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Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles
Catarina Pinto Reis, PhD,a Ronald J. Neufeld, PhD,b,4 Antonio J. Ribeiro, PhD,c Francisco Veiga, PhDa ´
Laboratorio Tecnologia Farmaceutica, Faculdade de Farmacia, Universidade de Coimbra, Coimbra, Portugal ´ ˆ ´ b Chemical Engineering Department, QueenTs University, Kingston, Ontario, Canada c Departamento Tecnologia Farmaceutica, I.S.C.S.N., Gandra, Paredes, Portugal ˆ Received 21 August 2005; Accepted 20 December 2005
Polymeric nanoparticles have been extensively studied as particulate carriers in the pharmaceutical and medical fields, because they show promise as drug delivery systems as a result of their controlled- and sustained-release properties, subcellular size, and biocompatibility with tissue and cells. Several methods to prepare nanoparticles have been developed during the last two decades, classified according to whether the particle formation involves a polymerization reaction or arises from a macromolecule or preformed polymer. In this review the most important preparation methods are described, especially those that make use of natural polymers. Advantages and disadvantages will be presented so as to facilitate selection of an appropriate nanoencapsulation method according to a particular application. D 2006 Elsevier Inc. All rights reserved.
Drug delivery; Nanoparticles; Preparation methods
Nanoencapsulation of drugs involves forming drugloaded particles with diameters ranging from 1 to 1000 nm. Nanoparticles are defined as solid, submicron-sized drug carriers that may or may not be biodegradable [1,2]. The term nanoparticle is a collective name for both nanospheres and nanocapsules. Nanospheres have a matrix type of structure. Drugs may be absorbed at the sphere surface or encapsulated within the particle. Nanocapsules are vesicular systems in which the drug is confined to a cavity consisting of an inner liquid core surrounded by a polymeric membrane . In this case the active substances are usually dissolved in the inner core but may also be adsorbed to the capsule surface . Nanoparticles are receiving considerable attention for the delivery of therapeutic drugs. The literature emphasizes the advantages of nanoparticles over microparticles  and liposomes [5,6]. The submicron size of nanoparticles offers
4 Corresponding author. Chemical Engineering Department, Dupuis Hall, Queens University, Kingston, Ontario, Canada K7L3N6. E-mail address: firstname.lastname@example.org (R.J. Neufeld). 1549-9634/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2005.12.003
a number of distinct advantages over microparticles, including relatively higher intracellular uptake compared with microparticles. In terms of intestinal uptake, apart from their particle size, nanoparticle nature and charge properties seem to influence the uptake by intestinal epithelia. Uptake of nanoparticles prepared from hydrophobic polymers seems to be higher than that of particles with more hydrophilic surfaces , thus more hydrophilic particles may be rapidly eliminated. Generally, nanoparticles based in hydrophobic polymers such as poly(styrene), uncharged and positively charged, provide an affinity to follicle-associated epithelia as well as to absorptive enterocytes, whereas negatively charged poly (styrene) nanoparticles show only low affinity to any type of intestinal tissues. In contrast, nanoparticles based on hydrophilic polymers, negatively charged, show a strong increase in bioadhesive properties and are absorbed by both M cells and absorptive enterocytes. A combination of both nanoparticle surface charges and increased hydrophilicity of the matrix material seem to affect the gastrointestinal uptake in a positive sense.
and poly(butylcyanoacrylate)  nanoparticles were produced by dispersion via surfactants into solvents such as cyclohexane (ICH. The method is classified into two categories. Methods Many methods have been developed for preparing nanoparticles. biodistribution pattern. the interfacial condensation method will be further discussed later in this review. these methods can be classified into two main categories according to whether the formulation requires a polymerization reaction or is achieved directly from a macromolecule or preformed polymer . consequently. There are now numerous preparation methods available for producing nanoparticles. Thus the hydrophilic PEG chains allow the control of protein and peptide absorption and. Thus the polymeric composition (hydrophobicity. This review covers all these methods including their detailed procedures and technological advantages. This classification will be crucial to evaluate the following methods and finally. and. alternative approaches are of greater interest. it is now possible to choose the best method of preparation and the best polymer to achieve an efficient entrapment of the drug. poly(ethylcyanoacrylate) (PECA). Biology. It is well known that the reticuloendothelial system. based on the use of an organic or aqueous continuous phase. Pinto Reis et al. the monomer molecule can be transformed into an initiating radical by high-energy radiation. and class 3 (solvents with low toxic potential). remove them from systemic circulation. generally conventional nanoparticles (no surface modification) and negatively charged particles can be rapidly opsonized and massively cleared by the fixed macrophages. Later. class 3). as well as providing several examples of encapsulants that are entrapped into or adsorbed to these particles. Nanoparticles can be also prepared directly from preformed synthetic or natural polymers and by desolvation of macromolecules.11]. surfactants. is a major obstacle to active targeting