Synthesis of micro- and nanoparticles of alginate and chitosan for controlled release of drugs

C H A P T E R 16

Synthesis of micro- and nanoparticles of alginate and chitosan for controlled release of drugs ´nica E. Manzano1, 2, Norma B. D’Accorso1, 2 Marı´a Natalia Pacho1, 2, Vero 1

Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Quı´mica Orga´nica, Ciudad Universitaria, Ciudad Auto´noma de Buenos Aires, Argentina; 2Centro de Investigaciones en Hidratos de Carbono (CIHIDECAR)-CONICET-UBA, Ciudad Auto´noma de Buenos Aires, Argentina

Chapter Outline List of abbreviations 364 1. Introduction 365 1.1 Micro and nanoparticles 365 1.1.1 Polymeric nanoparticles: advantages 367 1.1.2 Loading and realease of drugs 367 1.2 Nanoparticle sources: polymers 368 1.3 Alginate 371 1.4 Chitosan 373

2. Smart polymers in drug delivery 374 2.1 2.2 2.3 2.4

Temperature-responsive polymers pH-responsive polymers 378 Bioresponsive polymers 378 Field-responsive polymers 379

3. Preparation of nanoparticles

377

379

3.1 Dispersion of preformed polymers 380 3.1.1 Emulsificationeevaporation method (EEM) 380 3.1.2 Nanoprecipitation or solvent displacement procedure 380 3.1.3 Emulsification/solvent diffusion 381 3.1.4 Salting out 381 3.1.5 Dialysis 382 3.1.6 Supercritical fluid (SCF) technology 382 3.2 Polymerization methods 383 3.2.1 Emulsion polymerization 383 3.2.2 Interfacial polymerization 384 3.2.3 Controlled/living radical polymerization (C/LRP) 384 Natural Polysaccharides in Drug Delivery and Biomedical Applications. https://doi.org/10.1016/B978-0-12-817055-7.00016-9 Copyright © 2019 Elsevier Inc. All rights reserved.

363

364 Chapter 16 3.3 Ionic gelation or coacervation

385

4. Alginate and chitosan nanoparticles

385

4.1 Alginate nanoparticles 385 4.1.1 Nanoaggregates of alginate synthesized by self-assembly and complexation 385 4.1.2 Formation of alginate nanocapsules by complexation on the interface of emulsion droplets 4.1.3 Alginate nanosphere formation from water-in-oil emulsions 386 4.2 Chitosan micro- and nanoparticles 387 4.2.1 Chitosan nanofibers 389

5. Biomedical applications as drug delivery systems 5.1 Alginate nanoparticles 390 5.2 Chitosan nanoparticles 392 5.3 Chitosanealginate nanoparticles

394

6. Conclusion 394 References 395

List of abbreviations AgNPs ATRP bFGF BSA CasII-ia C/LRP CR CS CS NPs DDS DNA EEM EMA EPR FITC-dex FDA G GSH INH L LCST M MPS MS NMP NO NP O PAAc PCL

Silver nanoparticles Atom transfer radical polymerization Fibroblast growth factor Bovine serum albumin Casiopein III-ia Controlled/living radical polymerization Controlled release Chitosan Chitosan nanoparticles Drug delivery system Deoxyribonucleic acid Emulsificationeevaporation method European Medicine Agency Electron paramagnetic resonance Fluorescein isothiocyanate dextran Food and Drug Administration Guluronic acid Glutathione SH Isoniazid Drug loading capacity Lower critical solution temperature Mannuronic acid Mononuclear phagocytic system Mo¨ssbauer spectroscopy Nitroxide-mediated polymerization Nitric oxide Nanoparticle Oil Poly(acrylic acid) Polycaprolactone

390

386

Synthesis of micro- and nanoparticles of alginate and chitosan 365 PEG PEO PET PLLA PGA PLGA PMAAc PPO PVA PZA RAFT RESS RIF SAS SCF TPP UCST W

Poly(ethylene glycol) Polyethylene oxide Polyethylene terephthalate Poly(L-lactic acid) Polyglycolides Poly(lactic-co-glycolic acid) Poly(methacrylic acid) Polypropylene oxide Poly(vinyl alcohol) Pyrazinamide Reversible addition and fragmentation transfer chain polymerization Rapid expansion of supercritical solution Rifampicin Supercritical antisolvent Supercritical fluid technology Tripolyphosphate Upper critical solution temperature Water

1. Introduction 1.1 Micro and nanoparticles In the past decades, the scientific curiosity in nano- and microparticles has increased, because of the potential application in many fields, such as medical, optical, electronic, sensors, biotechnology, pollution control, and environmental areas. These attractive particles constitute a bridge between bulk and molecular materials, and consequently, their properties are different. For instance, they exhibit a big surface to mass ratio, interesting properties in the micro- and nanoscale, and ability to adsorb and transport compounds, such as drugs, probes, proteins, or antigens [1]. The difference between nanoparticles (NPs) and microparticles, lies in the size range, that is, the former are of nanometer size and the latter are of micrometer size [2]. In this sense, in terms of drug delivery applications, micro- and nanoparticles have become an attractive target. The ability to transport different types of drugs to varying areas of the body crossing natural and artificial membranes for sustained periods of time makes them a good choice for this application. Thus, the fact that these particles are in the scale of the nanometers allows efficient permeation through membranes in biological cells and makes them stable in bloodstream. Micro- and nanoparticles are usually designed and tailored so as to obtain specific compositions, size, and functionalities, making them useful for different applications [3]. It is important to highlight that for uses in nanomedicine, the design of systems is important, consisting of at least two components, composed of a pharmaceutically active compound and of a nanocarrier usually made of a polymer matrix. Many different drugs can be transported using carriers in the micro- and nanosize using different routes. These particles can be used to transport hydrophilic and hydrophobic active compounds, and

366 Chapter 16 Drug

10-1000nm

Nanoespheres

Nanocapsules

Figure 16.1 Nanosphere versus nanocapsule.

biomacromolecules, among others. Also, they can be engineered to deliver the pharmaceutical active ingredient to different targets such as lymphatic system, brain, arterial walls, and varied organs. Also, they may be designed to make them available for long periods in the systemic circulation [4]. Therefore, there exist a great number of protocols for the synthesis of nanoparticles depending on the drug type to be delivered and the desired target and route of administration. Once a procedure is selected, the parameters must be adjusted so as to generate the better characteristics for the particles, for instance, size, encapsulation efficiency, surface charge, and release features [4]. From now on, we will focus on the different nanoparticles in this chapter. It is desirable for polymeric nanoparticles (NPs) to be prepared from polymers that are proved to be biodegradable and biocompatible and of ranging between 10 and 1000 nm of diameter. Active or biological compound is, then, dissolved, captured, encapsulated, or attached to a nanoparticle matrix. This depends on the formulation method used for the synthesis of the nanoparticles, nanospheres, or nanocapsules (Fig. 16.1). In the case of nanocapsules, the drug is confined to a cavity surrounded by a polymer surface, whereas in nanospheres, the drug is dispersed in a uniform way in the matrix systems as a consequence of a physical distribution. In the past years, polymeric NPs, especially the ones derived from the biomass that are usually biodegradable, have emerged as potential drug delivery devices considering their applications in controlled release (CR) of drugs, the targeting ability to develop the drug to certain organs or tissues, the application as transporters of DNA, in applications such as gene therapy, and the capacity to carry the active drug through an oral route of administration [5]. In medical application, nanoencapsulation of active compounds increases the efficiency of the drug and enhances the acceptability and specificity, thus improving the therapeutic index of the drugs [6]. This work reviewed the basic principles of controlled release by intelligent polymers and nanoparticles made of calcium alginate and chitosan. It also includes their synthesis, as

Synthesis of micro- and nanoparticles of alginate and chitosan 367 well as the application of new syntheses developed for the CR of different drugs for the treatment of some diseases and disorders such as cancer, diabetes, and tuberculosis. 1.1.1 Polymeric nanoparticles: advantages Drug delivery systems (DDSs) are the chosen option if a specific drug or therapeutic is instable, making its administration difficult. These carriers must accomplish certain characteristics such as ability for drug association, enhanced physical and chemical stability, preservation of the drug/s encapsulated, and a regulated/sustained drug release profile. There are many advantages for the use of polymeric NP drug carriers; several of them are listed below [1,6,7]. As mention before, nanoparticles for drug delivery have certain advantages such as follows: • • • • • • • •

Protection of premature degradation Bioavailability Improved stability in volatile drugs Low cost of fabrication in large quantities Easy manufacture Improvements over traditional methods of administration in reference to efficiency Delivery of the agent to target locations in higher concentration Application together with other activities, for example, tissue regenerative engineering

It is important to highlight that the choice of the biopolymer used for the fabrication of the nanoparticles and the capacity of varying the way of releasing the drug make these nanoparticles ideal candidates for several applications such as cancer therapy, transport of vaccines, contraceptives, and antibiotics. 1.1.2 Loading and realease of drugs In the area of nanomedicine, especially in drug delivery, a successful system of nanoparticles is defined as the system having a high loading capacity so that it reduces the amount of the carrier necessary in the administration. Drug loading into the NP is accomplished by two different ways: the first one is incorporating the active compound at the same time of the production of the NP, and the second way of incorporation is by introducing the active compound after the formation of the nanoparticles. This is usually done by incubating the particles in the solution containing the drug. As a consequence, the drug entrapped is higher in the incorporation technique in contrast to the adsorption procedure. For the efficacy of NP/drug delivery system, it is important to have vital information such as drug binding capacity, which is obtained from adsorption isotherms, adsorption aptitude related to the hydrophobic balance in the polymer, and the specific area of the particles that usually followed a Langmuir mechanism [8].

