Fabrication of alginate microspheres for drug delivery: A review

Journal Pre-proofs Fabrication of alginate microspheres for drug delivery: a review Nguyen Thi Thanh Uyen, Zuratul Ain Abdul Hamid, Nguyen Xuan Thanh Tram, Nurazreena Binti Ahmad PII: DOI: Reference:

S0141-8130(19)36498-0 https://doi.org/10.1016/j.ijbiomac.2019.10.233 BIOMAC 13734

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International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

23 August 2019 16 October 2019 24 October 2019

Please cite this article as: N.T.T. Uyen, Z.A.A. Hamid, N.X.T. Tram, N.B. Ahmad, Fabrication of alginate microspheres for drug delivery: a review, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.233

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Fabrication of alginate microspheres for drug delivery: a review Nguyen Thi Thanh Uyen1, Zuratul Ain Abdul Hamid1, Nguyen Xuan Thanh Tram2 and Nurazreena Binti Ahmad1* 1

School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal, Pulau Pinang, Malaysia 2 Faculty of Materials Technology, Ho Chi Minh City University of Technology, VNU-HCM, Ho Chi Minh City, Vietnam *Corresponding Email: [email protected]

Abstract Alginate microspheres (AMs) have received much attention as a novel drug delivery system owing to various advantages of alginate such as inexpensiveness, nontoxicity, biocompatibility and biodegradability. The well-designed fabrication method is essential to achieve desired AMs suitable for specific drug delivery system. Reports on AMs preparation techniques have increased rapidly in the last decade. A number of synthesis parameters have been investigated for the improvement of physical, chemical and biological properties of AMs. Hence, this review summarizes the work to date on the fabrication techniques of AMs for drug delivery system, including spray-drying, extrusion and emulsification/gelation technique. Besides, the influence of various factors such as alginate concentration, oil phase, surfactant, cross-linker concentrations, cross-linking time, stirring speed, model drug and drug content on the morphologies, properties and encapsulation efficiency (EE) of AMs via extrusion and emulsification/gelation technique are summarized. Before embarking on the development of any drug delivery system, a thorough understanding of drug release mechanism and factors that impact the drug release profile are essential, which are also covered in this review. Keywords: Drug delivery system; Alginate microspheres; Spray-drying method; Extrusion method; Emulsification/gelation method; Drug release Contents: 1. 2. 3.

Introduction Drug delivery system and alginate microspheres Fabrication techniques of drug loaded AMs 3.1 Spray-drying technique 3.2 Extrusion technique 1

3.3 Emulsification/gelation technique 4. Factors influencing the properties of drug loaded AMs 4.1 Alginate concentration 4.2 Oil phase 4.3 Surfactant 4.4 Cross-linking concentration 4.5 Cross-linking time 4.6 Stirring speed 4.7 Model drug and drug content 5. Drug release profile 6. Conclusion 1.

Introduction

Controlled-release devices of drug delivery system (DDS) such as microspheres, nanoparticles, liposomes and micelles have been utilized to overcome the shortcoming of traditional drug administration in terms of therapeutic efficiency and patient compliance [1]. Among these carrier devices, alginate microspheres is one of the widespread studies because it offers both control of drug release rate and delivery of the drug to a specific treatment target. The AMs acts as a transient mask to protect the unstable encapsulated drugs (e.g. enzymes, proteins, peptides) [2, 3]. After the drug release, the alginate is degraded into water-soluble oligomers and further metabolized and eliminated from the body due to its biodegradability. Drug-loaded AMs are mainly applied for colon-specific drug delivery via oral administration. Recently, several drugs e.g. icariin [4], guar gum succinate [5], carboxymethyl starch [6], and mesalamine [7] have been encapsulated into AMs to treat inflammatory bowel diseases. AMs are formulated by three common techniques i.e. spray drying, extrusion, and emulsification/gelation method. Basically, in spray drying technique, alginate solutions are mixed with organic reagents and atomized by hot-air flow. However, this technique has low yield in laboratory scale due to the loss of product in the wall of the drying chamber and the emission of exhaust air contained fine particles (<2µm) [8]. In extrusion technique, alginate solutions are added drop wise to cross-linking solution [9-11]. Besides, emulsification/gelation technique involves the formation of stable polymer droplets in the emulsion system and the gelation of the droplets by the utility of cross-linking agent [7, 9, 12]. Extrusion and emulsification/gelation technique have been appeared to be potential methods for encapsulation

2

various types of drugs in the pharmaceutical and biotechnology industry [12, 13]. A well-design of extrusion and emulsification/gelation techniques enable us to obtain the AMs with desired particle size, encapsulation efficiency and controlled drug release rate by prevailing synthesis parameters such as alginate concentration, oil phase, surfactant, cross-linker concentrations, cross-linking time, stirring speed, model drug and drug content. In recent years, quite a number of reviews on AMs have been exploited by pharmaceutical researchers [2, 14, 15]. However, there are lacks of literature that specifically reviewed on the controlled properties of AMs via fabrication methods that have been enormously employed recently. On the other hand, the improvement in the AMs system for further clinical applications is still a challenge. Therefore, this review aims to provide a comprehensive overview of AMs as drug carrier from fabrication methods to some factors affecting the formation and properties of microspheres. In the first section of our review, we will summarize some aspects of the DDS and the utility of AMs. Meanwhile, preparation techniques, factors that influence the encapsulation of drugs into AMs as well as drug release rate, and the mechanism of drug release will be mentioned in the later part of this literature. 2.