because of its ability to recognize these systems. to apply a specific technique to a particular pharmaceutical application. are shown in Table 1. and the surfactants or emulsifiers are not needed. The single monomer methylmethacrylate (MMA) was used to . once in the bloodstream.C. Examples of drugs encapsulated with this system are triamcinolone . localization in the nanospheres by adsorption or incorporation) have a great influence on the drug absorption. charge. pilocarpine . and timolol . Alternatively. or coating modification with polymers  such as the attachment of poly(ethylene glycol) (PEG) chains to biodegradable polymer such as poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA). fluorescein . PMMA nanoparticles are suitable adjuvants for vaccines and are produced by a radical emulsion polymerization mechanism —generally without emulsifiers . Nanoparticles obtained by polymerization of a monomer Emulsion polymerization Emulsion polymerization is one of the fastest methods for nanoparticle preparation and is readily scalable . surface charge. As one of the first methods for production of nanoparticles. and there are two types of emulsion polymerization—organic and aqueous—depending on the continuous phase. n-pentane (ICH. in addition will allow regulation of cell behavior at the polymer surface. Additionally. and elimination. In the aqueous continuous phase the monomer is dissolved in a continuous phase that is usually an aqueous solution. Depending on the physicochemical characteristics of a drug. Recently these polymeric systems have been prepared by nebulization techniques. Phase separation and formation of solid particles can take place before or after termination of the polymerization reaction . because it requires toxic organic solvents. and Medicine 2 (2006) 8–21 9 The nanoparticle surface is also a very important consideration in targeting drug delivery. The most important methods of nanoparticle preparation. The polymerization methods can be further classified into emulsion and interfacial polymerization. The polymerization process can be initiated by different mechanisms. Surface modification of these nanoparticulate systems with hydrophilic polymers is the most common way to control the opsonization process and to improve the surface properties of the system. surfactants or protective soluble polymers were used to prevent aggregation in the early stages of polymerization . organic solvents assessed in this review are classified according to International Conference on Harmonization (ICH) classification according to their possible risk to human health and placed into one of three classes as follows: class 1 (solvents to be avoided). which are subsequently eliminated from the formed particles. / Nanomedicine: Nanotechnology. mainly the liver and spleen. avoid the effective delivery of the nanospheres to organs other than those of the reticuloendothelial system . Indeed. The continuous organic phase methodology involves the dispersion of monomer into an emulsion or inverse microemulsion. This procedure has become less important. As a result of the nonbiodegradable nature of this polymer as well as the difficult procedure. poly(methylmethacrylate) (PMMA). and the associated drug (molecular weight. any adjuvant substances. monomers and initiator. Moreover. and biodegradation profile) of the nanoparticles. class 2) as the organic phase. including g-radiation. as well as the encapsulants and the resulting approximate particle sizes. Polyacrylamide nanospheres were produced by this method [10. or ultraviolet or strong visible light. class 2 (solvents to be limited). Chain growth starts when initiated monomer ions or monomer radicals collide with other monomer molecules according to an anionic polymerization mechanism . Initiation occurs when a monomer molecule dissolved in the continuous phase collides with an initiator molecule that might be an ion or a free radical. or into a material in which the monomer is not soluble (nonsolvent). class 2). and toluene (ICH.
63]               [44. and –poly(alkylmethacrylate) Interfacial deposition Poly(lactic acid) Indomethacin 230 Salting-out Poly(lactic acid) Savoxepin b 1000 Other polymers: Poly(alkylmethacrylate) and ethylcellulose Emulsion/solvent diffusion Poly(lactic acid)–poly(glycolic acid) copolymer p-THPP 117 – 118 Doxorubicin b 1000 Poly(lactic acid) p-THPP 125 DNA b 300    [53. PVM/MA . poly(phenylesters) and polyurethanes Interfacial polycondensation Polyurethane and poly(etherurethane) a-Tocopherol b 500 Nanoparticles obtained directly from a macromolecule or a preformed polymer Nanoparticles prepared with synthetic/semi-synthetic preformed polymer Solvent evaporation b1000 Poly(lactic acid) Testosterone 100 or 120 Albumin 150 Tetanus toxoid ~300 Loperamide Poly(lactic acid)–poly(glycolic acid) copolymer DNA ~100 Cyclosporin A ~300 Other polymers: Poly(E-caprolactone) and poly(h-Hydroxybutyrate)  Solvent displacement ~168 Poly(lactic acid)–poly(glycolic acid) copolymer Indomethacin 274 Doxorubicin ~170 Cyclosporin A ~166 Valproic acid ~167 Ketoprofen ~187 Vancomycin ~105 – 170 Insulin Poly(lactic acid) Doxorubicin 270 Taxol ~260 Dexamethasone ~300 Vitamin K ~270 Poly(E-caprolactone) Cyclosporin A ~100 – 200 Other polymers: h-cyclodextrin inclusion complexes . poly(butylcyanoacrylate).44]       Poly(hexylcyanoacrylate) Poly(isohexylcyanoacrylate) Poly(dialkylmethylidene malonate) Continuous organic phase Polyacrylamide Poly(methylcyanoacrylate) Enzymes b1000 Triamcinolone 500 Fluorescein 800 – 1000 300 – 600 Pilocarpine Other polymers: Poly(ethylcyanoacrylate). / Nanomedicine: Nanotechnology.68.79]      (continued on next page) . and encapsulants Polymer Nanoparticles obtained by polymerization of a