368 Chapter 16 The parameter that characterized the drug loading efficiency is L (%w/w), which is defined as the percentage of drug entrapped in the polymer nanoparticle in comparison with the initial amount of drug.  w Final drug concentration  Drug concentration in the supernatant L % ¼ w Polymer final concentration Drug release (DR) from nanoparticles and the subsequent biodegradation of the matrix are key aspects in the development of successful formulations. The rates of releasing from the particles depend on several aspects, such as desorption of the drug bounded or adsorbed; diffusion or erosion, depending on the case, through the NP polymer wall or matrix. Thus, the profile for releasing the drug is governed by two different processes, diffusion and biodegradation, as mention before. Moreover, they are influenced by the type of polymer used as matrix for the drug delivery purposes. The polymeric drug carriers transport the drug by one of the following physicochemical mechanisms [9]. These mechanisms are summarized below: 1. The release of the drug is produced by the swelling with water of the NP, which as a consequence generates the liberation of the active compound. 2. The release of the active compound (drug) trapped in the polymeric matrix, in this case, involves an enzymatic reaction, which breaks or degrades the polymer in the target place. 3. The release involves a dissociation of the drug from the NP matrix. As mention before, when matrix polymeric NPs are used for drug delivery, the active compound is homogenously distributed (nanospheres) and the release occurs either by diffusion or by degradation of the polymer matrix, depending on the rate/velocity of each process. It is interesting to note that, in many cases, a fast initial release of the drug, which could be explained by superficial drug, which is adsorbed over the external layer, is observed. Then the rate of releasing is governed by the diffusion from the matrix, which causes an exponential delayed release rate. The release in nanospheres follows the firstorder kinetics. In contrast, release of the active compound from nanocapsules is through diffusion. As the drug core is covered with a polymeric layer, diffusion through this barrier is the mechanism governing the release. Theoretically, zero-order kinetics is followed.

1.2 Nanoparticle sources: polymers In the area of medical application, polymers gain considerable attention because they are a suitable material for the design of numerous and varied molecular systems that may be incorporated into some constructions in the nanoscale order. The composition of the

Synthesis of micro- and nanoparticles of alginate and chitosan 369 Table 16.1: Polymers used in drug delivery. Classification

Polymer Natural polymers

Proteins Polysaccharides

Gelatin, collagen, elastin, albumin Agarose, alginate, cellulose, dextran, cyclodextrins, and chitosan Cellulose derivatives (carboxymethyl cellulose, cellulose acetate) Synthetic polymers

Polyesters Polymers containing phosphorous Anhydrides Acrylic polymers Others

poly(lactic-co-glycolic acid), poly(εcaprolactone), poly(hydroxy butyrate) Polyphosphates, polyphosphonates Poly(sebacic acid), poly(adipic acid), poly(terephthalic acid), etc. poly(methylmethacrylate), polymethacrylates Polydimethylsiloxane, polyvinyl pyrrolidone, ethyl vinyl acetate, poly(cyano acrylates), polyurethanes

engineered NPs varied depending on the source material used. Nanomedicine formulation depends on the choice of suitable polymeric system with desirable characteristics such as high efficiency of encapsulation, enhancement of bioavailability, and retention time. The most common sources of NPs may be divided in the ones coming from biological origin such as polysaccharides and other biological polymers, and that of synthetic origin, either degradable or nondegradable, which are summarized in Table 16.1. As it is well known, there are a number of examples of the use of natural polymeric nanoparticles [10e12] used as vehicles for controlled release of drugs. Biodegradable NPs, especially those from the biomass, are frequently used to increase the therapeutic worth of some medicinal and pharmaceutical drugs (soluble or insoluble in water) and bioactive molecules. The most known vantage relies on the reduction of risks of toxicity and the patient costs. As mentioned in Section 1.1.1, there exist many advantages of encapsulating medicinal drugs, especially the protection of premature degradation and enhancement of absorption, among others. The polymeric matrix uses in nanomedicine must meet certain characteristics mentioned before (high encapsulation efficiency and bioavailability). These nanomedicines are accomplished by hit and trial method. It is important to note that when the polymeric matrix is chosen, the capacity of absorption is then established, because it depends on the polymer. In terms of control release, desired delivery place, and therapeutic impact, the

370 Chapter 16 nanoformulations of drugs are clearly superior compared with traditional nonnanoencapsulated medicine. The factor that influenced the delivery of the nanomedicines to the target place/organ is the size of the particle, surface charge, surface structure, and hydrophobicity. A significant feature in the design of these particles is the size and its polydispersity index, because these aspects will entirely determine the interaction with cell membranes and whether the NPs are capable of crossing physiological barriers [13]. Also, the surface charge is a key aspect for the internalization in the cell of NPs because it determines whether they will stay in the blood flow or will interact with cellular membranes oppositely charged [14]. Commonly, to increase the velocity of internalization, a surface positive charge (cationic) membrane is required, and thus it favors the interaction of the NPs with the cells. It is also an essential issue, the evaluation of the persistence of nanoparticles in the circulatory system. For instance, nanoparticles with hydrophobic surfaces are quickly opsonized and attacked by macrophages of the mononuclear phagocytic organs. As a result, many researchers focused their investigations on the modification of NP’s surface with different chemical structures so as to raise biodisponibility, which is increasing the time of circulation and blood persistence. An interesting approach is the coating with hydrophilic polymers, which generates a cloud of chains around the surface of the nanoparticle, which results in repulsion of proteins of the plasma [15]. To summarize, the performance of nanoparticles in vivo and in vitro is also affected by the morphology, the chemical characteristics of the surface, and the molecular weight. The antiadhesive properties of nanoparticles modified in it surface reduce the degree of degradation by liver macrophages, favoring the possibility of undergoing a permeation process because the modifications in the surface act as a barrier. As mentioned before, the polymer’s molecular weight used is directly related to the mechanism of release, so modifications in it may switch the mechanism involved. For instance, polymers with high molecular weight are used in in vitro release of drugs. Based on the aforementioned, it is crucial to determine the target and the route of administration when designing the delivery systems to avoid problems when treating with new active molecules [6]. In conclusion, biopolymers such as alginate and chitosan gained great attention in the past years as candidates as encapsulants in drugs in drug delivery systems. The reasons rely on the capacity for drug association, protection of encapsulated drugs, and delivery to target places, among the others mentioned in the previous section. Thus, the use of alginate and chitosan micro- and nanoparticles has emerged as an encouraging alternative for enhancing the delivery of molecules across biological surfaces [16]. Both biopolymers are biocompatible and biodegradable, permitted by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) [17]. These make

Synthesis of micro- and nanoparticles of alginate and chitosan 371 them perfect biomaterials for this purpose. Thus, the preparation as a nanoparticulate form is well known and studied and its remarkable properties (biocompatible, very low toxicity, among others) make it a nice candidate for drug delivery in biomedicine.

1.3 Alginate Alginates are defined as the salts of alginic acid typically extracted from brown that can be formed with Na, Ca, Mg, and K, among others, forming salts with different degrees of solubility in water, which confers varying degrees of viscosity. They are an important structural component of algae cells which gives rigidity, elasticity and flexibility, as well as the capacity of absorbing water [18]. Taking into account the chemical aspects, alginate is an unbranched anionic polysaccharide composed of the two monomers, b-D-mannuronate (M) and a-L-guluronate (G) (Fig. 16.2), linked 1 / 4. The polyanionic character to the carboxyl groups appears along the chain. As G and M blocks are diastereomers (epimers) in C5, a change in the conformation of the monomer chain is observed, leading to four possible glycosidic bonds at the molecular level. The blocks of MM and MG sequences are linked by glycosidic bonds b-(1 / 4), whereas the GG and GM blocks are linked by bonds a-(1 / 4). The flexibility or rigidity of the gel formed is influenced by the guluronic content, the distribution of the monomers in the polymer chain, the charge, and the volume of the carboxyl groups. The alginates are grouped in homopolymers of G-blocks (GGGG), M-blocks (MMMM), or heteropolymers in which the M and G blocks alternate (MGMG) (Fig. 16.3) [19]. Alginates obtained from different natural sources differ in the contents M and G and the length of each block. One of the alginate derivatives with commercial value is the one obtained by esterification of alginate with propylene oxide, known as propylene glycol alginate (PrGA).

Figure 16.2 Structure of monomers.

372 Chapter 16

Figure 16.3 Schematic representation of sodium alginate blocks.

New efforts and developments in alginate area are focused on the most outstanding characteristic of alginate, which is the sol/gel transition when multivalent cations are present in the solution, making the alginate unique compared with other gelling polysaccharides. This well-studied transition requires mild conditions and is generally independent of temperature. The formation of an alginate gel is determined by the affinity of these molecules with cations and their selectivity and cooperativity. The specific binding of the ions occurs strictly in the G-blocks. When two chains of G blocks are alienated, coordination sites are formed. Because of the looping of these chains, there are cavities between them that are sized to accommodate the calcium ion and are also coated with carboxylic groups and other electronegative oxygen atoms. After calcium cations are added, the alginate undergoes changes in the conformation, leading to what is known as “egg box” (Fig. 16.4). This is based on the dimerization of the chain and, finally, on the greater aggregation of the dimers. Additionally, it is interesting to note that at pH below the uronic acid’s pKa, alginates may form acid gels bearing the G-blocks and M-blocks.

Figure 16.4 “Egg-box” structure representation.

Synthesis of micro- and nanoparticles of alginate and chitosan 373 The physical properties of the alginate and its resulting hydrogels are determined by the composition (for instance, the ratio of M/G), the sequence, the length of the G block, and the molecular weight [18].

1.4 Chitosan Among the natural polysaccharides, one of the most abundant is the chitin. It is a hydrophobic, white, inelastic polymer extracted mainly as a by-product from the fishery industry [20,21] and, as a consequence, is the primary source of contamination in coastal areas [22]. As cellulose in plants and collagen in animals, chitin is a structural biopolymer of many crustaceans shells (shrimp, crab, lobsters, for instance), mollusks, and also some insects (spiders, beetles, ants, etc) and microorganisms such as fungus, spores, and algae. Chitin is a linear homopolymer composed mainly of units of 2-acetamido-2-deoxy-b-Dglucopyranose linked 1 / 4 (structure A in Fig. 16.5). This polymer is biorenewable, biocompatible, biodegradable, and biofunctional. Interestingly, this biopolymer has high molecular weight and a porous structure, which favors high water absorption [23]. Their properties range from the use in food, biomedicine, and cosmetics industry to the treatment of effluents, agriculture, and controlled agrochemical release [21]. Although it is one of the most abundant polymers, it has a great limitation for application and this is the high insolubility in water. The deacetylated derivative of chitin is chitosan (structure B in Fig. 16.5), which is a cationic, linear, nontoxic, adsorbable, biodegradable, and biocompatible polymer. Chitosan (A)

(B)

Figure 16.5 Deacetylaction of chitin to form chitosan.

