce large quantities of particles. It consists of a nozzle that has a coaxial air stream system parallel to it. When a polymeric solution is extruded through the nozzle, the airflow will aid in the detachment of the drop. This drop will fall in a coagulation bath, which promotes the crosslinking of the polymer [
7,
8]. This method is useful to produce large quantities of replicable microparticles. Alg particles have been produced with coaxial airflow for cell delivery [
9,
10] and drug delivery [
7,
8,
11]. Some studies used this process to produce GG particles [
12] but did not optimize the production process. It is important to understand what are the critical parameters that this technique has in the different types of particles that are developed. Thus, to have a replicable and reliable production method.
This article will focus on the development of GG:Alg microparticles made with the coaxial air-flow method. The optimization of the production method was carried out using a Design of Experiment (DOE). To the knowledge of the authors, no article focused on the optimization of GG:Alg microparticles using this method. The main goal of this work was to decrease the microparticle’s diameter (size) and to decrease dispersibility related to the diameter. After the production’s optimization, a characterization of GG:Alg microparticles for drug release was carried out in acidic and neutral environments. For drug release studies, methylene blue (MB) was chosen; it is a cationic dye used as a model drug, but also used in medical treatments. This was followed by a characterization of the particles for biomedical purposes.
2. Materials and Methods
2.1. Materials
High acyl gellan gum was purchased from Sigma-Aldrich (U.S.A) as Phytagel. Alginic acid sodium salt was purchased from BioChemica (Panreac Química SLU, Spain). Methylene Blue, in powder form, was purchased from Alfa Aesar (U.S.A.). Phosphate-buffered saline solution (PBS) with pH 7.4 was prepared following the following procedure: to 800 mL of Millipore water 4 g of sodium chloride (NaCl 100%) (J. T. Baker, Avantor U.S.A.), 0.1 g of potassium chloride (KCl ≥ 99%) (Sigma-Aldrich, U.S.A.), 0.72 g of disodium hydrogen phosphate (Na2HPO4) (Panreac Química SLU, Spain) and 0.12 g of potassium di hydrogen phosphate (KH2PO4) (Panreac Química SLU, Spain) were added. 200 mL of Millipore water were then added. For PBS with pH 6.5, the quantities were as follows: 8 g of NaCl, 0.2 g of KCl, 0.61 g of Na2HPO4 and 0.19 g of KH2PO4. Calcium chloride (Carl Roth, Germany) was used to prepare aqueous solutions of 3.5% w/v.
2.2. Production and Optimization of Particles using DoE
The particles were produced using an aqueous solution (2 % w/v) blend of alginate (Alg) and gellan gum (GG). A Nisco® Encapsulation Unit—VARJ1 [
13] was used to produce the particles via the coaxial air-flow method (
Figure 1). The solution was extruded into a 500 mL calcium chloride aqueous solution (3.5% w/v). The particles were left overnight in the bath and then filtered and washed with ethanol.
To optimize the production of GG:Alg particles a two-level fractional factorial design was used with 4 factors with a degree of fractionation 2 (24-1), with 3 replicates and a design resolution of IV. Design-Expert Software® Version 11 (Stat-Ease Inc., Minneapolis, MN, USA) was used to analyse the results. Four factors were chosen: A: GG:Alg Ratio within a 2% w/v aqueous solution; B: Gap bath-nozzle; C: Airflow; D: Pump flow. The diameter (size) of the particles without drying (wet stage) was defined as a response. To evaluate dispersibility, the Coefficient of Variation (COV) (standard deviation/mean) and normalized SPAN (=(d(0.9)-d(0.1))/d(0.5)) were both chosen as responses. The particle size was obtained using a Leica S9 Stereo Microscope and analysed via Image J software. At least 100 diameters were measured for each run.