monomer Emulsion polymerization Continuous aqueous phase Poly(methylmethacrylate) Poly(methylmethacrylate) copolymers Poly(methylcyanoacrylate) Poly(ethylcyanoacrylate) Poly(butylcyanoacrylate) Poly(isobutylcyanoacrylate) Encapsulant Size (nm) Reference Influenza antigen Doxorubicin Vinblastine Insulin Progesterone Ampicillin Antibodies hGRF Vincamine Ampicillin Doxorubicin Primaquine 130 300 200 – 300 b 500 250 40 – 80 120 140 – 170 200 30 – 80 300 b 1000                  [43. poly(vinylpyridine) and poly(acroleine) Interfacial polymerization Poly(ethylcyanoacrylate) Insulin ~151 220 240 Poly(isobutylcyanoacrylate) Indomethacin Darodipine 150 Insulin 150 – 300 Calcitonin b 1000 Octreotide 260 Poly(isohexylcyanoacrylate) Phthalocyanines 180 Other polymers: Polyamides. and Medicine 2 (2006) 8 – 21 Table 1 Examples of preparative methods. polymers. Pinto Reis et al.10 C. poly(styrene). Biology.
Encapsulation efficiencies reached 50% and 30% for the larger and the smaller capsules. / Nanomedicine: Nanotechnology. which is an acceptable excipient for oral administration. class 3) containing surfactant. thus avoiding heat and highenergy radiation. Sparingly water-soluble molecules such as progesterone . complexing with macromolecules. albumin and ethylcellulose Nebulization techniques Poly(lactic acid) 400 – 600 hGRF. poly(isobutylcyanoacrylate) [37.44]. The resulting colloidal suspension can be concentrated by evaporation under vacuum. Polymerization occurs at room temperature and requires neither g-irradiation nor a chemical initiator . p-THPP.46].38]. another method has been proposed [45. in that PACA is readily biodegradable . Interfacial polymerization Poly(alkylcyanoacrylate) nanoparticles One of the advantages of these polymers is their very rapid polymerization—occurring during seconds—initiated by ions present in the medium . organic solvents. the advantage of obtaining nanocapsules by this method is that the polymer is formed in situ. An advantage of interfacial polymerization techniques is high-efficiency drug encapsulation (eg. The final product was a suspension of nanocapsules in Migliol. produce PMMA nanospheres until 1986. pH changes. Besides the monomer. calcitonin . This mixture was then slowly extruded through a needle into a well-stirred aqueous solution. Insulin . salts. polymerization requires a chemical or physical initiation. As a result. with or without some ethanol (ICH. poly(dialkylmethylidene malonate) nanospheres were prepared at pH 6 . the main disadvantage is the use of organic solvents required for the external phase. mesotetra(hydroxyphenyl)porphyrin. To encapsulate highly watersoluble drugs. when the copolymerization of MMA. or chemical cross-linking. sonication. Pinto Reis et al. Hydrophilic drugs are encapsulated with good efficiency. Poly(diethylmethylidene malonate) nanospheres loaded with primaquine have also been prepared at pH 7. and second.4 . potentially toxic compounds were not used. First. indomethacin [43. The entrapment efficiency rose to 100% when the drug was added to the polymerization medium. thus no purification procedure was necessary. allowing the polymer membrane to follow the contours of the inner phase of an oil/water or water/oil emulsion . methacrylic acid. Biology. triamcinolone diacetate . Washing of solvents and replacement by water represents a timeconsuming and difficult procedure . 4 Induced by heat. and poly(isohexylcyanoacrylate)  were used in production of nanoparticles by this process.129]        Desolvation of macromolecules4 Gelatin Other polymers: casein. class 3) or in a liquid surfactant before being added to the aqueous polymerization medium. Nanocapsules are formed spontaneously by polymerization of cyanoacrylate after contact with initiating ions present in the water. and photoactivatable cytotoxic compounds used in photodynamic tumor therapy like phthalocyanines in an injectable vehicle . insulin with 95%) . and Medicine 2 (2006) 8–21 Table 1 (continued) Other polymer: Poly(E-caprolactone) Nanoparticles produced from natural macromolecules Serum albumin Gelatin Polysaccharides 11 Doxorubicin Mitomycin C Oligonucleotides Doxorubicin Insulin Tetanus and diphtheria toxoid Calcitonin DNA Cytostatics Insulin 200 – 1500 280 b1000 b1000 b1000 b1000 b1000 b1000   [128. doxorubicin) . The main difference is that the cyano group is replaced by an alkyloxycarbonyl group that is less reactive in the presence of the hydroxide ion initiator. can also be loaded in PACA nanospheres but must be dissolved in a solvent such as ether (ICH. PECA . and monoclonal antibodies  can also be incorporated into PACA nanospheres. poly(alkylcyanoacrylate) (PACA) nanoparticles seem to be a good alternative. PMMA nanospheres are not biodegradable . such as that for ampicillin  and doxorubicin . octreotide . human growth hormone–releasing factor. The polymerization mechanism is an anionic process. and ethylene glycol dimethacrylate was described to increase the hydrophilicity of the particles with the intention of modifying the distribution of the particles within the body . The preparation of PMMA nanospheres is simple and drugs can be successfully entrapped. darodipine . In this case. The percentage of drug incorporated or adsorbed decreases generally with the increasing amount of drug in the polymerization medium .40]. Various drugs or tracers were encapsulated into nanospheres with high entrapment efficiencies (eg. As well. . Cyanoacrylate monomer and drug were dissolved in a mixture of an oil and absolute ethanol . In addition.C. Examples of drugs encapsulated are insulin [36. growth hormone–releasing factors (GRFs) . hydroxypropylmethacrylate. respectively . class 3) or acetone (ICH. but two drawbacks have to be kept in mind.