Drug delivery system (DDS), novel drug delivery system (NDDS) and alginate microspheres

DDS is a system that transports therapeutic substance into the body to achieve a desired therapeutic effect. There are two main types of DDS, conventional drug delivery system (CDDS) and novel drug delivery system (NDDS). The drugs are delivered by CDDS through oral, buccal/sublingual, rectal, intravenous, subcutaneous and intramuscular route [16]. In CDDS, the concentration of therapeutic agents is not constant during the treatment and requires frequent dosage administration. Therefore, this explains the rapid increase in drug level in the blood above the toxic limit after each administration and then decreases below the therapeutic level until the next administration [17, 18]. The increase in drug concentration above the toxic limit results toxicity in body. Furthermore, the rise of frequent administration might add to the therapeutic non-compliance from the patient [19]. To overcome the above limitations of CDDS, the advanced technique, NDDS, was developed in which dosage forms were generated. From this, the rate of the drug is maintained within the therapeutically effective level with controlling

3

drug release in both speed and time. Therefore, continuous treatments were given to patients. Besides, the NDDS delivers drugs to the specific site on the body with optimum dose and declined toxicity effect as compared to the CDDS [19]. In NDDS, drug carrier is a substrate that enables drug to be transported to the target site, release the drug at a controlled rate and thus improving the bioavailability of the drug. Drug carriers e.g. microspheres, liposomes, nanoparticles and polymeric micelles are as important as the drug itself [20]. Among them, microspheres composed of biodegradable polymers constitute an important part in the pharmaceutical field by virtue of their small size and efficient carrier characteristic such as stability against forces generated during aerosolization, biocompatibility, targeting of specific sites, release of the drug in a predetermined manner, and degradation within an acceptable period of time [21, 22]. Microspheres are characterized as powders consisting of a natural or synthetic polymer, which are biodegradable in nature, having a spherical particles size range from 1 to 1000 µm and ideally having a particle size less than 200µm [18]. Fig. 1 shows an example on the morphology of AMs with an average diameter of 59µm [23]. In addition, with their spherical and small size, they can be easily injected into the body via several routes. Microspheres are used to protect drugs from environmental effects such as moisture, heat, oxidation and mask their horrible taste and odour [24, 25]. (a)

(b)

Fig. 1 The morphology of (a) drug-free alginate microspheres; (b) drug loaded alginate microspheres [23]. Copy right 2014. Reproduced by permission of Elsevier Science Ltd. Depending on how the encapsulation is done, microspheres are classified into two types, microcapsules and micromatrixes, as displayed in Fig. 2. Microcapsules are reservoir devices in which a drug core is coated by a polymeric material, whereas micro matrixes contain a drug which is uniformly dispersed in the polymeric matrix [26-28]. The fabrication of microcapsules 4

is much more difficult than micromatrixes type due to its architectures, core size and shell thickness are hard to control, and the core/shell material must be immiscible in each other [29].

Polymer Therapeutic agents

Microcapsule

Micromatrix

Fig. 2 Schematic structure of microcapsule and micromatrix Besides, the selection of polymers used for the preparation of microspheres plays a crucial role in the drug delivery process. The polymers are initial material for manufacturing, drug protection and enhancement of bioavailability [30]. There are two types of polymers including natural polymers and synthetic polymers [31]. Synthetic polymers include non-biodegradable polymers e.g. silicones, ethyl vinyl acetate (EVA) [32] and biodegradable synthetic polymers e.g. polylactic acid (PLA), polycaprolactone (PCL), polyglycolic acid (PGA) and polylactic-coglycolic acid (PLGA) [33, 34]. Meanwhile, natural polymers include protein (e.g. gelatine, collagen, lectins) and polysaccharide (e.g. alginate, pectins, and chitosan) [33, 35]. Natural polymers offer many advantages due to their natural abundance, low cost and a renewable resource in nature. Moreover, natural polymers are less toxic, biocompatible and biodegradable whereby they are degraded by enzymatic activity [34, 36]. Although synthetic polymers are safe and biocompatible they undergo hydrolytic degradation in the body and they tend to move away from the injection site. This in turn will induce embolism which eventually leads to damage on organ [33, 34]. Therefore, natural polymers are preferred to as potential candidates in microspheres fabrication. In recent years, natural polymers such as alginate, collagen and gelatine have been enormously investigated in the field of tissue engineering as well as drug delivery applications. However, gelatine micro-particles system has poor mechanical strength and rapid drug release [37, 38] while collagen is a high-cost polymer [39-41]. Alginate, on the other hand, has been utilized in 5

several applications from an additive in foods to wound dressing due to its inexpensiveness, nontoxicity, biocompatibility and biodegradability [42-45]. In the biomedical field, alginates are used for drug and cells entrapment, scaffolds in tissue engineering and controlling drug release based on the crosslinking with calcium salt [43]. Alginates, a natural polysaccharide, is a linear chain of (1,4)-linked β-D-mannuronic acid (M blocks) and α-L-guluronic acid (G blocks) in different proportions and sequential arrangements as shown in Fig. 3 [46, 47]. This polymer has been used to encapsulate variety of drugs, vaccine, proteins and deoxyribonucleic acid (DNA) as listed in Table 1. Alginate matrix is capable of forming a gel in the presence of a divalent cation such as Ca2+, Ba2+ or Sr2+ which is a simple and cost-effective process for microencapsulation of bio-active agents [48, 49].

1 α

1 β

4 1

α 1

β 4

Fig. 3 Structure of alginate showing the β-D-mannuronic and α-L-guluronic acids blocks Table 1 Representative studies (2007-2019) on the encapsulation of substances in AMs Loaded substance Soy isoflavone Keratin Retinonic Mesalamine Risedronate sodium Ibuprofen Metformin hydrochloride (MF) Blue dextran Metformin hydrochloride (MTF) Antigen fluorescein isothiocyanate conjugatedbovine serum albumin (FITCBSA) Ezetimibe Voglibose

Fabrication technique Extrusion Emulsification/gelation Spray-drying Emulsification/gelation Emulsification/gelation Extrusion Spray-drying Extrusion Extrusion

Year 2019 2018 2018 2018 2018 2018 2018, 2016 2017 2017

References [50] [13] [51] [7] [12] [10] [52, 53] [11] [54, 55]

Emulsification/gelation

2017

[48]

Extrusion Extrusion

2017 2017

[56] [44]

6

Curcumin Indomethacin 5-Florouracil Astaxanthin Icariin Guar gum succinate Carboxymethyl starch Piroxicam Blue dextran Risperidone Granisetron hydrochloride (GH) Cyanocobalamin Caffeine Immunoglobulin G (IgG) Probiotics Insulin Probiotic bacteria Diclofenac sodium Aceclofenac Glipizide Plasmid DNA (pDNA) Bacille Calmette-Guerin (BCG) 3.