The DOE had the aim of minimizing both the particle’s diameter (size) and dispersibility (analysing SPAN and COV) using the coaxial air flow method. The DOE focused on the production of the microparticles right after their production from the coaxial airflow technique at their wet stage. After DOE optimization, the particles were dried. After being overnighted in the CaCl2 solution, they were filtrated and subjected to a water-ethanol exchange. For this purpose, different Millipore water and ethanol blend solutions with increasing ethanol concentration were used: (water (% v/v): ethanol (% v/v)—90:10; 75:25; 50:50; 25:75; 10:90; 0:100). The microparticles were left for 1 h in each solution, starting from the 90:10 solution. Then the microparticles were dried in a vacuum pump.
2.3. Morphological Characterization
The morphology of dried and wet particles was analysed using a S9 Stereo Optical Microscope (Leica®, Germany) and a SEM Tabletop microscope TM3030 Plus (Hitachi®, Japan). Some of the optical microscopy images were also obtained in transmission mode, using an Olympus BX51 microscope (Olympus®, Japan), coupled with an Olympus DP73 CCD camera, and acquired with the Stream Basic v.1.9 Olympus software. A cold illumination source generated by a halogen lamp (KL 2500 LCD, SCHOTT) was used. All images were obtained and automatically scaled by the respective software. In all batches of particles, at least 100 diameters were measured before analysing.
2.4. Swelling
Swelling in mass (
Sw) was calculated using Equation 1. Batches with 0.02 g of particles were submerged in 10 mL of PBS with pH 6.5 and pH 7.4. At different time intervals, batches were filtered, weighted, and analysed to determine their diameter. In Equation 1,
m0 is the initial mass of the particles (dried state) and
mtw is the wet mass at each time.
2.5. In Vitro Degradation
The degradation was obtained by submerging particles in PBS solutions with pH 6.5 and pH 7.4, for different times within a maximum time range of 60 days (2 months). Different batches of particles were collected at different times. When collected, the particles were filtered, rinsed with water, and lyophilized. Then the mass loss was determined using the Equation 2, where
mtd is the particle’s dried mass at each time.
2.6. Encapsulation Efficiency and Loading Capacity
The encapsulation efficiency (EE%) and loading capacity (LC%) were determined by adding 0.02 g of particles to 5 mL of PBS with pH 6.4 and pH 7.4 with different concentrations of MB (10, 30, 60, 100, 140, 250 and 290 μg/mL). After 4 days at 37 °C on an orbital shaker, the concentration of the free MB in solution was determined using UV–VIS spectroscopy on a T90+ UV/VIS Spectrometer (PG Instruments Ltd, U.K.) at 664 nm, MB maximum absorbance. The calibration curves for MB concentration in PBS pH 7.4 and PBS pH 6.5 were determined to be abs = 0.1919conc + 0.0559 (R2 = 0.99) and abs = 0.1865conc + 0.0578 (R2 = 0.99), respectively (
abs being absorbance and
conc being concentration). EE% and LC% were determined using the following equations:
Being the mass of MB in the initial solution, the mass after the swelling of the particles, is the mass of encapsulated MB and the weight of the particles. Four replicas were used.
2.7. In Vitro Fourier-Transform Infrared Spectroscopy (FTIR) and Thermogravimetric Analysis (TGA)
FTIR analysis were carried out in a in a Perkin-Elmer–Spectrum Two (Waltham, USA). TGA analyses were performed on particles with and without MB to analyse any possible differences with the addition of MB. The analysis was carried out with a TGA-DSC–STA 449 F3 Jupiter equipment (Germany). It was performed in a temperature range of 25-800 °C with a 10 K/min heating rate under nitrogen atmosphere.
2.8. In Vitro Drug Release
For drug release tests, the MB concentration with the highest encapsulation efficiency was used to prepare the MB loaded particles. Batches with 0.025 g of MB loaded particles were loaded within a donor-recipient made of a permeable membrane. This recipient was then submerged in 50 mL of PBS solutions with pH 6.5 and pH 7.4. The systems were kept at 37 ºC with orbital agitation. At regular periods (0, 0.25, 1, 3, 6, 24, 48, 72, 144, 192, 240 and 312 hours (h)) 2 mL of the PBS was retrieved and replaced with fresh PBS. The 2 mL were then analysed by UV–VIS spectroscopy to determine the concentration of MB at each time. Five replicas were used.