whereas interfacial deposition forms only nanocapsules.12 C. leading to the precipitation of nanospheres. Nanoparticles obtained from preformed polymers With the exception of alkylcyanoacrylates and poly (dialkylmethylidene malonate). . and indomethacin . Interfacial polycondensation Polymeric nanoparticles can be also prepared by the interfacial polycondensation of the lipophilic monomer. Biology. A polymer organic solution containing the dissolved drug is dispersed into nanodroplets. Even though different types of emulsions may be used. To avoid these limitations. etc. This phase is injected into a stirred aqueous solution containing a stabilizer as a surfactant. Drugs or model drugs encapsulated were albumin . Frequently used polymers are PLA [52. generally PLA. phase separation is performed with a totally miscible solvent that is also a nonsolvent of the Fig 2. most of the monomers suitable for a micellar polymerization process in an aqueous phase lead to slowly biodegradable or nonbiodegradable polymers. To facilitate the formation of colloidal polymer particles during the first step of the procedure. The solvent is subsequently evaporated by increasing the temperature under pressure or by continuous stirring . Polyurethaneand poly(ether urethane)–based nanocapsules were synthesized by interfacial reaction between two monomers. inducing polymer precipitation as nanospheres. cyclosporin A [65. a fifth compound was introduced only on preparation of nanocapsules. this simplifies and thus improves process economics. requiring meticulous purification of the colloidal material. Polymer deposition on the interface between the water and the organic solvent. viscosity of organic and aqueous phases. Solvent displacement involves the precipitation of a preformed polymer from an organic solution and the diffusion of the organic solvent in the aqueous medium in the presence or absence of a surfactant [68-71]. this method can only be applied to liposoluble drugs. During the second step polymer solvent is evaporated. nucleic acid . oligomer. texanus toxoid .53]. diethylenetriamine. PLGA . and poly(h-hydroxybutyrate) (PHB) [59. surfactant. polyurethane polymer and poly (ether urethane) copolymers were chosen and successfully applied as drug carriers for a-tocopherol . In this case. The first step requires emulsification of the polymer solution into an aqueous phase (see Figure 1). A modified interfacial polycondensation method was also developed. leads to the instantaneous formation of a colloidal suspension . such as phtaloyldichloride and the hydrophilic monomer. **Surfactant is optional. The polymer.) can be more or less toxic. The polymer precipitates in the form of nanospheres in which the drug is finely dispersed in the polymer matrix network. oil/water emulsions are of interest because they use water as the nonsolvent. The size can be controlled by adjusting the stir rate. caused by fast diffusion of the solvent. methods using preformed polymers instead of monomers have been proposed. These nanoparticles were smaller than 500 nm. However. in a nonsolvent or suspension medium such as chloroform (ICH. because it eliminates the need for recycling. Schematic representation of the solvent displacement technique.63]. facilitating the washing step and minimizing agglomeration . poly(E-caprolactone) (PCL) [56-58]. ethylcellulose (EC) . and temperature . using a dispersing agent and high-energy homogenization .60]. is dissolved in a water-miscible solvent of intermediate polarity. testosterone . Synthetic preformed polymers Emulsification/solvent evaporation Emulsification-solvent evaporation involves two steps. residual molecules in the polymerization medium (monomer. and Medicine 2 (2006) 8 – 21 Fig 1. Schematic representation of the emulsification-evaporation technique. ***In interfacial deposition method. class 3). and limitations are imposed by the scale-up of the high energy requirements in homogenization . / Nanomedicine: Nanotechnology. loperamide [53. Pinto Reis et al. cellulose acetate phthalate . class 2) or ethyl acetate (ICH. However. prazinquantel . Solvent displacement and interfacial deposition Solvent displacement and interfacial deposition are similar methods based on spontaneous emulsification of the organic internal phase containing the dissolved polymer into the aqueous external phase (see Figure 2). in the presence and absence of the surfactant . type and amount of dispersing agent. solvent displacement forms nanospheres or nanocapsules.66]. In addition.