Extrusion Extrusion Emulsification/gelation Extrusion Emulsification/gelation Extrusion Extrusion Extrusion Emulsification/gelation Extrusion Emulsification/gelation Extrusion Spray-drying Extrusion Emulsification/gelation Spray-drying Extrusion Emulsification/gelation Extrusion Extrusion Emulsification/gelation Emulsification/gelation

2016 2016 2016 2016 2016 2016 2016 2016 2016, 2014 2014 2014 2014 2014 2013 2013 2013 2012 2011 2011 2010 2008 2007

[57] [58] [59] [60] [4] [5] [6] [61] [23, 62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75]

Fabrication techniques of drug loaded AMs

A thorough understanding on fabrication techniques of drug loaded AMs are crucial. There are three commonly reported techniques to fabricate AMs which are the spray drying techniques, extrusion and emulsification/ gelation techniques. Each of these methods is felicitous for a particular delivery formulation and extensively discussed in this section. 3.1 Spray-drying technique Spray-drying is a rapid, continuous and one-step process that combines drying and encapsulation to produce microspheres in large quantities [8, 76]. The working principle of a spray dryer, it encompasses four major stages to be taken to form microspheres: (i) dissolving, emulsifying or dispersing the drug in alginate solution, (ii) atomization of the liquid feed into droplets using the nozzle, (iii) drying of the droplets in the drying gas and formation of dry microspheres, and (iv) the separation and harvesting of the dried microspheres from the drying gas [77, 78]. This

7

technique is feasible for the encapsulation of both aqueous drug solution [52] and micronized drug particles [66] in the alginate phase, resulting in the formation of microcapsule. Alginate is one of the natural polymers suitable for the encapsulating shell of microcapsule using the spraydrying method. This approach is capable of producing drug encapsulated AMs with high encapsulation efficiency (EE). For instances, Bagheri et al. [66] investigated the formation of caffeine-loaded AMs using a Büchi B-191 mini spray dryer (Büchi, Flawil, Switzerland). The results showed that AMs with a mean diameter of around 4µm and EE of 70% were obtained. The spray drying condition used: inlet drying air temperature of 140°C, outlet drying air temperature of 80°C, feed spraying pressure of 7 bars, and nominal feeding rate (pump power) of 10%. Also, spray-drying technique was used to encapsulate metformin hydrochloride (MF) drug into AMs [52]. Marta Szekalska et al. showed that the obtained microspheres have a narrow size distribution in the range between 1.6 and 5.7 µm and a high production yield of 40-80 %. It is worth mentioning that the EE of all formulated microspheres was higher than 100%. This is explained due to the partial loss of the polymer phase during the formation of microspheres which led to the decrease of theoretical polymer mass and changed theoretical drug content to higher than the previewed ones. In addition, the spray-drying method was used to produce microspheres that have a narrow size distribution (less than 10µm) [8, 69, 79]. The particles size depends on numerous parameters such as the type of nozzle, feed rate, air flow rate, inlet temperature, aspirator rate and properties of materials [52, 66]. The homogeneous size and microspheres can be obtained by modifying the conventional spray drying atomizer which is capable of producing uniform droplets, resulting in eliminating the large quantities of hot air [1]. The drying system in spray dryer is more beneficially economical than freeze drying in the fabrication of AMs due to their low energy consumption involved to dry sample. On top of that, the solvent contamination in emulsion method can also be overcome by tailoring the air flow rate and inlet temperature. However, certain drawbacks e.g. high cost, viscosity limitation, thermal instability and low yield for small batch production have limited the utility of spray drying method in the fabrication of drug-loaded AMs. For examples, the highest possible viscosity of the alginate solution using in this technique is around 300 cps [8]. 8

3.2 Extrusion technique Extrusion technique is the simplest and widest technique used in drug-loaded AMs fabrication by ionic gelation of alginate which involves a simple diffusion and cross-linking reaction by Ca2+ [10, 80]. Therefore, the extrusion method is also known as ionic gelation method. Briefly, the core drug is dispersed in the alginate solution and then this mixture is drip into a gelling bath which contains calcium chloride (CaCl2) through a simple dripping tool as shown in Fig. 4. Here, the Na+ ions in alginate structure are replaced by the Ca2+ ions and harden the membrane to form particles. This method involves easy experimental setup with a simple dripping tool (e.g. syringe or pipette) or a complex tool e.g. stainless steel needles [77, 81]. Dripping tool Drug + Alginate solution

Alginate micro beads

Gelling solution

Fig. 4 Schematic illustration of the extrusion method In extrusion technique, the gelation of alginate is highly dependent on the concentration of CaCl2 due to Ca2+ ions are capable of binding to the carboxylic group of alginate, leading to the formation of a thermo-stable gel [57, 70]. Lin et al. [60] successfully encapsulated 90% of astaxanthin into alginate beads by adjusting CaCl2 concentrations, processing conditions of stirring rate and time, and needle size. In their study, it was found that the role of Ca2+ ions in the enhancement of drug stability in alginate matrix, more than 90% of the original amount of astaxanthin remained after 21 days of storage at 25°C. The large size particles, ranging from hundreds of micrometers to millimeters has become the main drawback of the extrusion method [70, 82, 83]. The size of microspheres depends on the alginate solution concentration, the nozzle diameter and the distance from the outlet to hardening bath [9]. Kuo-Yu Chen and Si-Ying Zeng [11] successfully encapsulated a water-soluble model drug, blue dextran (BD), into AMs by using the extrusion method through a needle with an inner gauge diameter of 0.17mm. They concluded that all the obtained microspheres had a spherical 9