374 Chapter 16 differs from chitin in the improved reactivity and solubility in dilute acidic solutions (formic, acetic, citric, tartaric, and dilute mineral acids, among others) because of the presence of the free amino groups. The presence of these basic groups also affects the properties of the chitosan, which vary depending on the pH. Chitosan is most commonly prepared by deacetylation under concentrated alkaline aqueous conditions from native chitin at high temperature (100e160 C) and also by enzymatic treatment with deacetylase. The resulting chitin is deacetylated in a 50%e90% depending on the reaction conditions. As a consequence of the partial deacetylation, chitosan is a copolymer of glucosamine and N-acetylglucosamine. There exist several methods to get complete deacetylation, which involves several steps, as reported by Dutta et al. [22]. Beyond the chemical properties of chitosan, some of the remarkable include the high nitrogen content, high hydrophilicity, crystallinity and viscosity, and insolubility in water and organic solvents. It is used as chelating, complexing agent and also as a flocculant of charged molecules [21]. Other properties include the use as adhesives because of the ability to form films and as antiacid and blood anticoagulants. It is important to highlight that although the polymer is a weak base, the deprotonated amino group acts a powerful nucleophile. Thus, the reactive amino side groups and secondary hydroxyl groups offer great possibilities for chemical modifications, which makes chitosan a suitable biopolymer for the development of new biomaterials.

2. Smart polymers in drug delivery The great limitation of most pharmaceutical and biological therapeutics is the short halflives, bad bioavailability, and poor physical and chemical stability. It is understood that as physical instability the alteration of protein structure is usually extremely ordered. This leads to reactions of denaturation, aggregation, and/or precipitation which are not desirable [24]. Many drugs may degrade as a consequence of oxidations, hydrolysis (of esters and amides), and racemization reactions. Subsequently, the joint work of two separate areas such as the polymeric (material) science and the pharmaceutical industry makes a great contribution as they design and develop novel DDSs. With polymeric devices, temporal and/or spatial control may be achieved for drug delivery applications. The choice of the polymer matrix is an interesting task, and the great variety of existing structures require a detailed understanding of the morphology of the surface and bulk properties to achieve the desirable physicochemical, mechanical, and biological functions. Among the physical properties, those to which we must pay special attention are durability, permeability, and degradability. It is vital when choosing a polymer to prove it is safe, that is, to have a fully biochemical characterization and to provide preclinical assays. To prove biocompatibility with blood and tissues, it is vital to study the lubricity,

Synthesis of micro- and nanoparticles of alginate and chitosan 375 Table 16.2: Factors influencing biodegradation of polymers. Factors influencing biodegradability Composition and chemical groups of the polymer’s backbone Physical and chemical features (ionic exchange and strength, pH, size, defects in the chain) Morphologic aspects (amorphous, semicrystalline, crystalline, microstructure) Degradation mechanisms (enzymatic, hydrolysis, microbial) Distribution of molecular weight Conditions of processing and sterilization Route of administration and site of action

hydrophilicity, and surface energy. Swelling and hydrolytic degradation depends directly on the capacity of absorbing water. Moreover, matrix structural properties such as the micromorphology and pore size, which regulate the mass transport into (water) and out (active compound) of the polymer, are very important. In reference to biodegradable polymers, it is crucial to identify that degradation involves a chemical process that depends on multiple factors, as stated in Table 16.2. Degradation can take place either on the surface or in the bulk, depending on the structure and chemical groups present in the polymer chain. Surface degradation occurs when the rate of degradation of the polymer exceeds the rate of permeation of water into the core of the nanoparticle. This type of erosion is desired because the erosion kinetics and rate of drug release are greatly reproduced [25]. In contrast, bulk erosion takes place when the velocity of permeation of water into the bulk of the matrix is faster than the rate of degradation of the polymer; hence, complex degradation kinetics is observed. Commonly, the biopolymers applied in drug delivery devices suffer bulk degradation. In the biomedicine area, the formulations of nano- and microparticles used show similar degradation kinetics (bulk and surface). Additionally, the erosion process can be modulated by varying the surface area of the device or by changing the hydrophobic/hydrophilic balance in the polymer [25]. Among the great variety of polymers, the ones that are stimuli-responsive are promising as drug delivery platforms as they deliver the active compound at a controlled rate and keep the biological active form. A polymer that suffers drastic changes in its physical properties because of environmental stimulus is called stimuli-sensitive or smart polymer. These macromolecules are known as smart polymers based on the fact that no change occurs until the polymer reaches the target place, where a trigger activates the change. It is interesting to note that the change is generally reversible and when the stimulus ceased their original shape is restored [24,26,27]. The nonlinear response of these polymers by a minor stimulus is an exclusive characteristic, which results in a noticeable macroscopic modification in the structure and

376 Chapter 16 Polymeric Matrix DRUG

Temperature

Bioresponsive pH

Field

Stimuli Change in temperature

Molecule Pendant acid/basic group

Electrid field

DRUG RELEASE

Figure 16.6 Stimulus responsible for the drug release in smart polymer systems.

varies from swelling, contracting to disintegration. As shown in Fig. 16.6, there are various stimuli that can be used for controlling the release of drugs from these intelligent polymeric devices. As mentioned before, some transitions are reversible, such as the physical transitions, solubility, and conductivity. These changes in the polymers occur as a result of the neutralization of the charges, by changes in pH or changes in hydrogen bonding caused by changes in the temperature. The major advantages of drug delivery devices made with smart polymer comprise the reduction of the frequency of the dose, the simplicity of preparation, and the preservation of therapeutic drug concentration with only one administration. Also, the release of the pharmaceutical can be tailored to be sustained in the time reducing side effects and improving the stability [28e31]. As stated before, there are many types of stimuli that can response in smart polymers. The response obtained by physical or chemical stimuli in a smart polymeric solution is restricted to the formation/disruption of forces in which hydrogen bonding, dipoleedipole, van der Waals, and electrostatic interactions are included. The most common type of chemical responses is generated by reactions of reduction/oxidation, acidebase, and hydrolysis of lateral groups bonded to the polymer backbone. Sometimes, changes in the conformation and degradation in the polymer structure, caused by an external stimulus, are observed as a response to irreversible bond breakage. Some important features to consider, when using a smart polymer as well as any drug delivery device, are the biodegradability and biocompatibility; the profile of release; the drug loading capacity; and absence of harmful secondary effects such as toxicity, immunogenicity, and carcinogenicity.

Synthesis of micro- and nanoparticles of alginate and chitosan 377 Table 16.3: Different smart polymers for drug delivery. Stimulus

Advantages

Temperature

Simple incorporation of active moieties Easy manufacturing and formulation

pH

Appropriate for thermolabile drugs

Light

Easy control of the trigger mechanism Precise control over the stimulus

Electric field

Pulsatile release with changes in electric current

Limitations Problems during the injection Low mechanical strength, biocompatibility issues Not referred for thermolabile drugs Lack of toxicity data Low mechanical strength

Low mechanical strength of gel Possible leaching of chromophores attached monovalently Inconsistent responses to light Need of surgical implantation Requirement of external stimulus by an adequate equipment Complex optimization of the electric current

Examples Cellulose, chitosan Poly N-alkylacrylamides Poly N-vinylcaprolactams

Polyvinylimidazoles Polymethacrylic acids Alginate and chemically modified carboxymethyl chitosan Modified polyacrylamides

Sulfur polystyrenes Poly(thiophene)s

To increase and improve the application areas, scientist focused their attention on the development of novel polymers with enhanced biocompatibility and biodegradability. The design of new smart polymers with precise and programmable responses will result in enhanced DDSs. In this sense, the versatility of smart polymers makes them a candidate because their properties can be easily tailored and tuned. It is worthy to mention that it is possible to adjust the sensitivity of the polymer to a narrow range of stimulus. The great disadvantage of external stimuli is the slow response. Table 16.3 summarizes the different stimuli observed in smart polymers. In addition, the advantages and disadvantages as well as examples of polymers are presented.

2.1 Temperature-responsive polymers The polymers that are thermosensitive are those which their solubility changes drastically as a result of slight changes in temperature. These aqueous polymeric solutions show a behavior that is dependent on the temperature and reversible solegel transitions. Thus, the release of drugs together with the physical and chemical stability is controlled by this

378 Chapter 16 aspect. Usually, hydrophilic to lipophilic relation of moieties in backbone of the polymer governs this phenomenon. Most thermoresponsive polymers present alkyl moieties (i.e., methyl, ethyl, and propyl) attached to the polymer chain. Two of the parameters that need to be taken special attention are the lower critical solution temperature (LCST) and the upper critical solution temperature (UCST) [32e35]. It is defined as LCST, the temperature above which the one-phase polymer solution turns into a two-phase system as a result of the hydrophobicity and insolubility of the polymer in the solvent, whereas below that temperature the polymers are soluble. These kind of polymers may be classified into three categories (negative, positive, and reversible) depending on the response to the change in the temperature. Some examples of LCST thermoresponsive polymers used in biomedical devices include poly-N-isopropylacrylamide, poly-N-vinylcaprolactam, modified natural polysaccharides, chitosan, and triblock copolymers made of PLGAePEGePLGA [34].

2.2 pH-responsive polymers Most polymers that are responsive to pH have a basic or acidic pendant group attached to the polymeric chain, which confers the property of accepting or releasing protons (Hþ) as a consequence of changes in pH. These polymers contain many polyelectrolytes, that is, ionizable groups, and are grouped into two categories: polyacids and polybases. The polymers that accept protons are the weak polyacids and they do so at low pH, whereas at neutral or high pH, they release them, which is the main difference with polybases, as one should expect. This variation in the charge of the polymer leads to the alteration in the structures of the molecules, giving rise to the release of the trapped drug. Some of the most used polymers as pH-responsive are the alginate, poly(methacrylic acid) (PMAAc), polyvinylimidazoles, and poly(acrylic acid) (PAAc). As mentioned before, pH is involved in many processes such as the preparation, purification, and hydrogel device formation. Also, it governs the rate of swelling, releasing, and degradation of the polymer net. For instance, for the alginate, the carboxylic groups are responsible for the remarkable sensitivity to pH. At pH below 3.4 (below its pKa), the carboxylic acid groups are protonated, which makes the structure insoluble. Above 4.4 units of pH, the carboxylic group became deprotonated (COO), giving rise to electrostatic repulsions, which cause an expansion in the backbone of the polymer and swelling of the hydrophilic polymeric matrix, having it highest capacity at pH equal to 7.4.