4. Discussion
The particle’s diameter significantly decreased with higher air flows, whilst it increased with higher pump flows. However, pump flow did not impact the particle’s size as the air flow did. In an atomization process, Chan et al. [
22] also found that an increase in volumetric liquid flow rate (equivalent to pump flow in this work) also produced larger particles. Using a coaxial airflow device, Workamp et al. [
23] found similar results where polyacrylamide and gelatine particles decreased their diameter with increasing volumetric airflow rates. Nastruzzi et al. [
24] also found that air flow was also significant to their microparticles size, with a negative effect. In this study, height (equivalent to Gap bath-nozzle) and flow (equivalent to Pump flow) also did not significantly affect the particle’s size.
Higher SPAN and COV values were obtained with higher airflows. Chan et al. [
22] found that a lower air-to-liquid mass flow rate ratio resulted in lower particle size distribution. In terms of t-value effects, airflow had a higher effect on SPAN than on COV. The ratio of the GG:Alg (A) had the second-highest effect on dispersibility. Being a positive effect, higher dispersibility occurred at the higher level of 25%:75% (with a higher concentration of alginate and less gellan gum). This was attributed to viscosity. Higher contents of gellan gum increased the viscosity of the solutions, so using lower viscosity solutions increased the dispersibility of the particles.
The main goal was to minimize the size and dispersibility of the particles. For the GG:Alg ratio (A), 50%:50% was selected since it decreased dispersibility. For factor B, the Bath-nozzle gap did not affect the microparticle’s size or dispersibility therefore, the intermediary level of 15 cm was chosen. Airflow (C) was the most significant factor in all responses. Using higher airflows, smaller diameters were obtained, however, an increase in dispersibility also occurred. On the other hand, airflow had a much higher effect on microparticle size than on COV and SPAN. Thus, this factor had a much higher impact on size than on dispersibility. So, the higher level, 5 L/min, was chosen to obtain smaller particles. Regarding Pump flow (D), the lower level of 5 mL/h was chosen since it affected the diameter of the particles with a positive effect and had no effect on dispersibility. The wet microparticles produced in these conditions had a diameter between 400 and 450 μm.
Higher swelling indexes were obtained with neutral pH. This was attributed to the fact that both polymers are anionic polysaccharides. Carboxylic acid groups of both polymers undergo deprotonation in more basic environments, leading to more negatively charged groups and a decrease in the strength of intermolecular hydrogen bonds [
25,
26]. The increase in the negativity of the chains leads to repulsive electrostatic charges between chains, thus promoting the spaces between the chains and promoting the penetration of the liquid. Also, at lower pH, calcium ions dissociate from the structure, promoting the formation of hydrogen bonds, leading to a more closely packed structure, and preventing higher swellings [
27]. These results are in accordance with previous studies that also found that particles with alginate [
26] and with gellan gum [
14,
28] also had less swelling with lower pH.
Regarding encapsulation efficiencies with MB, due to the deprotonation at neutral pH, there will be more anionic moieties in both polymers at pH 7.4. Being MB a cationic drug, at pH 7.4 there will be more electrostatic interactions between MB and the anionic moieties of the polysaccharides, than at pH 6.5 [
29]. At a more acidic pH, there is also a higher concentration of H
+ protons that will compete with MB for the vacant anionic moieties [
30]. Also, at neutral pH the microparticle’s swelling is more pronounced which helped the trapping of MB [
26]. Othman et al. [
3] used alginate particles for MB removal from residual waters. The adsorption capacity of the particles improved with an increase in pH. These results are in accordance with our results. Following the MB entrapment, the GG:Alg microparticles were then dried. The particles suffered no agglomeration but did not retain the spherical form that they previously had.
In TGA, the microparticles with MB did not lose as much mass as the microparticles without the model drug. Since MB did not fully degrade at the final temperature, its presence within the microparticles reduced the mass loss. The difference in mass loss observed between particles loaded at pH 6.5 and pH 7.4 might be explained by loading capacity (
Figure 6b). Higher loading capacities were obtained with pH 7.4, thus there was more MB within these particles. Hence, less mass was lost with microparticles loaded within PBS pH 7.4. Also, the difference might be due to an interaction between MB and the polymers, leading to more stable complexes. Temeepresertkij et al. [
29] found that the carboxylates of alginate interacted with the N, C and S of MB, leading to more stable alginate/MB complexes. Gellan gum also has carboxylates in its structure, thus there also might be a similar interaction between MB and gellan gum [
31].