shape with a smooth surface. The average diameter of microspheres was large in size of 212.9µm and drug EE was 52%. Another extrusion-based study is a process of incorporating poorly watersoluble curcumin reported by L. Hu et al. [57]. In their study, they reported that the large micro bead sizes were formed up to millimeter size as using a syringe needle gauge 18 with an inner gauge diameter approximately 0.83mm. The micro beads presented regular spherical with high alginate concentration at 1.5% and 2% w/v, whereas irregular spheres were obtained at lower alginate concentration at 1% w/v. In addition, the micro bead size decreased slightly with the increase of CaCl2 concentration. This was due to the shrinkage of micro beads induced by a higher degree of cross-linking between alginate solution and CaCl2. The improvement in shape and size of drug-loaded alginate particle has been investigated by applying an electrostatic field (e.g. 7 to 10 kV) between the positively charged needle and ground collecting solution [84-86]. 3.3 Emulsification/gelation technique The emulsification/gelation technique is feasible to fabricate microspheres at the micro-scale with that particle sizes can be less than 100µm, low cost and relatively easy experimental set up. Generally, emulsification/gelation technique involves two steps; the formation of stable polymer droplets in the emulsion system and the hardening of the droplets [9]. In this method, the gelation occurs when the alginate solution collides with the cross-linker ions. There are two types of gelation: internal and external gelation as depicted in Fig. 5. The external gelation mechanism is referred to the gelation in extrusion method in which the ions from external source diffuse into alginate droplets. This method is the most common and easiest for the encapsulation of both soluble and insoluble drugs [87]. However, external gelation produces heterogeneous alginate gels due to the gelation on the surface occurs prior to core gelation, giving the particles a rigid outer surface and soft core [9, 15]. Besides, the particle size distributions of AMs tend to be wide and they are often clustered. In contrary, the internal gelation has weak gelation so the particles are often soft and tend to have high agglomeration, leading to lower encapsulation efficiency and faster release profile [9, 88-90].

10

(a)

Ca2+

Ca2+

(b)

Ca2+

H+

● = CaCO3

Ca2+

Fig. 5 Mechanism of (a) external gelation; (b) internal gelation [88]. Copy right 2009. Reproduced by permission of Elsevier Science Ltd. The schematic of emulsification/external gelation and emulsification/internal gelation method for preparation of AMs are displayed in Fig. 6. For emulsification/external gelation process, the drug is thoroughly dispersed in aqueous alginate solution before emulsion processing. Then, the mixture is added into the oil phase which contains the suitable surfactant to form water in oil (w/o) emulsion under continuous stirring. The chemical cross-linking agent, CaCl2, is dropped slowly into the emulsion and stirring is maintained during cross-linking time. Finally, the microspheres are washed to remove the oil phase, harvested by filtering and dried at room temperature. On the other hand, in the internal gelation, an insoluble calcium salt is presented inside the alginate-drug solution before the gelation takes place and Ca2+ ions are liberated by acid in the oil phase, giving the cross-linking between Ca2+ ions and alginate. Viewing in term of particle size and size distribution aspect, Song et al. successfully produced alginate

beads

entrapping

yeast

cell

by

both

emulsification/internal

gelation

and

emulsification/external gelation technique [68]. It was reported that a narrow size distribution ranging from 35-373 µm and an average diameter of 151.1µm of beads were obtained by emulsification/internal gelation method. Meanwhile, emulsification/external gelation method produces beads with a broader size distribution ranging from 35µm to 863µm and an average diameter of 325.4µm. The bead size distributions are affected by the size of the emulsion droplets or the emulsifier e.g. the amount of emulsifier, its adsorption and concentration. Furthermore, by using emulsification/external gelation technique, the larger average diameter of 11

beads was attributed by the collision of two or more droplets of alginate and CaCl2. Vandenberg and De La Noüe studied on the EE and release profile of a model protein, bovine serum albumin (BSA) from chitosan-AMs produced by emulsification/external and internal gelation method [91]. From their results, BSA loss during the microspheres manufacturing from internal gelation (a)

Drug + Alginate solution (Water phase)

Cross-linker

Oil phase + surfactant

Emulsification Gelation

(b)

Washing Drug + Alginate solution + insoluble calcium salt (Water phase)

Acid + oil

Drying

Emulsification Gelation

Alginate droplets

Drug loaded AMs

Oil phase + surfactant

Fig. 6 Schematic illustration the emulsification/gelation method (a) external gelation; (b) internal was significantly higher than that of external gelation, at 62.4% and 4.7%, respectively. Thus, it can be seen that the drug encapsulation efficiency of microspheres via internal gelation are higher than those formed via external gelation. In addition, by using an internal gelation method, the release of BSA during 24 hour acid incubation period was significantly higher (16.3%) than that of external gelation method (2.1%). It was suggested that the homogeneity as well as smaller in size of AMs formed by internal gelation method leads to the higher release of BSA. Generally, the size of microspheres formed by emulsification/gelation method was much smaller than that produced from extrusion method. For example, Eamy Nursaliza Yaacob et al. reported AMs loaded model antigen fluorescein isothiocyanate conjugated-bovine serum albumin (FITCBSA) with the mean diameter less than 83 ± 5 µm [48] or AMs containing cystamine with size less than 1µm in diameter produced by Kyeongnan Kwon and Jin-Chul Kim [62]. While, by 12

using the extrusion method, the size of the beads was large up to millimeter size, as mentioned in section 3.2. Therefore, depending on the specific requirements in an individual application, fabrication techniques enable us to control the particle size of AMs. 4.

Factors influencing the properties of drug loaded AMs

Particles size and size distribution of drug-loaded AMs are the crucial parameters for patient safety during the application period as these features will potentially influence the product injectability, biodistribution, microsphere degradation as well as their drug release kinetics and therapeutic efficiency. Indeed, the large particles may clog small blood vessels and lead to an embolism, and drug release rate is found to be dependent on the cross-linking agent [92]. The encapsulation of drug into AMs via emulsification/gelation method involves lots of formulation variables and operation condition in order to get the desired products. Thus, it is essential to acquire an in-depth understanding of the effects of synthesized parameters on the morphologies, properties and EE of AMs. In this section, we are summarizing recent literature regarding factors that influenced the formation and characteristics of AMs, including alginate concentration, oil phase, surfactant, cross-linker concentrations, cross-linking time, stirring time, model drug and drug content in extrusion and emulsification/gelation method. 4.1 Alginate concentration Alginate concentration is referred as the most important factor that effects the characteristics of AMs e.g. the morphology, particle size as well as drug EE. It can be summarized that the mean particle size and drug EE of the AMs increase with the increase of alginate concentration [58, 60, 93-96]. The increase of alginate concentration leads to increase in viscosity of the alginate solution, which rises the interfacial tension between alginate droplets and the oil phase. Hence, larger microspheres are formed [93, 95]. For examples, Lin et al. [60] investigated the effect of alginate concentration on the particle size and drug EE by extrusion method. It was found that increasing of alginate concentration from 1% to 3% w/v led to the rise of greater particle size of microspheres from 728.7µm to 1053.4µm, respectively. The results also showed that high alginate concentration is effective in improving the drug EE which was 100% at 3% w/v of alginate concentration. These findings demonstrate that the particle size and drug EE are significantly affected by alginate concentration. For the same purpose, Bose et al. [58] produced 13