2.3 Bioresponsive polymers In the area of biomedicine, biologically responsive polymer systems stand out and are gaining special attention. As these stimuli are inherently present in the natural system,

Synthesis of micro- and nanoparticles of alginate and chitosan 379 they constitute the main advantage of the use of this type of polymeric systems. The clue to understand these types of responsive polymers is that their response is a result of the interaction of common functional groups with biologically relevant species. Bioresponsive polymers depend on the target species with which they interact, for instance, antigen, glucose, and enzyme responsive polymers.

2.4 Field-responsive polymers The polymers that respond to an electric, magnetic, sonic, or electromagnetic field are called field-responsive polymers. The major vantage that makes them excelled in contrast to conventional stimuli-sensitive polymers is their fast response time, anisotropic deformation, and precise control when releasing the drug. These features may be achieved because of the fact that the stimuli may be directionally applied and the easy way of modulating the signal point. Among the natural polymers used for the synthesis of these thermoresponsive systems are hyaluronic acid, alginate, and chitosan, whereas synthetic polymers include allylamine, vinyl alcohol, acrylonitrile, methacrylic acid, and vinylacrylic acid, for example. In addition, mixtures of natural and synthetic polymers can be used for such purposes. It is well known that many of these electrosensitive polymers have charges (they are usually polyelectrolytes), which undergo deformation upon an electric field based on the anisotropic swelling or deswelling, which makes the movement of the ions toward the cathode or anode. There is a big stress observed around the anode and a smaller surrounding the cathodes, which promotes a deformation of the gel under these conditions [24].

3. Preparation of nanoparticles In the past years, many methods and procedures have been settled for preparation of NPs. There are different classes depending on the mechanism implicated in the formation of the NPs: (1) if they are formed directly from a polymer or macromolecule (dispersion of the preformed polymers) or (2) if a 2(polymerization of monomers). If the preparation of the polymer is by modification of an existing one, then polymers used for this aim should meet certain requirements such as nontoxicity, nonantigenicity, biodegradable, and biocompatible. As stated before, some of the natural polymers used for the preparation of NPs are chitosan, gelatin, albumin, and sodium alginate as molds. Some examples of synthetic polymers also for this aim are polycyanoacrylates, polycaprolactone, polylactic acid (PLA), polyglycolic acid (PGA), and poly(lactic-co-glycolic) acid (PLGA), to mention some [8]. In the next paragraph, the different methods used for the preparation of NPs from either preformed polymers or by means of a reaction of polymerization are detailed.

380 Chapter 16

3.1 Dispersion of preformed polymers 3.1.1 Emulsificationeevaporation method (EEM) Conventionally, two strategies are most commonly used in the formation of emulsions by EEM: single and double emulsion. In the first case (single emulsion), an oily phase (O) is poured into an aqueous phase (W), whereas in double emulsion, an emulsion is mixed into another emulsion, which is a multiphase system consisting of (water-in-oil)-in-water ((W/ O)/W). Both require ultrasonication or high-speed homogenization, and the organic solvent is evaporated through continuous magnetic stirring or using reduced pressure, as it is detailed below. In this method (EEM), the polymer and the drug are dissolved both in an organic solvent such as dichloromethane (DCM), chloroform, or ethyl acetate as shown in Fig. 16.7. Then, the mixture is poured into an aqueous solution and it emulsifies, resulting in an oil (O)-in-water (W) emulsion with the help of a surfactant/emulsifying agent as gelatin or poly(vinyl alcohol) (PVA). When the stable emulsion is formed, the mixture is vigorously mixed and the organic solvent is removed. The elimination process can be divided into two stages: first, either the extraction or the diffusion to the continuous phase of the organic solvent that is forming droplets is carried out, followed by the evaporation of said solvent. Both involve mass transfer phenomena. Finally, the nanoparticles are collected by ultracentrifugation, and to remove the surfactants or other undesirable products, the particles are washed with distilled water and lyophilized [9]. Even though these methods are proved to work well in a laboratory scale, they could not be able to scale into pilot production [8]. 3.1.2 Nanoprecipitation or solvent displacement procedure In this method, polymer and drug are dissolved in an organic solvent, which is soluble or partially soluble in water. Then, the nanoparticles are precipitated in an aqueous medium Ultrasound or High Pressure HomogenizaƟon

Organic phase Polymer and drug soluble

Solvent evaporation

Emulsification

Aqueous phase Stabilizer

Emulsion

Figure 16.7 Schematic illustration of the EEM.

Nanoparticles suspension

Synthesis of micro- and nanoparticles of alginate and chitosan 381 by the diffusion of the organic solvent into the aqueous medium. Sometimes, it may require the use of a stabilizer as a surfactant. This method leads to the precipitation of nanospheres. A colloidal suspension is formed by diffusion of the organic solvent into the aqueous phase, a phenomenon that occurs in the interphase. This solvent diffusion leads to the precipitation of the nanoparticles. It is important to mention that this technique is suitable if a small volume of nontoxic oil is included into the organic phase. In general, it is a simple technique, but it is limited to water-miscible solvents and lipophilic drugs, because it is not an efficient method to encapsulate water-soluble medicines [9]. The methods mentioned before require the use of organic solvents, which can be dangerous for living organisms and for the environment. To avoid the use of an organic solvent, there are two methods of preparing NPs: emulsificationesolvent diffusion and salting-out method. 3.1.3 Emulsification/solvent diffusion This method is based on the emulsification of an organic solution of a polymer and a drug (most commonly reported 2-butanone and ethyl acetate as a solvent saturated with water) in an aqueous solution containing a stabilizing agent or mixtures (saturated with solvent) in continuous agitation, followed by the application of vacuum and/or temperature to generate the displacement of the solvent by rapid evaporation. In this technique, the solvent diffuses quickly from the internal to the external phase, thus promoting the aggregation of the drug and the polymer. As a consequence of the quick displacement, areas of local supersaturation are formed in the place where the nanoparticles are formed. The main difference with EEM is that there is no need of homogenization of the emulsion to obtain the nanoparticles, a drop of emulsion will form several nanoparticles. This technique has several advantages: (1) the use of organic solvents accepted by the pharmaceutical industry; (2) the option of reusing the solvent; (3) adaptability to various biodegradable or nonbiodegradable polymers; (4) absence of high energy sources; and (5) high reproducibility in a lab scale. 3.1.4 Salting out This is a modified method of emulsion process. To avoid the use of surfactants and chlorinated solvents, polymeric NPs are prepared from aqueous dispersions using an emulsion technique, which involves a salting-out process. Then, the preparation consists of

382 Chapter 16 incorporating a saturated aqueous solution of an electrolyte or an unsaturated aqueous solution of electrolyte containing polyvinyl alcohol (PVA), which acts as an agent that increases the viscosity. This solution is poured into an acetone polymer solution under continuous stirring. Salt aggregate prevents acetone from mixing with water through a salting-out process. Finally, the formation of nanospheres is induced by the addition of large amounts of water that allow the complete diffusion of the acetone present in the aqueous phase [36]. 3.1.5 Dialysis To obtain NPs of a narrow range of size, dialysis is the chosen method as it is simple and effective. It consists of the transport of solvent through a physical barrier (semipermeable membrane) from a phase composed of pure solvent to solution containing the polymer. In a dialysis tube, an organic solvent is placed with the polymer dissolved in it. As the solvent moves from one side to the other, the polymer starts to aggregate as a consequence of the diminishment of the solubility, which results in a homogeneous suspensions of nanoparticles [9]. 3.1.6 Supercritical fluid (SCF) technology In recent years, the boom in the utilization of supercritical fluids as a convenient alternative to organic solvent has increased. This is not only because the use does not damage the environment but also because it is a profitable method as the NPs are obtained in high purity and in the absence of organic solvents. There are two different methods reported in bibliography that uses SCF; they are summarized in the next paragraph. 1. Rapid expansion of supercritical solution (RESS): As the name states, the polymer and drug are solubilized in a supercritical fluid and then a quick expansion using a nozzle is performed. In this condition, the SCF precipitates and a uniform distribution of drug in the polymer matrix is obtained. The limitation of the techniques relies on the necessity of using polymers of low molecular weight (<10,000), which are soluble in the SCF, whereas highemolecular-weight polymers are insoluble. 2. Supercritical antisolvent (SAS): In this technique, the solute is dissolved in an appropriate organic solvent and the supercritical fluid is charged. The solute will precipitate when exposed to high pressures, which will make the antisolvent enter the liquid phase. When the desired final pressure is arrived, after the precipitation has already occurred, the antisolvent will displace the residual solvent [8].