The microparticles had a higher mass loss with neutral pH than with pH 6.5, having a mass loss of around 50% at the end of 60 days. Alginate degrades at a faster rate with pH 7.4. Deprotonation of the carboxylic acid may lead to a faster dissolution of the alginate structure. Also, ionically linked alginates lose the divalent cations that form the structure and degrade at neutral pH [
32,
33,
34]. In gellan gum, FTIR analysis made by Zhao et al. [
35] confirmed that under acidic conditions (in their case pH 1.2) hydrogen ions diminished the electrostatic repulsions of the chains and thus promoting the formation of the doubled helices structure, thus, forming more resistant structures. This is also supported by Picone et al. [
36], that, with rheological studies, found that at neutral pH the formed gellan gum gels were more fragile and deformable than the ones formed at acid mediums. Su et al. [
37] studied an ionically crosslinked alginate scaffold, that was submersed in a PBS solution (pH 7.4). At the end of 25 days, 90% of the initial weight had been lost. In another study [
38], 3D printed ionically crosslinked alginate scaffolds lost half of their weight within 7 days. Regarding gellan gum, this polysaccharide is more stable in PBS. Zu et al. [
39] analysed gellan gum with different types of crosslinking. In PBS (pH 7.4) gellan gum only lost around 20 % in weight at the end of 42 days. In another study, Reis et al. [
40] also had similar results with the immersion of ionically crosslinked gellan gum in PBS, with also a loss of 20 % at the end of 30 days. Comparing the two polymers, alginate generally degrades faster than gellan gum. The weight that was lost during the 60 days in the GG:Alg particles might be due to a more pronounced alginate degradation than gellan gum. In an artificial urine solution, Barros et al. [
41] compared the degradation of alginate and gellan gum. At the end of 60 days, alginate had lost all the weight, whilst gellan gum maintained more than 50% of the weight.
Using the variations of KP and SP models with the T
lag, better mathematical fits were obtained for the release profiles of MB (
Table 2). The introduction of T
lag to the models has been proven in earlier studies to be more adequate for swelling particles [
17,
18]. However, both models revealed that a Fickian diffusion profile dominated the release (
n,
m <0.43). Using the Weibull model, a Fickian release profile was also obtained. But when compared to the modified KP and PS models the Weibull model had a reduced R
2adj, making a not so good fit as the others. Thus, the release profile was more dominated by the concentration gradient than by swelling. Even though the particles with pH 7.4 had higher swelling indexes, the particles with pH 6.5 had a higher cumulative release profile. In
in vitro drug release studies, Jana et al. [
4] had their microspheres with a good fitting with the KP model with a Fickian release mechanism. With alginate particles [
42], morin was released via a Fickian diffusion profile also with the KP model. Tu et al. [
43]. prepared alginate particles via spray coagulation and then loaded with MB. A good fit with the Higuchi model was obtained, where the release was also controlled by diffusion. On the other hand, Voo et al. [
44] prepared alginate particles with higher stiffness and then loaded them with MB. Unlike the previous study, the release profile from the particles was found to be non Fickian by the KP model. In a different study [
45], gellan gum beads loaded with methotrexate had a release profile with a good fit with the KP model with a Fickian release profile.
The pH of the solution contributed to a faster release of MB. A faster release of MB under acidic mediums can be explained by a less pronounced interaction between the drug and the anionic moieties at pH 6.5. In gelatin/chitosan films, Koc et al. [
46] studied the release profiles of MB in different pH (pH 1.5 and pH 8). Similarly, higher release profiles were obtained at the more acidic pH than at the more basic, also due to non-protonated groups in gelatin and chitosan at the more basic pH. The relation between the mainly lead diffusion release profile and the pH of the release medium contributed to the GG:Alg particle’s MB release profile.