indomethacin loaded AMs by using the extrusion method. In this study, the particle size of microspheres increased from 379.3µm to 386.3µm with increasing alginate concentration from 1% to 2% w/v, respectively due to the concurrent increase of alginate viscosity. It was reported that increasing alginate concentration leads to increasing amount of entrapped drugs. This could be explained by the enhancement of gelling between cross-linking and sodium alginate. Similar finding was also reported in the study of Shukla et al. [93] in which diloxanide furoate (DF) loaded AMs were prepared by emulsification/gelation method. The results showed an increase in particle size from 417.8µm to 453.9µm when the alginate concentration was increased from 3% to 5% w/v, respectively. The higher the alginate concentration, the more viscous dispersion becomes, which forms larger alginate droplets and consequently larger microspheres. The drug EE increased from 48.6% to 63.9% with the increase of alginate concentration. Rajinikanth et al. [94] successfully encapsulated metoprolol tartrate in AMs by emulsification/gelation method. In the report it was found that the particle size of microspheres was proportionally increased from 55.3µm to 84.5µm as the alginate concentration increased from 2% to 6% w/v, respectively. Furthermore, high concentration of alginate (6% w/v) resulted in the formation of discrete microspheres, while aggregation of small microspheres were formed at low alginate concentration at 2% w/v. The drug EE also was found to be proportional to alginate concentration, and reached 91.8% at 6% w/v of alginate concentration. In another study, Silva et al. [95] prepared insulin-containing AMs via emulsification/gelation method. It was concluded that an increase of alginate concentration from 2% to 3% w/v resulted in a significant increase in microspheres mean size from 53.0µm to 112.2µm, respectively, but it was not in the case of drug EE. Kailas et al. [96] reported an increase in particle size of domperidone encapsulated AMs from 57.6µm to 65.3µm with increasing of alginate concentration from 5% to 7% w/v, respectively due to the formation of large microspheres. It can be concluded that the mean particle size and drug EE of AMs are increased as alginate concentration increased as summarized in Table 2.

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Table 2 Effect of alginate concentration on the mean particle size and drug EE of AMs Alginate concentration (% w/v) 1 2 3 1 2 3 4 5 2 4 5 6 2 2.5 3 5 6 7

Drug

Astaxanthin Indomethacin Diloxanide furoate

Metoprolol tartrate

Insulin

Domperidone

Mean particle size (µm)

Encapsulation efficiency (%)

728.7 ± 194.3 885.6 ± 112.6 1053.4 ± 99.7 379.3 ± 2.6 386.3 ± 2.1 417.8 ± 3.3 424.6 ± 3.4 453.9 ± 3.2 55.3 ± 18.9 72.7 ± 21.7 79.8 ±16.7 84.5 ± 20.8 53.0 ± 12.9 97.1 ± 44.3 112.2 ± 79.1 57.6 ± 2.1 63.2 ± 4.3 65.3 ± 3.1

82.5 ± 4.2 93.3 ± 2.0 100.0 ± 4.2 64.2 ± 2.8 79.6 ± 1.3 48.6 ± 1.1 47.6 ± 0.6 63.9 ± 0.5 77.3 86.8 88.1 91.8 75.8 ± 1.8 77.2 ± 1.5 77.9 ± 2.8 25.4 ± 0.5 38.4 ± 0.4 50.7 ± 0.4

Ref.

[60] [58] [93]

[94]

[95]

[96]

4.2 Oil phase Various oils e.g. liquid paraffin [7, 59, 97, 98], soybean oil [99], olive oil [100, 101], sunflower oil [102, 103] have been reported in the fabrication of AMs via emulsification/gelation method. Among them, liquid paraffin is the widest oil phase used in fabrication of AMs. However, the effect of oil phase on the characteristics of AMs has not been widely investigated. It was reported that the viscosity of the oil phase affects the size and uniformity of microspheres. For instance, Wang et al. [104] reported that the obtained particles were larger in size with broader size distribution with the present of olive oil (the oil phase in water-oil emulsification) as compared to liquid paraffin oil due to the viscosity of olive oil was higher compared to liquid paraffin oil. Thus, the study demonstrated that the size and size distribution of the microspheres is dependent on the viscosity of oil phase.

15

Moreover, rapeseed oil (RPS) and rapeseed methyl ester (RME) with different viscosity were used as oil phase to fabricate alginate beads [105]. The results showed that smaller beads were produced by using RME rather than RPS. This was due to the lower viscosity of RME as compared to the RPS. For instance, the kinematic viscosity at 25oC of RME and RPS are 6.49 mm s-2 and 62.63 mm s-2, respectively. However, in the case of a constant viscosity ratio between oil phase and water phase, the emulsion droplet diameter did not change and consequently particle sizes of the microspheres did not change [106]. Therefore, these studies demonstrate that the particle size and size distribution of AMs can be tailor by the ratio of oil phase viscosity to water phase viscosity. 4.3 Surfactant In emulsification/gelation and extrusion processes, surfactants play a crucial role in the formation of the microspheres. The role of surfactant is to lower the interfacial tension between hydrophilic and hydrophobic molecules, leading to stable emulsions and prevent the emulsion droplets from coalescing for the production of discrete microspheres [107]. An effective way of picking the proper emulsifier is through hydrophilic-lipophilic balance (HLB) indicator. Emulsifiers with HLB in the range of 8-18 are hydrophilic and therefore, it is suitable for oil in water emulsion. While the water in oil emulsifiers where the HLB values ranging from 3.5 to 6 is known as lipophilic surfactants [108]. Fig. 7 (a) shows the components of emulsifier that contains a lipophilic group (tail) and a hydrophilic group (head). Thus, emulsifiers are attracted to both polar and nonpolar components. When a lipophilic surfactant was added to water in oil emulsion, nonpolar tails may extend outward into the oil phase, while polar head groups remain in contact with the water phase [9, 108] (Fig. 7(b)). Consequently, the droplets are stable in the emulsion and prevent them from agglomeration. Additionally, the surfactant also helps to modify and smoothen the surface of microspheres [13].