Synthesis of micro- and nanoparticles of alginate and chitosan 383

3.2 Polymerization methods As mentioned before, NPs can also be synthesized by the polymerization reaction of monomers. In this method, the addition of the drug can be done before the addition of the monomer or at the end of the polymerization reaction, by dissolving it in a suitable solvent. The resulting NP suspension usually needs to be purified by ultracentrifugation or by resuspending the particles in a medium free of isotonic surfactant. The size and molecular mass of NPs depend on the reaction conditions such as concentration of monomer, pH of the polymerization medium, speed of stirring, the type and concentration of the stabilizer (i.e., dextran-70, dextran-40, dextran-10, poloxamer188, poloxamer-184), and/or surfactant (i.e.,.polysorbate-20, polysorbate-40, or polysorbate-80). 3.2.1 Emulsion polymerization Usually, for the synthesis of nanoparticles, the polymer is formed by an emulsion polymerization. This type of polymerization is characterized by small reaction time and the easy way of scaling. The main disadvantages are the need to eliminate toxic organic solvents, surfactants, monomers, and initiators in the final step. In this methodology, the continuous phase may be organic or aqueous, and on this basis, it is classified into two categories. When a continuous organic phase is used, then the monomer is dispersed in an emulsion or in a phase in which it is insoluble. In this method, the use of surfactants or soluble protective polymers is required to prevent the formation of aggregates in the polymerization. In contrast, in an aqueous continuous phase, the monomer is dissolved in an aqueous, which is the continuous phase, and there is no requirement of surfactants or emulsifiers. There are many different ways of initiating a polymerization process, and it depends on the polymer that is desired to be synthesized. The formation of the solid particles can occur before or after the polymerization is finished. Among the emulsion polymerizations, two different methods could be distinguished: 1. Miniemulsion polymerization: This method uses water, monomer mixture, costabilizer, surfactant, and initiator. The main contrast with a typical emulsion polymerization relies on the fact that it uses a substance of low molecular mass as a costabilizer and a high shear device to have a stable state in this type of polymerization and reach an interfacial tension greater than zero. 2. Microemulsion polymerization: This is a novel and useful method to synthesize NPs. In the microemulsion, a water-soluble initiator is introduced into the aqueous phase of a microemulsion containing swollen micelles. Then, the polymerization starts from a

384 Chapter 16 thermodynamically stable state formed spontaneously from high quantities of surfactant systems, which have an interfacial tension at the O/W interface close to zero. Some of the critical factors in determining the kinetics of the polymerization as well as the properties are the reaction temperature and the types and concentrations of initiator, surfactant, and monomer. Although both methods (emulsion and microemulsion) differ in the kinetic, they do generate colloidal polymer particles of high molecular mass. In terms of particle size and average number of chains per particle, microemulsion has the smaller values. 3.2.2 Interfacial polymerization This polymerization is a step polymerization involving two dissolved reactive monomers, one in a continuous phase and the other in a dispersed phase. Then, at the interface of the two liquid phases, the polymerization reaction takes place. When nanometer-sized hollow particles are needed, then interfacial cross-linking reactions are used. For instance, some of these reactions are the polyaddition and polycondensation or radical ones. If a nanocapsule containing oil is desired, a microemulsion polymerization with the monomers at the O/W interface is used. In this case, the organic solvent, which must be completely miscible with water, functions as a monomer vehicle and the interfacial polymerization of this occurred on the surface of the droplets formed in the emulsification. By this method, you can synthesize nanospheres and nanocapsules by varying the solvent. The utilization of aprotic solvents such as acetone and acetonitrile stimulates the synthesis of nanocapsules, whereas protic solvents, such as ethanol, n-butanol, and iso-propanol, encourage the formation of nanospheres. In addition, nanocapsules can be synthesized through interfacial polymerization of monomers in W/O microemulsions. 3.2.3 Controlled/living radical polymerization (C/LRP) In a controlled/living free radical polymerization, the active end of the polymer chain is a free radical; then it is a type of living polymerization. This type of polymerization has the advantage of producing NPs with a narrow particle size and with control in the size distribution. The main disadvantages of LRP consist of the lack of control over the molar mass, the molar mass distribution, the end functionalities, and the macromolecular architecture. As it is impossible to avoid the terminations reactions, most of the limitations come from these reactions. The methods so far used are as follows: 1. nitroxide-mediated polymerization (NMP), 2. atom transfer radical polymerization (ATRP), and 3. reversible addition and fragmentation transfer chain polymerization (RAFT).

Synthesis of micro- and nanoparticles of alginate and chitosan 385 As of other polymerization, it is vital to control the concentration of the monomer and additives such as control agent and initiator, and emulsion type as they determined the particle size of the final product.

3.3 Ionic gelation or coacervation The method implicates a mixture of two aqueous phases, one formed by the cationic polymer from the biomass, such as chitosan, and other formed by a synthetic polymer, a diblock copolymer such as ethylene oxide (PEO) or propylene oxide (PPO). The other phase is formed by a polyanion sodium tripolyphosphate. In this method, the positively charged polymer interacts with the negatively charged tripolyphosphate, resulting in an aggregate of nanometric size. Although electrostatic interactions are the driving forces for the formation of the coacervates, the transition from liquid to gel owing to ionic interaction is based on ionic gelation [9].

4. Alginate and chitosan nanoparticles 4.1 Alginate nanoparticles Alginate nanoparticles are synthesized primarily by two methods: •

•

Complexation: For the complexation of alginate, a cross-linker such as calcium is used. This divalent cation is obtained from calcium chloride, which may occur in aqueous solution or at the interface of a drop. The first results in the synthesis of nanoaggregates, whereas the second in the nanocapsule alginate. It can also be carried out by mixing alginate with a polyelectrolyte with opposite charge, such as chitosan. Alginate-in-oil emulsification is followed by external or internal gelation in the drop, leading to the synthesis of alginate nanospheres [37].

Then, depending on the synthesis method of the alginate nanoparticles, nanoparticles, nanospheres, or nanoaggregates can be selectively obtained (Fig. 16.8). 4.1.1 Nanoaggregates of alginate synthesized by self-assembly and complexation Rajaonarivony et al. described a new method for the synthesis of nanoaggregates of alginate (250e850 nm) as a carrier drug [38]. The particles were synthesized in a solution containing alginate in the sodium form by addition of calcium chloride (CaCl2). Both concentrations were inferior than those typically required in the formation of alginate gel, so that disperse nanometric aggregates were formed in a continuous water phase (state before gel formation). Then, an aqueous polycationic solution, such as poly-L-lysine, chitosan, or Eudragit E100, was added and resulted in a coating of the polyelectrolyte complex of these alginate nanoparticles. In addition, alginate aggregates were prepared by mixing with chitosan or calcium ions with the alginate solution.

386 Chapter 16 Dripping/Extrusion Co-axial laminar air flow Generator of electrostatic beads Vibrating nozzle Mechanical seccional or cutting Air

Spinnig disk atomization Emulsification

0

1

2

3

4

5

6

7

8

9

10

Particle size (μm)

Figure 16.8 Comparison of the size obtained for different methods of preparation of alginate microparticles.

Also Rajaonarivony et al. [38] determined that the size and properties of the nanoaggregates were affected by the concentration of alginate and cationic polymer and also of the calcium chloride. Another determination factor is their molecular weight and the order in which the components (CaCl2 and polycationic polymer) were added to the solution of alginate. Also, when it is desired to synthesize nanoaggregates of alginate, the conditions of high shear during the synthesis are relevant. 4.1.2 Formation of alginate nanocapsules by complexation on the interface of emulsion droplets The preparation of nanocapsules of alginate is carried out by depositing said polymer at the interface of a template drop, with the consequent elimination of the solvent. After deposition at the interface of the droplets, by covalent or physical intermolecular crosslinking, the polymer layer is then stabilized. In this technique, the alginate and the drug in an organic solvent are dissolved, which will act as the internal oil phase of the capsules. Then, an aqueous solution of alginate containing an additional surfactant (i.e., Tween 80) is slowly added to this organic phase. Then, an O/W emulsion is formed by sonication and the aqueous suspension of the nanocapsule is allowed to equilibrate for some time before the removal of the solvent. 4.1.3 Alginate nanosphere formation from water-in-oil emulsions Alginate-in-oil emulsification is followed by external or internal gelation in the drop, resulting in the formation of alginate nanospheres. These have been used to synthesize microspheres and nanospheres of alginate. The alginate nanospheres are synthesized by

Synthesis of micro- and nanoparticles of alginate and chitosan 387 the emulsification together with the external or internal gelation by a process of two stages. For the first, a water/oil emulsion is formed from an alginate solution containing the substance to be encapsulated and an oil phase, and then, for the second, it is melted by the addition of a cross-linker, frequently calcium (Caþ2) from a solution of CaCl2. Moreover, Reis et al. described a method consisting of two stages for the production of alginate nanospheres by emulsification followed by internal gelation [39]. Firstly, a solution of alginate with an insoluble calcium source (e.g., calcium carbonate) is emulsified in an oily phase, giving a W/O emulsion. Therefore, an acid soluble in the oil phase is added, decreasing the pH inside the alginate particles and inducing the release of Caþ2 from the insoluble salt, and thus triggers the gelation in situ of the particles of alginate. The properties and applications of the alginate particles depend on the method of preparation of the gels, either by internal or by external gelation. Based on this, those prepared by external gelation as a concentration gradient of the cations is generated (high concentrations at the surface and low concentrations in the core) tend to have a denser structure with smaller pores on the surface of the particle. In contrast, those prepared through internal gelation present a more uniform distribution of the cations throughout the particle, giving rise to a more homogeneous particle.

4.2 Chitosan micro- and nanoparticles In the past years, in the drug delivery, the use of polymeric NPs has increased greatly, based on the ability of overcoming physiological barriers without degradation of the encapsulated drug and on the site-specific delivery [40,41]. As mentioned above, chitosan is a candidate biopolymer for this application, not only for its remarkable properties but also for the previous applications in the pharmaceutical field. To use chitosan derivatives in the pharmaceutical industry as drug carriers, it is essential to develop different techniques to obtain micro- and nanoencapsulation of drugs using these polymers. It is worthy to mention that nanoparticles are particles of size ranging between 1 and 1000 nm, whereas microparticles are particles of size ranging between 0.1 and 100 mm in size. These particles will increase surface/volume relation, providing a close interaction with different biological surfaces, and also will retain the biological activity of the molecule encapsulated till the final place of release. All the methods for the production of micro- and nanoparticles involved assembly of molecules in greater structures with different polydispersity. Fan et al. [42] reported that colloids of big polydispersity and poor stability are detrimental to the effectiveness of the particle in drug delivery applications. Thus, molecules of bigger size have a high drug