In fact. reducing encapsulation efficiency. / Nanomedicine: Nanotechnology. spontaneous emulsification is not observed if the coalescence rate of the formed droplets is sufficiently high . even though some water-miscible solvents produce a certain instability when mixed in water. according to its boiling point. miscible with the solvent of the polymer but immiscible with the mixture. to produce the precipitation of the polymer and the consequent formation of nanoparticles. this one is efficient in encapsulating lipophilic drugs Fig 4. Subsequently. An aqueous solution is used as the dispersing medium. The polymer deposits on the interface between the finely dispersed oil droplets and the aqueous phase. no need for homogenization. Then. Emulsification/solvent diffusion Emulsification/solvent diffusion (ESD) was proposed in the literature based on the use of organic solvents. acetone. high loading efficiencies are generally reported for lipophilic drugs when nanocapsules are prepared . it is necessary to promote the diffusion of the solvent of the dispersed phase by dilution with an excess of water when the organic solvent is partly miscible with water or with another organic solvent in the opposite case.79]. Schematic illustration of the ESD technique. and Medicine 2 (2006) 8–21 13 Fig 3. Disadvantages are the high volumes of water to be eliminated from the suspension and the leakage of water-soluble drug into the saturated-aqueous external phase during emulsification. the dichloromethane increases the mean particle size  and is considered toxic. The main difference is that polymers such as PLA are dissolved together with the drug in a solvent mixture (eg. Highly loaded nanoparticulate systems based on amphiphilic h-cyclodextrins to facilitate the parenteral administration of the poorly soluble antifungal drugs bifonazole and clotrimazole were prepared according to the solvent displacement method . because entrapment efficiencies as high as 98% were obtained . high batch-to-batch reproducibility. class 2) are used to dissolve and increase the entrapment of drugs. and phospholipids) . polymer . and then it was adapted to the following salting-out procedure. This method has been applied to various polymeric materials such as PLA . forming nanocapsules . Interfacial deposition is a process used for the production of nanocapsules. because the solvent and the nonsolvent of the polymer must be mutually miscible. This technique presents several advantages. of oil nature. The progressive addition of the polymer solution to the nonsolvent generally leads to the formation of nanospheres close to 200 nm in size. benzyl benzoate. it seems difficult to choose a drug/polymer/ solvent/nonsolvent system in which particles would be formed and the drug efficiently entrapped. In interfacial deposition. Polymer deposition occurs at the interface between water and benzoyl nanodroplets. The usefulness of this simple technique  is limited to water-miscible solvents. simplicity.C. In fact. acetone/dichloromethane (ICH. Pinto Reis et al. . according to the oil-to-polymer ratio. a fifth compound is introduced. resulting in the deposition of the polymer in the form of nanoparticles of about 230 nm in size [44. forming nanocapsules with a shell-like wall . leading to solvent diffusion to the external phase and the formation of nanospheres or nanocapsules. however. This mixture is injected slowly into a stirred aqueous medium. the polymer-water saturated solvent phase is emulsified in an aqueous solution containing stabilizer.77]. This technique was well adapted for the incorporation of cyclosporin A. The encapsulating polymer is dissolved in a partially watersoluble solvent such as propylene carbonate (ICH not given) and saturated with water to ensure the initial thermodynamic equilibrium of both liquids. The procedure is illustrated in Figure 3. the solvent is eliminated by evaporation or filtration. Finally. As with some of the other techniques. The solvent displacement technique allows the preparation of nanocapsules when a small volume of nontoxic oil is incorporated in the organic phase . This method is basically applicable to lipophilic drugs because of the miscibility of the solvent with the aqueous phase . This phenomenon has been explained by local variations of the interfacial tension between the two immiscible liquids due to the mutual diffusion of the third liquid. Although. and narrow size distribution. and poly(methyl vinyl ether-comaleic anhydride) (PVM/MA) [76. Schematic of the salting-out technique. ease of scale-up. PLGA . in which the diffusion rate is enough to produce spontaneous emulsification. and it is not an efficient means to encapsulate water-soluble drugs. Nanoparticles seem to be formed by a mechanism comparable to the bdiffusion and standingQ process found in spontaneous emulsification. this is not a polymerization technique but an emulsification/solidification technique. Biology. Considering the oil-based central cavities of the nanocapsules. PCL . such as high encapsulation efficiencies (generally N70%).68.