16

(a)

(b)

Oil phase

Lipophilic (tail) Alginatedrug droplet Hydrophilic (head)

Fig. 7 (a) Schematic structure of surfactant and (b) Mechanism of the lipophilic surfactant Various hydrophobic emulsifiers such as Span 80, Span 85, Polyglycerol polyricinoleate (PGPR) are reported as surfactants in AMs fabrication. The dissolubility in oil of emulsifiers indicates a need for thorough understanding of physical, chemical properties of surfactant before involving into the fabrication. For instance, PGPR is well dissolved in sunflower and corn oil [103], whereas, Span 80 is the most common surfactant in liquid paraffin because Span 80 effectively dissolve in liquid paraffin [13, 98, 109]. Viewing from particle size aspect, most of the literature showed that increased in surfactant concentration reduced the droplet sizes, consequently, smaller sizes and size distribution of microspheres were formed [9]. M. Alnaief et al. evaluated the effect of Span 80 concentration on the size of droplet emulsion for AMs using emulsification/gelation process [109]. They concluded that the average particles size significantly decreased from 909µm to 22µm when increased the surfactant content from 0 % to 3 % w/v. Increasing the Span 80 concentration led to a decrease of the interfacial tensions between alginate and oil phase of the emulsion and thus the average size of the microspheres decreased [109]. A similar finding was reported by Márquez et al. in which the smaller droplet diameter are produced with higher surfactant concentration [110]. 4.4 Cross-linking concentration

17

As mentioned in section 1, divalent cations including Ca2+, Sr2+, Ba2+ ions have been reported as cross-linker for the formation of AMs. However, Sr2+ and Ba2+ ions are mildly toxic, whereas Ca2+ ions are nontoxic. Thus, Ca2+ ions are the most widely used agent to cross-link alginate [15, 111]. When Ca2+ ions are introduced to alginate polymer solution, Ca2+ ions are located between electronegative alginate molecules and form a reticulated structure which is known as the ‘eggbox’ model. Hence, it is easier to control the release profile of active agents in a sustained release system for various drugs, proteins, and cells by tailoring the cross-linking concentration [62, 98]. CaCl2, Ca-EDTA or CaCO3 is used as the source of Ca2+ ion in the emulsification/gelation process [99]. The effect of the CaCl2 concentration on the characteristics of blue dextran loaded AMs was investigated by Baimark and Srisuwan [23] via emulsification/gelation method. It was found that the microspheres were agglomerated at low Ca2+ concentration of 0.5% and 1.25% because the cross-linking concentration is not enough to harden the microspheres. Average particle size also slightly decreased from 123µm to 110µm as the Ca2+ concentration increased from 2.5% to 10% w/v, respectively. A sensible reason for this outcome can be elucidated by the high cross-linking concentration led to a lower swelling of the alginate during the gelation process. It was also reported that there was a rise in EE from 77% to 82% when CaCl2 concentration increased from 2.5% to 10%, respectively. This may be explicated by the higher Ca2+ concentration inducing faster hardening of the AMs which may hinder the diffusion of drug out of the alginate droplets during crosslinking process. Rastogi et al. [98] have prepared isoniazid-loaded AMs by emulsification/gelation method. In the report, it was emphasized that the cross-linking concentration of 7.5% was the optimum condition for the encapsulation of isoniazid drug because further increase in CaCl2 concentration resulted in decreasing the EE. This was explicated by the instant gelling of sodium alginate in addition of CaCl2 and squeezing out of the aqueous phase along with the hydrophilic drug from the gel. Hu et al. [57] investigated the effect of CaCl2 concentration on curcumin loaded alginate microbeads by extrusion method. It was found that the effect of cross-linking concentration on the particles size of alginate beads was similar to that of using emulsification/gelation method [98]. The study also demonstrated that the particle sizes of micro-beads slightly reduced with the increase of CaCl2 concentration. This may be due to the shrinkage of beads at a high degree of 18

cross-linking between the alginate and CaCl2. In case of EE, it was reported that high CaCl2 concentration of 25.0% has a positive effect on the rise of EE of up to 98.8% due to the presence of more cross-linked network. Cho et al. [112] successfully encapsulated resveratrol in AMs via extrusion method. The mean particle size of microspheres decreased from 244.0µm to 217.3µm as CaCl2 concentration increased from 0.5% to 1%, respectively due to the rigid gel network. In summary, increasing of CaCl2 concentration leads to increase mean particle size and drug EE, but high concentration of CaCl2 may reduce the EE of AMs as shown in Table 3. Table 3 Effect of CaCl2 concentration on the mean particle size and drug EE of AMs

Method

Drug

Emulsification/gelation

Blue dextran

Emulsification/gelation

Isoniazid

Extrusion

Curcumin

Extrusion

Resveratrol

CaCl2 concentration (% w/v) 2.5 5 10 7.5 10 20 2.0 13.5 25.0 0.5 1

Mean particle size (µm)

EE (%)

123 ± 42 119 ± 58 110 ± 46 244.0 217.3

77 81 82 79.9 50.5 44.9 98.1 98.7 98.8 96.3 98.5

Ref.