388 Chapter 16 loading capacity, whereas lesser ones have a better efficiency in the delivery of them to the target sites. There are many reports [7,17,43e46] reviewing the different techniques and methods to encapsulate drugs using chitosan as encapsulant in micro- and nanosize. The most common methods used to form microparticles of chitosan are emulsion cross-linking, coacervation, spray drying, sieving method, and ionic gelation. 1. Emulsion cross-linking or emulsion solvent diffusion: It is a versatile method based on the immiscibility of an organic solvent (O) with water (W). The organic phase contains the hydrophobic drug, whereas the aqueous solution is formed by the chitosan and a stabilizer. This technique involves the dispersion in the form of small droplets of an aqueous solution containing the polymer in an oil phase having the active product (encapsulant). An appropriate surfactant is used to stabilize the aqueous droplets. To give hardness to the droplets, a cross-linking agent is used, which possesses aldehyde groups that react with the amine group of chitosan. The result is the formation of an emulsion and exposed to high pressure where NPs are precipitated, as a consequence of the decrease of the solubility owing to the diffuse of the organic solvent into the aqueous phase. 2. Coacervation/precipitation: The coacervation is an electrostatically driven separation, which results from association of oppositely charged macroions. In the case of chitosan, it utilizes the insolubility of this polymer in alkaline pH medium. An acid solution of chitosan is blown into an alkaline pH medium where it precipitates. There are also other factors that can be used in the coacervation technique such as changing temperature, physical conditions, biopolymer ratios, or ionic strengths. Varying the ionic strengths is a technique that will be discussed later. 3. Spray drying: In this technique, the drug is dispersed or dissolved in an acidic solution containing the chitosan and a suitable cross-linking agent. Then, the solution is atomized in a steam of hot air. 4. Sieving method: Nanoparticles are prepared by cross-linking with glutaraldehyde, and as result of this, a nonsticky glassy hydrogel is achieved. Further purification involves the passage of the product obtained through a sieve of an adequate mesh size. 5. Polyelectrolyte complexation or ionic gelation: It is important to highlight that chitosan achieves a high grade of protonation because of the amine groups and is able to produce hydrogels when certain polyanions are presented. This property is the basis of this method. The interaction of oppositely charged macromolecules, for instance, the cationic charged chitosan and tripolyphosphate (TPP), is used to form the chitosan micro- and nanoparticles by inter- and intramolecular cross-linkages. The TPP is chosen based on the fact that it is nontoxic, multivalent, and gels forming polymer.

Synthesis of micro- and nanoparticles of alginate and chitosan 389 As it was mentioned before, the smaller the particle, the better the interaction with the target cell. In this sense, the techniques that achieved better results in the synthesis of nanoparticles were emulsionedroplet coalescence, emulsion solvent diffusion, ionic gelation, reverse micellar method, polyelectrolyte complexation, and desolvation for the synthesis of nanoparticles. In the next pages, a summary of the techniques that were not described before is presented. 1. Emulsionedroplet coalescence: It is a combination of cross-linking and precipitation. The difference relies on the fact that, in a first stage, an emulsion of chitosan and drug in a liquid paraffin oil is mixed under high-speed stirring with a stable aqueous chitosan and NaOH solution. As a consequence, droplets of each emulsion will collide and then precipitate. 2. Reverse micellar method: A reverse micelle is a W/O droplet, which differs from common micelles that are formed in an O/W system. Thus, a lipophilic surfactant is required. The basis of this technique is the formation of the NPs in the aqueous core, followed by cross-linking with glutaraldehyde. It is important to highlight that increasing in the cross-linking results in bigger particles [47]. 3. Desolvation: It is also denoted as a simple coacervation or phase separation that comprises an aggregation of the macromolecules. As precipitating agents, substances such as sodium sulfate and solvents nonmiscible with water are used. The competing agent (of larger hydrophilicity such as a solution of sodium sulfate) is poured into the solution containing the chitosan. When the salt contacts the aqueous environment, the water surrounding the chitosan is eliminated and precipitation by insolubilization occurs. The first researcher who reported the use of nanoparticles of chitosan, by emulsification, in drug delivery was Ohya et al. [48]. They immobilized 5-fluorouracil (an anticancer drug) by a cross-linking technique and studied the controlled release and effective targeting of the drug to the target place. 4.2.1 Chitosan nanofibers A nanofiber is defined as a type of nanomaterial fiber with cross-sectional diameters that ranges from 10 to 100 nm. These nanofibers have gained attention because of their unique physicochemical properties and characteristics; thus they possess high specific area and area to volume ratio [49]. There exist a variety of methods to prepared nanofibers such as, solegel procedure, chemical vapor deposition, thermal oxidation, and electrospinning, among others. Electrospinning has excelled over the others in the preparation from natural resources [46]. Electrospinning is a method that enables the production of ultrafine fibers by means of the charge and ejection of melt or a solution of the polymer through a spinneret, which works under a high-voltage electric field. As soon as the polymer passes through the mentioned

390 Chapter 16 spinneret, it solidifies or coagulates forming a filament. In this sense, electrospinning is a good technique to obtain nanofiber of chitosan because this polymer is protonated in acidic medium, turning it into a polyelectrolyte [43]. One of the first researchers who synthesized nanofibers from chitosan was Ohkawa et al. [50,51] whose survey focused on the influence of the solvent, viscosity, and concentration on the morphology of the fiber. A problem related to electrospinning of chitosan is the formation of beds. To overcome this difficulty, the use of chitosan in conjunction with other synthetic polymers (blend) constituted a great solution. Many blends of chitosan together with PVA, PVO, and PET, among others, have been reported [52]. This opens a new possibility for the application of nanofiber blends of chitosan in biomedical applications.

5. Biomedical applications as drug delivery systems 5.1 Alginate nanoparticles As mentioned above, alginate is a biomaterial that has been extensively used in biomedicine as drug delivery systems, which releases the drug when exposed to an external stimulus. In some responsive (smart) polymers, the activation could occur by interaction of nanostructured magnetic particles, which were introduced in the NPs of alginate with an external magnetic field. To control the velocity of releasing of the biopolymer in this attempt of field-responsive polymers, there are two features that must be studied: (1) the way the nanoparticles respond to the magnetic field and (2) physicomechanical properties of the polymer net. Thus, controlling the dimension is critical for the success of a device in the medical area, and that is the reason for the development of methodologies for the preparation of magnetic alginate NPs. Numerous researchers focused their investigations on the use of divalent ions (Caþ2, Srþ2, Baþ2, etc.) to generate cross-linking with guluronic acid units of alginate [53]. Only less studies use Feþ3, because alginate interacts with Feþ3 in a weird way, which is not fully understood. This is important to know, because many samples are prepared in an aqueous solution, which contains small amount of iron oxide that results in nucleation between the polymer chains. On the basis of the size and the magnetic type, the particles could stand in the superparamagnetic state. Some researchers stated that as the alginate principal chain becomes more restricted, in a conformational way, in contact with iron, resulting in aggregation. Sreeram et al. [54] realized that the ions of Fe(III) bounded to the special places in the alginate led to the formation of spatially separated centers of iron(III) on the polymer matrix refusing the theory of the formation of a covering of FeOOH that made the polymer precipitate. Nesterova et al. [55] developed clusters of iron(III,) which were superparamagnetic and which led to ferrihydrite that is poorly crystalline. They studied

Synthesis of micro- and nanoparticles of alginate and chitosan 391 several polymers such as PVA, polyacrylic acid, and alginate. As a result of their studies, they demonstrate that iron particles are superparamagnetic, and when putting them together with the polymer, they exert great organization and stability. To study these complexes, many techniques are used, such as electron paramagnetic resonance (EPR), 57Fe Mo¨ssbauer spectroscopy (MS), and magnetization measurements. Finotelli et al. [56] synthesized ferric alginate beads by mixing an aqueous solution of alginate into solutions of FeCl3 in concentration that goes from 0.1 to 0.5 M. This group demonstrated that iron is absorbed in alginate in the form of Fe(III) by means of MS and EPR spectroscopy. The EPR results suggest that the iron(III) takes the place of the sodium. During this process, these superparamagnetic particles of iron hydroxide are precipitated inside the polymer matrix. It is well known that the addition order of cations to the aqueous solution of alginate influences directly the size range of alginate NPs. Nevertheless, some studies stated that there were some advantages for adding a step involving complexation with a polyelectrolyte in the procedure [57]. In this sense, Sarmento et al. made insulin-loaded NPs by ionotropic pregelation of alginate and adding a step of complexation with chitosan. The particles obtained in this way were in the nanometer size range and have a moderate loading capacity (14.3%). Other researchers used other complexing polymers, for instance, dextran, and they also loaded insulin into the NPs of alginateedextran. In this case, this group used a nanoemulsion followed by in situ gelation to form the nanospheres, which have a size ranging from 267 nm to 2.76 mm [39]. The resulting NPs have an efficiency of encapsulating insulin of 82.5% [58]. Another interesting task is the chemotherapy in which new delivery devices are continuously searched. A typical complication in antitubercular chemotherapy is that the patient does not respect the long-term multidrug regimens. Based on studies of treatments of mycobacterial infections that used modified-release drug delivery devices, Zahoor et al. [59] developed nanoparticles of alginate, which they loaded several pharmaceutics such as isoniazid (INH), pyrazinamide (PZA), and rifampicin (RIF). These new alginate NPs were formed using the cation-induced gelification method, and they were fully characterized and studied [38]. The protocol involves the incorporation of calcium chloride to a solution of sodium alginate having diverse concentrations of INH, PZA, and RIF. In the next step, a solution of chitosan was added and the final mixture was kept steady and stored at room temperature. Final NPs were obtained by means of centrifugation. In the same way, drugfree nanoparticles were also prepared. By this manner, they were able to obtain NPs in the nanosize, with encapsulation efficiencies that vary from 70% to 90% for INH and PZA, and 80%e90% for RIF. It is worthy to mention that in comparison with the drug-free NPs, the ones with the drug showed better bioavailabilities and they showed better results in inhalable chemotherapeutic treatments than the ones with no NP. As a result of this study,

392 Chapter 16 inhalable alginate NPs are emerging as promising system for the controlled release of antitubercular drugs [59]. Other study of the same group demonstrated the ability of alginate NPs, to encapsulate different drugs. In this case, they loaded the alginate NPs with azole antifungal and antitubercular drugs, which are used for the treatment of murine tuberculosis [60]. Several studies involving alginate nanoparticles with different drugs encapsulated are available in the literature [61,62]. Finally, Joshi et al. [63] developed a new template made of gelatin NPs and CaCO3, which were embedded in microspheres. This is interesting; thus, it is something nano in a micro (nano-in-micro) size. For this purpose, gelatin nanoparticles loaded with dexamethasone, fluorescein isothiocyanate dextran (FITC-dex), nanoparticles of CaCO3, and a “nano-inmicro”system of alginate were synthesized and fully characterized. The study of the release of dexamethasone from these nanoparticles and from the “nano-in-micro” system showed a decrease in the order of releasing: 24% for uncoated, 18% for coated, a 17% in a ratio of 1:4 for the “nano-in-micro” system. On the other hand, “nano-in-micro” showed the longer releasing times (14 days). Thus, “nano-in-micro” systems are likely possible for use as drug release devices and biosensors.