The main advantage of salting out is that it minimizes stress to protein encapsulants . because it can play an important role in the encapsulation efficiency of the drug. A technique was proposed based on the desolvation of natural macromolecules. indocyanine .81]. iso-octane emulsions. The preparative steps of this procedure are described in Figure 4. / Nanomedicine: Nanotechnology. doxorubicin-loaded PLGA nanoparticles . Pinto Reis et al. this technique does not offer much advantage over the other techniques .3-butadiene or formaldehyde. or non -electrolytes such as sucrose) and a colloidal stabilizer such as polyvinylpyrrolidone or hydroxyethylcellulose. characterized by the method of stabilization. nanoparticles were produced emulsifying serum albumin aqueous solution in cottonseed oil at 258C . thus inducing the formation of nanospheres. Production of nanoparticles from natural macromolecules Albumin nanoparticles produced in an external-oily emulsion Two main methods are used in the preparation of albumin microspheres. including mesotetra(hydroxyphenyl) porphyrin-loaded PLGA (p-THPP) nanoparticles [80. Polymer and drug are initially dissolved in a solvent such as acetone. and magnesium acetate. Gelatin nanoparticles produced in an external-oily emulsion Emulsified gelatin solution droplets were hardened by cooling the emulsion below the gelation point in an ice bath. thermal treatment at elevated temperatures (958–1708C) or chemical treatment in vegetable oil. Schematic representation of the emulsification–internal gelation technique using alginate.out is based on the separation of a watermiscible solvent from aqueous solution via a salting-out effect. This oil/water emulsion is diluted with a sufficient volume of water or aqueous solution to enhance the diffusion of acetone into the aqueous phase. calcium chloride. . or aqueous medium. Both the solvent and the salting-out agent are then eliminated by cross-flow filtration . This mixture was cooled and diluted with ethyl ether to reduce the viscosity of the oil phase to permit separation by centrifugation. For this reason. Heat treatment of albumin is applicable only to drug molecules that are not heat sensitive. In this case albumin nanospheres were formed by homogenizing the oil phase containing the albumin droplets and thermally stabilized by heating at 1758 to 1808C for 10 minutes . The particles were stirred. may be useful when heatsensitive substances have to be processed . The salting-out procedure can be considered as a modification of the emulsification/solvent diffusion. Other techniques involve slight modification of either of the two methods . . therefore. The greatest disadvantages are exclusive application to lipophilic drugs and the extensive nanoparticle washing steps . The emulsified macromolecule is subsequently cross-linked with glutaraldehyde and washed. Salting out with synthetic polymers Salting . cyclosporin (Cy-A)-loaded gelatin and cyclosporin (Cy-A)-loaded sodium glycolate nanoparticles . but the purification step remains the main problem with the elimination of the cottonseed oil . Particles released the drug doxorubicin much faster than particles formed by heat treatment . Biology. Because of the need for chlorinated solvents. coumarin-loaded PLA nanoparticles . and Medicine 2 (2006) 8 – 21 Fig 5. which simplifies the purification step . such as magnesium chloride. and EC nanospheres leads to high efficiency and is easily scaled up . isolated by centrifugation. As a modification of this method . Salting out does not require an increase of temperature and. and dried by lyophilization. plasmid DNA-loaded PLA nanoparticles . which is subsequently emulsified into an aqueous gel containing the salting-out agent (electrolytes. This technique used in the preparation of PLA. poly(methacrylic) acid. The selection of the saltingout agent is important. an aqueous solution of albumin was emulsified in chloroform containing hydroxypropylcellulose and EC as stabilizers.14 C. then denaturing the albumin by resuspending the particles in ether containing the cross-linking agents 2. Several drug-loaded nanoparticles were produced by the ESD technique.
these are difficult to remove and may cause toxicity . vaccines . This technique is applicable to heat-sensitive drugs. Another interesting system for drug delivery systems could be nanoparticulate carriers from bioacceptable macromolecules. / Nanomedicine: Nanotechnology. and this point must be carefully considered. results in a diameter of 400 nm. Agarose nanoparticles Agarose nanoparticles were developed for the administration of therapeutic proteins and peptides . insulin . Compared with the previously described method. and cross-linked with formaldehyde . cross-linking increases significantly the size of the particles. Gelled nanodroplets were filtered. Chitosan nanoparticles obtained by formation of a spontaneous complex between chitosan and polyanions such as tripolyphosphate  have small diameters (200 –500 nm) and show a quasi spherical shape under transmission electron microscopy. Gliadins possess the ability to interact with epidermal keratin as a result of their richness in praline. This methodology requires the preparation of an agarose . The preparation of alginate nanoparticles via this method does not require specialized equipment and can be performed at ambient temperature. inert. The hydrogel. and biocompatible. forms a suitable matrix for proteins and peptides that can be entrapped in the gel during formation . but new strategies have been devised. In this context. Alginate particles are usually produced by dropwise extrusion of sodium alginate solution into calcium chloride solution. The particle size ranged between 100 and 600 nm with a mean of 280 nm . anticancer agents . being hydrophilic. a number of drugs can be covalently bound to the gelatin by formaldehyde treatment. however. which constitutes a disadvantage . Chitosan nanoparticles produced by a promoting gelation in an emulsification-based method as illustrated in Figure 6. resulting in gelation of the gelatin droplets. In an additional advance. Additionally. alginate particles have been produced by using a modified emulsification/internal gelation method  as illustrated in Figure 5. Biology. obtained by air atomization . The preparation of alginate nanoparticles was first achieved in a diluted aqueous sodium alginate solution in which gelation was induced by the addition of a low concentration of calcium. The methods proposed to prepare chitosan nanoparticles are based on the spontaneous formation of complexes between chitosan and polyanions  or the gelation of a chitosan solution dispersed in an oil emulsion . Schematic