[23]

[98]

[57] [112]

4.5 Cross-linking time Generally, the cross-linking time had no significant influent on the morphology of AMs in both extrusion and emulsification/gelation method [70, 96]. However, the particle size of AMs increased with the increase of cross-linking time [12, 96]. This can be due to the extent of crosslinking between Ca2+ ions and the guluronic acid units of sodium alginate, leading to the formation of larger alginate particles. In addition, the cross-linking time had a strong influence on the EE of drug-loaded AMs. For example, Ziyaur Rahman et al. [113] reported a significant decrease in EE from 84.31% to 46.52% as the cross-linking time increased from 10 to 30min, respectively. The reason for this is that increasing gelation time allowed more Ca2+ ions to diffuse into the microspheres and these ions displace the drug, hence decreasing drug EE. On the

19

other hand, short cross-linking time resulted in incomplete gelling of alginate solution and consequently, the drug EE was decreased [98]. Lin et al. [60] have prepared astaxanthin encapsulated alginate beads by extrusion method. It was reported that there was an increment in mean particle size of beads from 1029.2µm to 1112.9µm as the cross-linking time increased from 15 to 60 min, respectively. On the contrary, by evaluating the effect of cross-linking time on the characteristics of Lactobacillus acidophilus loaded alginate beads using extrusion method, Lotfipour et al. [70] found that the cross-linking time had no significant effect on the size of beads. For instance, as cross-linking time increased from 15min to 60min, the beads size rose from 1.34mm to 1.35mm, respectively. Rajinikanth et al. [94] successfully encapsulated metoprolol tartrate in AMs using emulsification/gelation method. The results showed that the mean particle size of the microspheres increased from 55.3µm to 74.5µm with increasing cross-liking time from 5min to 25min, respectively. This can be explained by the extent of cross-linking between Ca2+ ions and the COO- groups of sodium alginate as increasing the cross-liking time. Consequently, the viscosity of the alginate phase increases and leading to the formation of large microspheres. The drug EE showed an inverse behaviour with increasing cross-linking time. For instance, as crosslinking time increased from 5min to 25min, the drug EE decreased from 77.3% to 49.9%, respectively. This may be explained by the prolong cross-linking time, the microspheres size is increased, however, the increased or decreased of drug EE depends on the type of drug encapsulated as well as the fabrication method of AMs. 4.6 Stirring speed The stirring speed is often used to control particle sizes and size distribution of AMs. In general, high stirring speed produced finer microspheres due to more energy is provided for the dispersion between the oil phase and the water phase in the emulsion [67, 93]. For instance, Ahmed et al. affirmed that stirring speed is the most crucial parameter which controlled microspheres size from the drug-alginate solution in the oil phase. They elaborated that at low stirring speed of 200 rpm produces large size microspheres (890-630 µm), while at higher stirring speed of 600 rpm produces finer sized microspheres (160-100 µm). It was explained that increasing the stirring speed resulted in stronger sheer force of the stirrer blade and increases 20

turbulence in the emulsion and subsequently, producing smaller size emulsion product [71]. Similarly, the finding of particle size was reported by M. Alnaief et al [109] and they explicated that increasing the stirring speed from 200 rpm to 1400 rpm gave high energy input to form larger interfacial surface area, thus the dispersed droplets became finer and the microspheres were smaller in size as shown in Table 4. On the other hand, it was found that the EE decreases from 73.1% to 64.8% with increasing stirring speed from 1000 to 1500 rpm [93]. This was because a higher stirring rate led to more diffuse of curcumin from alginate phase into the oil phase; hence the entrapment efficiency was decreased. Table 4 Variation of particle size of AMs at different stirring speeds [109] 200 rpm

400 rpm

600 rpm

1400 rpm

D32 (µm)

168 ± 16

177 ± 16

145 ± 16

34 ± 4

D10 (µm)

67 ± 10

80 ± 22

56 ± 6

15 ± 4

D50 (µm)

463 ± 46

508 ± 46

381 ± 70

60 ± 10

D90 (µm)

1139 ± 140

1003 ± 50

1066 ± 150

151 ± 6

Average (µm)

547 ± 32

534 ± 44

486 ± 100

75 ± 10

4.7 Model drug and drug content Over the past 10 years, various types of drugs have been incorporated successfully in AMs system as shown previously in Table 1. Most of the drugs have high water solubility and therefore it is easier to obtain homogeneous dispersion of the drug in alginate solution prior to emulsification. Furthermore, highly water-soluble drugs will prevent the diffusion of the drug from the alginate phase to oil phase during fabrication, leading to enhance the drug entrapment efficiency [114]. However, it was also reported that some slightly hydrophilic drugs e.g. curcumin and DF were also able to be encapsulated in AMs with high EE [57, 93]. In general, the drug content is another important factor which impacts on the drug EE. An increase in drug content will improve drug encapsulation efficiency. However, a high proportion of drug content results in a decline of EE. This is because when the drug amount exceeds the loading capacity in microspheres system, the overloading drugs will transfer to the oil phase [9, 115]. By using emulsification/gelation method, Shukla et al. [93] reported that the entrapment 21

efficiency of DF loaded AMs decreased from 73.8% to 51.4% when DF content increased from 100 mg to 200 mg. They also stated that the size of AMs could be tailored by changing the DF content. Salunkhe et al. [64] also demonstrated the influence of drug-loaded on the morphology of AMs by using emulsification/gelation method whereby a smooth microsphere was changed into a rough surface after the introduction of granisetron hydrochloride (GH) drug as displayed in Fig. 8. Table 5 summarizes the factors that can be tailored to obtain the desired properties of AMs for specific drug delivery during the fabrication process.

(a)

(b)

Fig. 8 SEM images of (a) blank alginate microspheres and (b) GH-loaded microspheres [64]. Copy right 2014. Reproduced by permission of Springer Nature.