5.2 Chitosan nanoparticles To use chitosan nanoparticles (CS NPs) for drug delivery applications, it is important to study the best conditions to get the smaller particles that, as already mentioned, will be the best candidates for this purpose. It is also key fact to study the degree of entrapment of drugs to use as biocarriers. In this sense, Katas et al. [64] synthesized CS NPs by ionic interaction with dextran sulfate and they determined that the concentration of CS and dextran along with pH controlled the particle size. The smallest particles were obtained at pH 4, in a ratio of 0.5:1 of chitosan: dextran. They also demonstrated they could get a 98% of entrapment efficiency when bovine serum albumin (BSA) or double-stranded siRNA was loaded into the nanoparticles. A remarkable characteristic of chitosan, which was exploited by many researchers, was the ability as a transporter for mucosal delivery based on its mucoadhesivity. Zhang et al. [65] prepared CS NPs of controlled size by ionic gelation method, encapsulated BSA, and demonstrated that the release of this protein occurred conditions similar to intestinal fluid at pH 7.5 over a period of 6 days. Thus it may be concluded that small CS NPs of homogeneously sized are desirable for delivery systems, which targets mucoses. Another interesting approach was presented by the group of Cha´vez de Paz [66], who synthesized various CS nanoparticle complexes of different molecular weight and degrees of deacetylation and then evaluated the antimicrobial effect. It is interesting to remark that

Synthesis of micro- and nanoparticles of alginate and chitosan 393 nanocomplexes formed from chitosan of high molecular weight showed lower antimicrobial activity (20%e25% of cells damaged) in comparison with those of low molecular weight (>95% of cells damaged). In the field of tissue engineering, CS NPs also appeared as an interesting candidate as specific delivery vehicles. Specially, a recent research explored the use of CS NPs for the delivery of growth factors [67]. In this approach, the authors fabricated particles loaded with fibroblast growth factor (bFGF) and also with bovine serum albumin, which were incorporated in a nanocomposite scaffold of CS and gelatin to enhance their biological properties. They demonstrated that this composite could release the growth factor sustainable in time and, therefore, enhanced the proliferation of fibroblast cells. Chitosan nanoparticles were also tested as delivery systems to carry and deliver therapeutics for systemic and dermal administration. Pelegrino et al. [68] reported the encapsulation of a nitric oxide (NO) precursor, glutathione SH (GSH), into an ultrasmall chitosan nanoparticle. NO is a significant molecule synthesized by some skin cell and participates in processes such as dilatation of blood vessels, wound healing, anticancer activities, and immune response, to mention some. These authors were able to load GSH with 99.6% efficiency and by nitrosation of GSH with nitrous acid, which results in the formation of the donor NO, they proved a sustained release of the active drug. The levels of NO in epidermis increased significantly by topical administration of the NPs. The potential of chitosan nanoparticles is huge that it has even been used for pulmonary drug delivery [69]. When administrating nanoparticles through the lung route, it is important to take into account that nanoparticles must be administrated as droplets or powdered particles, which involved the development of technologies for its administration. In fact, Calvo et al. [70,71] reported the synthesis of chitosanePEOePPO nanoparticles as auspicious vehicle for the administration of several drugs and pharmaceuticals (therapeutic proteins, genes, antigens, etc.) by various routes, including nasal and ocular. It is interesting to note that the NPs were prepared by ionic gelation, in which one phase contained CS and a diblock copolymer of ethylene oxide and propylene oxide (PEO-PPO) and, the other phase was a solution of sodium tripolyphosphate (TPP). The loading efficiency was bigger than 80%, using BSA as a prototype protein. The use of CS NPs on tumor growth inhibition was tested by Miranda-Caldero´n et al. [72] that loaded nanoparticles with casiopeins, a group of anticancer agents, specifically casiopein III-ia (CasII-ia.) The nanoparticles were prepared by a coacervation process. Although the percentage of encapsulation was low due to the hydrophilicity, mice transplanted with B16 melanoma tumors and treated with the nanoparticles showed an increased life span compared with the free drug. Also nanoparticles formed by CS and folic acid and CasII-ia inhibited tumor growth up to nine times.

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5.3 Chitosanealginate nanoparticles As mentioned previously, sometimes better results are obtained if two different polymers are used. In this sense, the use of chitosan together with alginate is a promising candidate for uses in biomedicine for all the advantages (biodegradability, biocompatibility, nontoxicity, etc.) mentioned before. Therefore, many researchers have studied this blend. For instance, there are studies that use NPs of alginateechitosan to get a porous antimicrobial composites and biosynthesized silver nanoparticles (AgNPs). By means of the capacity for forming pores, chitosan and alginate were the chosen polymers, whereas AgNPs were selected for the antimicrobial property. The antimicrobial activity was evaluated against Staphylococcus aureus and Escherichia coli. The result of the bacterial filtration efficiency of CSealginateeAgNPs was 1.5 times better than that of the CSealginate complex. This development is relevant for applications in antimicrobial filtration and cancer treatment. Other approaches utilize the chitosanealginate polyelectrolyte complex as wound dressing. Based on the extraordinary features of the CSealginate polyelectrolyte complex hydrogels among which we can mention the high biocompatibility, degradability, and nontoxicity, this is a promising material that can successfully improve the wound healing. Thus, CSealginate-based composites with silver and pharmaceuticals were applied for wound dressing, antimicrobial, bone tissue engineering, anticancer, and dental applications.

6. Conclusion This chapter summarizes the new advances in drug delivery systems based on naturally occurring polymers. It also details the different categories in smart polymers, because the new advances in drug delivery devices are focused on this sense. Specially, it is focused on nanoparticles of alginate and chitosan. It also presents different ways, techniques, and procedures used for the preparation of nanoparticles of alginate and chitosan and the way the drugs may be loaded and released from the polymer matrix. As a conclusion, alginate and chitosan are promising polymers for the development of nanovehicles in biomedicinal area as drug delivery devices and for immobilization of enzymes. Based on their extraordinary properties such as biodegradability, biocompatibility, and nontoxicity, they are excellent candidates in the biological field. Also, they are low in costs and easily and feasibly available, and the property of forming hydrogels in the presence of cations makes them attractive. It is important to highlight that these polysaccharides form part of the renewable biomass, and their manipulation is friendly with the environment, so they are an interesting material to apply in the medicinal applications.

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References [1] Kohane DS. Microparticles and nanoparticles for drug delivery. Biotechnol Bioeng 2007;96(2):203e9. [2] Kreuter J. Nanoparticles and microparticles for drug and vaccine delivery. J Anat 1996;189(Pt 3):503e5. [3] Wang EC, Wang AZ. Nanoparticles and their applications in cell and molecular biology. Integr Biol (Camb) 2014;6(1):9e26. [4] Hans M, Lowman A. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002;6(4):319e27. [5] Langer R. Biomaterials in drug delivery and tissue engineering: one laboratory’s experience. Acc Chem Res 2000;33(2):94e101. [6] Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces 2010;75(1):1e18. [7] Grenha A. Chitosan nanoparticles: a survey of preparation methods. J Drug Target 2012;20(4):291e300. [8] Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE. Biodegradable polymeric nanoparticles as drug delivery devices. J Control Release 2001;70(1):1e20. [9] Nagavarma BVN, Yadav HKS, Ayaz A, Vasudha LS, Shivakumar HG. Different techniques for preparation of polymeric nanoparticles- a review. Asian J Pharmaceut Clin Res 2012;5(Suppl. 3):16e23. [10] Silva GA, Coutinho OP, Ducheyne P, Shapiro IM, Reis RL. Starch-based microparticles as vehicles for the delivery of active platelet-derived growth factor. Tissue Eng 2007;13(6):1259e68. [11] Janes KA, Calvo P, Alonso MJ. Polysaccharide colloidal particles as delivery systems for macromolecules. Adv Drug Deliv Rev 2001;47(1):83e97. [12] Teijeiro-Osorio D, Remun˜a´n-Lo´pez C, Alonso MJ. New generation of hybrid poly/oligosaccharide nanoparticles as carriers for the nasal delivery of macromolecules. Biomacromolecules 2009;10(2):243e9. [13] Brannon-Peppas L, Blanchette JO. Nanoparticle and targeted systems for cancer therapy. Adv Drug Deliv Rev 2004;56(11):1649e59. [14] Feng S-S. Nanoparticles of biodegradable polymers for new-concept chemotherapy. Expert Rev Med Devices 2004;1(1):115e25. [15] Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Adv Drug Deliv Rev 2002;54(5):631e51. [16] Prabaharan M, Mano JF. Chitosan-based particles as controlled drug delivery systems. Drug Deliv 2004;12(1):41e57. [17] Wang Y, Li P, Truong-Dinh Tran T, Zhang J, Kong L. Manufacturing techniques and surface engineering of polymer based nanoparticles for targeted drug delivery to cancer. Nanomaterials 2016;6(2):26. [18] Manzano VE, Pacho MN, Tasque JE, D’Accorso NB. Alginates: hydrogels, their chemistry, and applications. In: Hasnain S, Nayak AK, editors. Alginates versatile polymers in biomedical applications and therapeutics; 2019. p. 745. [19] Lupo B, Gonza´lez C, Maestro A. Microencapsulacio´n con alginato en alimentos. Te´cnicas y aplicaciones. Rev Venez Cienc Y Tecnol Aliment 2012;3(1):130e51. [20] Islam S, Bhuiyan MAR, Islam MN. Chitin and chitosan: structure, properties and applications in biomedical engineering. J Polym Environ 2017;25(3):854e66. [21] Zargar V, Asghari M, Dashti A. A review on chitin and chitosan polymers: structure, chemistry, solubility, derivatives, and applications. ChemBioEng Rev 2015;2(3):204e26. [22] Dutta PK, Ravikumar MNV, Dutta J. Chitin and chitosan for versatile applications. J Macromol Sci Polym Rev 2002;42(3):307e54. [23] Ma´rmol Z, Pa´ez G, Rinco´n M, Araujo K, Aiello C. Quitina y Quitosano Polı´meros Amigables. Una Revisio´n de Sus Aplicaciones [Chitin and chitosan friendly polymer. A review of their applications]. Rev Tcnocientifica URU 2011:53e8. August 2016. [24] Priya James H, John R, Alex A, Anoop KR. Smart polymers for the controlled delivery of drugs e a concise overview. Acta Pharm Sin B 2014;4(2):120e7. [25] Pillai O, Panchagnula R. Polymers in drug delivery. Curr Opin Chem Biol 2001;5(4):447e51.