representation of chitosan nanoparticles preparation by the emulsification technique. Agarose nanoparticles were produced using an emulsion-based technology as illustrated in Figure 7. The main difficulty of this method is the nanoparticle washing step to eliminate the residual oil droplets. Pinto Reis et al. The smallest particles produced had a minimum size of 1 to 5 Am. Various methods for producing chitosan nanoparticles are described in the literature [105. to efficiently encapsulate lipophilic substances such as a-tocopherol .C. vegetable protein fractions termed gliadins have been chosen from wheat gluten.101]. For this reason. Thermal gelation results from the formation of helicoidal structures responsible for a three-dimensional network in which large amounts of water can be entrapped. Alginate nanoparticles Sodium alginate is a water-soluble polymer that gels in the presence of multivalent cations such as calcium . and nucleic acids [100. Agarose aqueous solution forms thermally reversible hydrogels while being cooled below the gelling temperature (318–368C). it would be of interest to study carefully the influence of different formulation and process parameters in this process according to the application. Chitosan considerably enhanced the absorption of peptides such as insulin and calcitonin across the nasal epithelium . This leads to the formation of invisible clusters of calcium alginate gels. this technique has a major disadvantage of involving organic solvents during the isolation of the particles. this property leads to a desired controlled release of drug. and Medicine 2 (2006) 8–21 15 Fig 6. tetanus and diphtheria toxoid . washed. Chitosan nanoparticles Chitosan nanoparticles have been developed to encapsulated proteins such as bovine serum albumin. Furthermore. Alginate particle size depends on the size of the initial extruded droplet.106]. a significant disadvantage of the cross-linking agent relates to its toxicity.
Techniques based on supercritical or compressed fluid can be an interesting tool for preparation of nanoparticulate and microparticulate products . but unfortunately the yield is comparatively low. The liquid nanodroplets then gel to protein-containing agarose hydrogel nanoparticles. it is necessary to incorporate some additives. Many variables can affect the stability of nanoparticles.115] have been applied to produce nanoparticles. ineffective drug release. or by addition of a desolvating agent (ethanol or concentrated inorganic salt solutions). Among other requirements. Recent reports outline the important use of gelatin as drug delivery systems for DNA  and cytostatics . / Nanomedicine: Nanotechnology. cross-flow filtration. it is very difficult to dissolve strong polar substances in supercritical CO2. and the solute particles eventually precipitate. because the precipitated solute is free of solvent. Nanoparticles were prepared using the process of reversible swelling of macromolecules [90. Nanoparticles constitute a relatively stable physical system because of their colloidal nature. and the solution is expanded through a nozzle. such . Chemical integrity of drug is also a fundamental aspect of the overall stability evaluation of the nanoparticles. ultracentrifugation. Pinto Reis et al. may be useful when heatsensitive drugs are used . In fact. recently. The main advantage is that this process does not require an increase in temperature and. therefore. This technique is clean. Gelation of agarose is then induced by diluting the emulsion with cold corn oil under agitation at 58C. and casein  as the macromolecular materials. However. The small size of the dispersed aqueous nanodroplets is achieved by homogenization. Finally. a colloidal suspension is stable and does not tend to separate as a result of slow deposition due to the mixing tendencies of diffusion and convection. In the case of gelatin. bovine serum albumin . some agglomeration can occur. However. and Medicine 2 (2006) 8 – 21 Fig 7. and elevated operating pressures requiring highpressure equipment.111] using gelatin . Variations in nanoparticle production by the desolvation process were described . drug degradation or denaturation. the use of cosolvents and/or surfactants to form microemulsions makes it possible to dissolve polar and ionic species. New techniques based on supercritical or compressed fluids Some of the techniques described above are complex. low drug loading. Protein drugs such as insulin were encapsulated in poly(ethylene glycol)/poly(l-lactide) (PEG/ PLA) nanoparticles by this technique . Nanoparticles produced by desolvation of macromolecules Another technology applicable to a wide range of polymers is based on desolvation by charge and pH changes. The most commonly reported procedures are gel filtration. To prevent a complete precipitation. Biology. nanoparticles should be easily stored and administered. solution in corn oil emulsion at 408C . Some parameters are crucial for the stability. and. Generally. The necessity for and degree of purification are dependent on the final purpose of the formulation developed. It has been demonstrated for numerous applications involving protein drug delivery systems. The supercritical fluid is evaporated in the spraying process. this new process requires a high initial capital investment for equipment. Schematic illustration of agarose nanoparticles preparation by the emulsification technique.16 C. but the use of potentially toxic compounds such as glutaraldehyde and desolvating agents requires subsequent purification . Another requirement relates to the sterilization of the formulation. Peptides and proteins to be encapsulated are initially added to the agarose solution. or unsuitable physical and morphological properties . the drug and the polymer are solubilized in a supercritical fluid. In addition. dialysis. compressed supercritical fluids require elaborate recycling measures to reduce energy costs. However. human serum albumin . This process offers the advantage of producing nanoparticles directly in aqueous suspension. Finally. different methods such as the two-step desolvation method [114. and the products may often be characterized by high residual solvent content. supercritical CO2 has solvating properties characteristic of both fluorocarbons and hydrocarbons. they must be free of impurities. In this technique. It also provides advantages such as suitable technological and biopharmaceutical properties and high quality. The choice of the sterilizing treatment depends on the physical susceptibility of the system. Stability upon storage of the nanoparticles Nanoparticles are intended to be administered as pharmaceutical dosage forms in humans.