Table 5 Summary of factors that influence the formation and properties of AMs Factors Alginate concentration

Brief description Low alginate concentration: an agglomeration of microspheres Increasing alginate concentration resulted in: - increased in particle sizes - broadening of the size distribution - increased in EE

References [58, 60, 93-96]

Oil phase

Depend on the viscosity of types of oil Affect to the uniformity and size of microspheres Improvement characteristic of microspheres surface and stable emulsion Increasing surfactant concentration resulted in: - decreased in particle sizes - narrowed of the size distribution

[104-106]

Surfactant

22

[9, 13, 107-110]

Cross-linking (Ca2+) concentration

Low Ca2+ concentration: agglomeration of microspheres Had no significant influent on the particles size Increasing Ca2+ concentration resulted in: - increased slightly in EE - then decreased in EE at Ca2+ concentrationoverloaded

[23, 57, 98, 112]

Cross-linking (Ca2+) time

No significant influent on the morphology of microspheres Increasing Ca2+ time resulted in: - increased in particle sizes - significantly decreased in EE

[12, 60, 70, 94, 96, 98, 113]

Stirring speed

Increasing stirring speed resulted in: - decreased in particle sizes - decreased in EE

[67, 71, 93, 109]

Model drug and drug content

Encapsulation both water-soluble and water-insoluble drug Increasing initial drug content resulted in: - increased in EE - then decreased in EE at drug-overloaded - minor changes in particle sizes

[9, 57, 93, 114]

5.

Drug release profile

Drug release of AMs is mainly dependent on the properties of the alginate matrix as well as the encapsulation of drug in polymer system, including polymer microcapsules and polymer micro matrix. In the polymer microcapsules system, the polymer membrane with a specific permeability controls the drug release. This is due to the drug is encapsulated within a polymeric membrane as a form of solid or liquid or suspension, as shown in Fig. 3. Therefore, the mechanism of drug release from alginate microcapsules system was divided into two main pathways: drug release via the degradation of alginate network and the diffusion of drug through alginate network [116]. The degradation of alginate network could cause a dramatical release of the drug; consequently, it is not suitable for controlling the release of the drug. Hence, alginate microcapsules have widely been applied in the drug delivery system based on the diffusion pathway, where the drug diffuses out through the swell able polymer network [116, 117].

23

In the polymer micro matrix system, the drug release mechanism is mainly based on the combination of diffusion and degradation of polymer matrix. When AMs are exposed to dissolution medium, the diffusion of the drug occurs through matrix swelling and results in the dissolution/erosion at the edge of the microspheres [117]. In the microspheres system of sodium alginate cross-linked with CaCl2, the swelling behaviour is a result of the ionic exchange of Ca2+ ions with Na+ ions, which is presence in the medium. It increases the electrostatic repulsion of the carbon group and the relaxation of the polymer chains, as a consequence of the swelling and water absorption [118, 119]. In addition, the drug release kinetics and the release mechanism from drug loaded AMs can find out by fitting the release results into different models including zero order, first order and Higuchi model. For instant, the zero-order model ( Qt = K0 t ,where Qt is the amount of curcumin release in time t, K0 is the zero-order release constant and t is the time) describes the release of drug is only a function of time. This means the data obtained were plotted as the cumulative amount of drug released versus time and the release rate constant is independent of drug concentration. In the first-order model ( log C = log C0 -

Kt , where C0 is the initial 2.303

concentration of curcumin, K is the first-order rate constant and t is the time), the plots were plotted as log cumulative percentage of drug remaining versus time and the release rate constant is dependent on drug concentration. The Higuchi model ( Q = K H t , where Q is the amount of curcumin release in time t, KH is the release constant of Higuchi and t is the time) describes the drug release from the matrix system as a cumulative percentage drug release versus the square root of time, presenting a linear function. This model is indicating that drug release from AMs followed by diffusion controlled mechanism [19, 25, 120]. According to literature, drug release rates as well as time administration are known to be impacted by various factors, including drug and polymer characteristic, particle size and size distribution of microspheres, drug-polymer ratio and release medium [9, 121]. Besides, crosslinking has also been displayed to prolong drug lifetime within the body [121]. For example, the release rates of drug in AMs were found inversely proportional to the microsphere sizes. Since particle sizes decreased leading to the increase in surface area between particles, subsequently the release rates of drug out of the microspheres increased [9]. Soni et al. [122] showed that 24

increased in the alginate concentration resulted in the prolonged release of theophylline from alginate particles due to an increase in particle size as shown in Fig. 9. A study conducted by S. Mandal et al [123] showed that drug release behavior was affected by CaCl2 cross-linking concentration. It was found that a greater sustained release profile was achieved with the increased of CaCl2 concentration due to the formation of a harder gel network.

Fig. 9 Percentage cumulative in vitro Theophylline release from AMs in pH progression medium at different alginate concentration of 2, 4, 6, 8 % w/v, which designated as F1, F2, F3, F4 at, respectively, [122]. Copy right 2010. Reproduced by permission of Elsevier Science Ltd. 6. Conclusion AMs appear to be a potential biomaterial in novel drug delivery system due to alginate’s biocompatibility, degradable properties and gel-forming ability. Controlled and targeted drug delivery of AMs can be achieved via well-designed formulation as well as synthesis parameters involved in the fabrication process. Hence, this review approaches AMs through understanding some related characteristics of AMs and recent strategies in fabrication technique. Extrusion technique is a simplest and widest technique in alginate fabrication but micro beads obtained are large in size, ranging from hundreds of micrometers to millimeters. Spray-drying technique produces particles of small average diameter (less than 10µm) with a narrow size distribution. 25

However, the main drawback of this method is it is costly when compared to extrusion and emulsification/gelation technique. Whereas, emulsification/gelation method is the most feasible to fabricate and control microspheres at the micro-scale with that particle sizes can be less than 100µm at low cost and relatively easy experimental set up. During the fabrication process, the morphologies, particle sizes and size distribution and drug EE of AMs are influenced by numerous parameters e.g. alginate concentration, type of oil phase, surfactant and cross-linker concentrations, cross-linking and stirring time and model drug and drug content. The rate and extent of drug release, for instance, are controlled by drug and polymer characteristic, particle sizes and size distribution of microspheres, drug-polymer ratio and release medium. Additionally, cross-linking has also been shown to prolong drug lifetime in the body. With the comprehensive understanding of fabrication techniques, microsphere-based alginate is expected to contribute enormously to the drug delivery application. Acknowledgments This work is financially supported by Universiti Sains Malaysia Bridging Grant (Grant No. 304.PBAHAN.6316318) and partially supported by Japan International Cooperation Agency (JICA) through AUN/SEED-Net project (Grant No. 304.PBAHAN.6050388.J135). References [1]

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