396 Chapter 16 [26] Ward MA, Georgiou TK. Thermoresponsive polymers for biomedical applications. Polymers 2011;3(3):1215e42. [27] Kikuchi A, Okano T. Intelligent thermoresponsive polymeric stationary phases for aqueous chromatography of biological compounds. Prog Polym Sci 2002;27(6):1165e93. [28] Hoffman AS, Stayton PS, Bulmus V, Chen G, Chen J, Cheung C, Chilkoti A, Ding Z, Dong L, Fong R, et al. Really smart bioconjugates of smart polymers and receptor proteins. J Biomed Mater Res 2000;52(4):577e86. [29] Singh KA-T, Smart J. Polymer based delivery systems for peptides and proteins. Recent Patents on Drug Delivery & Formulation. 2007. p. 65e71. [30] Kumar A, Srivastava A, Galaev IY, Mattiasson B. Smart polymers: physical forms and bioengineering applications. Prog Polym Sci 2007;32(10):1205e37. [31] Bawa P, Pillay V, Choonara YE, du Toit LC. Stimuli-responsive polymers and their applications in drug delivery. Biomed Mater 2009;4(2):22001. [32] Singh S, Webster DC, Singh J. Thermosensitive polymers: synthesis, characterization, and delivery of proteins. Int J Pharm 2007;341(1):68e77. [33] Jeong B, Kim SW, Bae YH. Thermosensitive solegel reversible hydrogels. Adv Drug Deliv Rev 2012;64:154e62. [34] Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv Drug Deliv Rev 2001;53(3):321e39. [35] Aguilar MR, Roma´n JS, editors. Smart polymers and their applications. Woodhead Publishing; February 7, 2014, ISBN: 9780857097026. p. 584. [36] Alle´mann E, Gurny R, Doelker E. Preparation of aqueous polymeric nanodispersions by a reversible salting-out process: influence of process parameters on particle size. Int J Pharm 1992;87(1):247e53. [37] Paques JP, Van Der Linden E, Van Rijn CJM, Sagis LMC. Preparation methods of alginate nanoparticles. Adv Colloid Interface Sci 2014;209:163e71. [38] Rajaonarivony M, Vauthier C, Couarraze G, Puisieux F, Couvreur P. Development of a new drug carrier made from alginate. J Pharm Sci 1993;82(9):912e7. [39] Reis CP, Ribeiro AJ, Houng S, Veiga F, Neufeld RJ. Nanoparticulate delivery system for insulin: design, characterization and in vitro/in vivo bioactivity. Eur J Pharm Sci 2007;30(5):392e7. [40] Devalapally H, Chakilam A, Amiji MM. Role of nanotechnology in pharmaceutical product development. J Pharm Sci 2007;96(10):2547e65. [41] Singh R, Lillard JW. Nanoparticle-based targeted drug delivery. Exp Mol Pathol 2009;86(3):215e23. [42] Fan W, Yan W, Xu Z, Ni H. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf B Biointerfaces 2012;90:21e7. [43] Divya K, Jisha MS. Chitosan nanoparticles preparation and applications. Environ Chem Lett 2018;16(1):101e12. [44] Agarwal M, Nagar DP, Srivastava N, Agarwal MK. Chitosan nanoparticles based drug delivery: an update. Int J Adv Multidiscip Res. 2015;2(4):1e13. [45] Rampino A, Borgogna M, Blasi P, Bellich B, Cesa`ro A. Chitosan nanoparticles: preparation, size evolution and stability. Int J Pharm 2013;455(1e2):219e28. [46] Zhao LM, Shi LE, Zhang ZL, Chen JM, Shi DD, Yang J, Tang ZX. Preparation and application of chitosan nanoparticles and nanofibers. Braz J Chem Eng 2011;28(3):353e62. [47] Banerjee T, Mitra S, Kumar Singh A, Kumar Sharma R, Maitra A. Preparation, characterization and biodistribution of ultrafine chitosan nanoparticles. Int J Pharm 2002;243(1e2):93e105. [48] Ohya Y, Shiratani M, Kobayashi H, Ouchi T. Release behavior of 5-fluorouracil from chitosan-gel nanospheres immobilizing 5-fluorouracil coated with polysaccharides and their cell specific cytotoxicity. J Macromol Sci Part A 1994;31(5):629e42. [49] Kenry, Lim CT. Nanofiber technology: current status and emerging developments. Prog Polym Sci 2017;70:1e17.

Synthesis of micro- and nanoparticles of alginate and chitosan 397 [50] Ohkawa K, Cha D, Kim H, Nishida A, Yamamoto H. Electrospinning of chitosan. Macromol Rapid Commun 2004;25(18):1600e5. [51] Ohkawa K, Minato K-I, Kumagai G, Hayashi S, Yamamoto H. Chitosan nanofiber. Biomacromolecules 2006;7(11):3291e4. [52] Jia Y-T, Gong J, Gu X-H, Kim H-Y, Dong J, Shen X-Y. Fabrication and characterization of poly (vinyl alcohol)/chitosan blend nanofibers produced by electrospinning method. Carbohydr Polym 2007;67(3):403e9. [53] Stokke BT, Smidsroed O, Bruheim P, Skjaak-Braek G. Distribution of uronate residues in alginate chains in relation to alginate gelling properties. Macromolecules 1991;24(16):4637e45. [54] Sreeram KJ, Yamini Shrivastava H, Nair BU. Studies on the nature of interaction of iron(III) with alginates. Biochim Biophys Acta Gen Subj 2004;1670(2):121e5. [55] Nesterova MV, Walton SA, Webb J. Nanoscale iron(III) oxyhydroxy aggregates formed in the presence of functional water-soluble polymers: models for iron(III) biomineralisation processes. J Inorg Biochem 2000;79(1):109e18. [56] Finotelli PV, Morales MA, Rocha-Lea˜o MH, Baggio-Saitovitch EM, Rossi AM. Magnetic studies of iron(III) nanoparticles in alginate polymer for drug delivery applications. Mater Sci Eng C 2004;24(5):625e9. [57] Sarmento B, Ribeiro AJ, Veiga F, Ferreira DC, Neufeld RJ. Insulin-loaded nanoparticles are prepared by alginate ionotropic pre-gelation followed by chitosan polyelectrolyte complexation. J Nanosci Nanotechnol 2007;7(8):2833e41. [58] Hamidi M, Azadi A, Rafiei P. Hydrogel nanoparticles in drug delivery. Adv Drug Deliv Rev 2008;60(15):1638e49. [59] Zahoor A, Sharma S, Khuller GK. Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis. Int J Antimicrob Agents 2005;26(4):298e303. [60] Ahmad Z, Sharma S, Khuller GK. Chemotherapeutic evaluation of alginate nanoparticle-encapsulated azole antifungal and antitubercular drugs against murine tuberculosis. Nanomed Nanotechnol Biol Med 2007;3(3):239e43. [61] Boissie`re M, Allouche J, Chane´ac C, Brayner R, Devoisselle J-M, Livage J, Coradin T. Potentialities of silica/alginate nanoparticles as hybrid magnetic carriers. Int J Pharm 2007;344(1):128e34. [62] Pal A, Esumi K. Photochemical synthesis of biopolymer coated aucore-agshell type bimetallic nanoparticles. J Nanosci Nanotechnol 2007;7(6):2110e5. [63] Joshi A, Keerthiprasad R, Jayant RD, Srivastava R. Nano-in-micro alginate based hybrid particles. Carbohydr Polym 2010;81(4):790e8. [64] Katas H, Raja MAG, Lam KL. Development of chitosan nanoparticles as a stable drug delivery system for protein/SiRNA. Int J Biom 2013;2013. [65] Zhang H, Oh M, Allen C, Kumacheva E, Zhang H, Oh M, Allen C, Kumacheva E. Monodisperse chitosan nanoparticles for mucosal drug delivery monodisperse chitosan nanoparticles for mucosal. Drug Deliv 2004;5(6):2461e8. [66] Chavez de Paz LE, Resin A, Howard KA, Sutherland DS, Wejse PL. Antimicrobial effect of chitosan nanoparticles on Streptococcus mutans biofilms. Appl Environ Microbiol 2011;77(11): 3892e5. [67] Azizian S, Hadjizadeh A, Niknejad H. Chitosan-gelatin porous scaffold incorporated with chitosan nanoparticles for growth factor delivery in tissue engineering. Carbohydr Polym December 15, 2018;202:315e22. [68] Pelegrino MT, Weller RB, Chen X, Bernardes JS, Seabra AB. Chitosan nanoparticles for nitric oxide delivery in human skin. Medchemcomm 2017;8(4):713e9. [69] Grenha A, Al-Qadi S, Seijo B, Remun˜a´n-Lo´pez C. The potential of chitosan for pulmonary drug delivery. J Drug Deliv Sci Technol 2010;20(1):33e43.

398 Chapter 16 [70] Calvo P, Remun˜a´n-Lo´pez C, Vila-Jato JL, Alonso MJ. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci 1997;63(1):125e32. [71] Calvo P, Remun˜an-Lo´pez C, Vila-Jato JL, Alonso MJ. Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines. Pharmaceut Res 1997:1431e6. [72] Miranda-Caldero´n JE, Macı´as-Rosales L, Gracia-Mora I, Ruiz-Azuara L, Faustino-Vega A, Gracia-Mora J, Bernad-Bernad MJ. Effect of casiopein III-ia loaded into chitosan nanoparticles on tumor growth inhibition. J Drug Deliv Sci Technol December 2018;48:1e8.