there are several problems that remain to be solved. especially in cases in which the storage conditions are unfavorable. The process is not suitable to all drugs. Sometimes cryoprotectants like glucose. the postpreparative steps. Freezing is the most aggressive step of the freeze-drying operation for colloidal operations. / Nanomedicine: Nanotechnology. Simple. Other difficulties remain. Such excipients are indeed cryoprotectants that facilitate the aqueous reconstitution of the freeze-dried product. In some systems the ease of redispersion depended on the manufacturing process. inadequate stability of certain active components. and residual solvent analysis must be extensively investigated. usually used for purposes of isotonicity (eg. Lyophilization (freeze-drying) seems to be a highly stabilizing process. it is now possible to choose the best method of preparation and the best polymer to achieve an efficient entrapment of the drug. are summarized in Table 2. Efficient drug entrapment and transition to large scale are of utmost importance to industrial applicability. There are now numerous preparation methods available for producing nanoparticles. or even in the refrigerator. such as purification and preservation. and sorbitol were added to ensure redispersibilty or to allow vitrification of the suspension during the cooling and to avoid crystallization of the liquid suspension. It is generally applied to enhance the physicochemical stability of the nanoparticles to achieve a pharmaceutically acceptable product. It is thus important to improve the nanoparticle resistance by addition of a cryoprotectant to avoid alteration of the suspension . nanoparticles are obtained in the form of a dry powder that is easy to handle and store. Finally. and reproducible techniques are now available to prepare drug-loaded nanospheres and nanocapsules. as the duration of contact with the aqueous environment when the drug is water soluble. and Medicine 2 (2006) 8–21 Table 2 Polymeric nanoparticles: general advantages and drawbacks of the preparation methods Method Polymerization of monomers Emulsion polymerization Organic Aqueous Interfacial polymerization Preformed polymers Synthetic Emulsification/solvent evaporation Solvent displacement and interfacial deposition Salting out Emulsion/solvent diffusion Natural Albumin Gelatin Polysaccharides Alginate Chitosan Agarose Desolvation Simplicity of procedure Need for purification Facility scaling-up EE (%) 17 Safety of compounds Low High Low High High High Medium NR NR High High Medium NR High High High Low NR High Medium High High Medium Medium High High NR High Medium Low NR High High NR NR High High NR NR Low High High Medium High High High Medium Medium High High NR Low Low Medium Low Medium Medium Low Medium Low Low High High High Low EE. together with their advantages and disadvantages. no reference available. the development of a technique that allows the incorporation of biomolecules without affecting their activity constitutes a fundamental goal for nanotechnology. The evolution of nanoparticle preparation methods has been marked by three aspects: need for less toxic reagents. particularly important for nanocapsules. and important technological advances have been achieved. and light exposure when the drug is light sensitive. In this respect. Biology. After complete desiccation. In most cases the freeze-dried particles are readily dispersible in aqueous solutions. Pinto Reis et al. trealose. or in a laboratory desiccator. This technique involves the freezing of the suspension and subsequent elimination of its water content by sublimation under reduced pressure. The chosen method should minimize loss of the drug or its pharmacological activity. mannitol. glucose) or stabilization (eg. Ultrasonication was applied by some authors to ensure complete redispersion of nanoparticles. nonreproducible or predictable release charac- . NR. safe. Nevertheless. Depending on the physicochemical characteristics of a drug. nanoparticles can be stored in sealed vials at room temperature. Summary and conclusions The most important methods for the preparation of nanoparticulate drug carriers. and optimization to improve yield and entrapment efficiency. especially for temperature-sensitive drugs. encapsulation efficiency. the surrounding pH when drug degradation is pH dependent. such as the formation of an incomplete or discontinuous film. It is also important to be aware of the presence of pharmaceutical excipients. simplification of the procedure to allow economic scale-up. In addition. Stability studies are thus important and can be performed according to the drug and to the polymer properties. dextran and surfactants).C. There are some methods to increase the stability of the nanoparticles.
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