Arch. Pharm. Res. (2014) 37:423–434 DOI 10.1007/s12272-014-0342-4

REVIEW

Polymeric vehicles for topical delivery and related analytical methods Heui Kyoung Cho • Jin Hun Cho • Seong Hoon Jeong Dong Chul Cho • Jeong Hyun Yeum • In Woo Cheong

•

Received: 11 August 2013 / Accepted: 22 January 2014 / Published online: 11 February 2014 Ó The Pharmaceutical Society of Korea 2014

Abstract Recently a variety of polymeric vehicles, such as micelles, nanoparticles, and polymersomes, have been explored and some of them are clinically used to deliver therapeutic drugs through skin. In topical delivery, the polymeric vehicles as drug carrier should guarantee nontoxicity, long-term stability, and permeation efficacy for drugs, etc. For the development of the successful topical delivery system, it is of importance to develop the polymeric vehicles of well-defined intrinsic properties, such as molecular weights, HLB, chemical composition, topology, specific ligand conjugation and to investigate the effects of the properties on drug permeation behavior. In addition, the role of polymeric vehicles must be elucidated in in vitro and in vivo analyses. This article describes some important features of polymeric vehicles and corresponding analytical methods in topical delivery even though the application span of polymers has been truly broad in the pharmaceutical fields. Keywords Polymeric vehicles  Topical delivery  Skin permeation  Block copolymers

H. K. Cho  J. H. Cho Cosmetic Research Center, Coway Co. Ltd., 459-11 Gasan-dong, Geumcheon-gu, Seoul, South Korea S. H. Jeong College of Pharmacy, Dongguk University, Seoul, Gyeonggi 410-820, South Korea D. C. Cho  J. H. Yeum  I. W. Cheong (&) Department of Applied Chemistry, Kyungpook National University, 80 Daehak-ro, Buk-gu, Taegu 702-701, South Korea e-mail: [email protected]

Abbreviations ASMs BSA CAB CBZ CLSM CAC CMC CsA DEE DTO–SA EPM FDC FITC HDV HEMA HLB HPLC LRP MAMA Mn MW Nagg NR NSAID OMC PAMAM PAsn-g-PCL PCL PEEP–PCL– PEEP PEG PGA

Amphiphilic star-like macromolecules Bovine serum albumin Cellulose acetate butyrate Carbamazepine Confocal laser scanning microscopy Critical aggregation concentration Critical micelle concentration Cyclosporin A Drug encapsulation efficiency Desaminotyrosyl tyrosine octyl esters– suberic diacids Enalapril maleate Franz diffusion cell Fluorescein isothiocyanate Hydrodynamic volume 2-Hydroxyethyl methacrylate Hydrophile–lipophile balance High performance liquid chromatography Living radical polymerization 9-(Methylaminomethyl)anthracene The number average molecular weight Molecular weight Aggregation number Nile red, dye Non-steroidal anti-inflammatory drugs Octyl methoxycinnamate Polyamidoamine Poly(aspargine)-graft-poly(caprolactone) Poly(e-caprolactone) Poly(ethyl ethylene phosphate)–poly (E-caprolactone)–poly(ethyl ethylene phosphate) Poly(ethylene glycol) Poly(glycolic acid)

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PHB PLA PLGA PMM PPG PPO PS PTX ROP SC SDS Sn(Oct)2 ST TEWL VE

H. K. Cho et al.

Polyhydroxybutyrate Poly(lactic acid) Poly(lactic-co-glycolic acid) Poly(methylidene malonate) Poly(propylene glycol) Polypropylene oxide Poly(styrene) Paclitaxel Ring opening polymerization Stratum corneum Sodium dodecyl sulfate Stannous octoate Styrene Transdermal water loss Viable epidermis

Introduction Topical/transdermal delivery system is a method to administrate the therapeutic agents through the animal or human skin. It has received considerable attention from the cosmetics, pharmaceutics, and biotechnology; because the delivery systems offer several important advantages over traditional methods, such as painless and non-invasive drug dosage, bioavailability improvement, side effect reduction, and so on. Of course, some problems (e.g., inflammation, erythema, and itching) can be caused by drugs or other excipients in the topical delivery formulation. For a longtime the effective penetration of drug through SC in human skin has been a major target in the topical delivery. The SC located on the outermost layer of the skin provides a main barrier against drug transport because it has highly organized crystalline lipid lamellae. It consists of hydrophilic bundles of keratin (i.e., corneocyte) and continuously tortuous crystalline layer of ceramides, free fatty acids, cholesterol, cholesterol sulfate, etc. (Wertz 2000). Physical techniques, such as iontophoresis (the application of a low electrical potential gradient across the skin) (Fan et al. 2008; Fang et al. 1999), electroporation (use of high-voltage pulses) (Banga et al. 1999), and sonophoresis (use of ultrasonic energy) (Hikima et al. 2009), have been aimed to disrupt and weaken the SC layer in an attempt to enhance the cutaneous delivery; however, these strategies are more suitable to hydrophilic or water-soluble drugs since VE layer under SC is known as a hydrophilic one (Honeywell-Nguyen and Bouwstra 2005). For hydrophobic drugs, the most suitable method can be encapsulation, and which has been developed in pharmaceutical fields to modify the transport and release properties of drugs. The classes of encapsulant can be lipids,

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surfactants, polymers, inorganics, etc. Among them, polymers have received great attention during the past decades due to their versatility (Olvera-Martinez Blanca et al. 2005; Wu et al. 2009a). There are two methods to encapsulate drugs: drug conjugation (chemical method) and wrapping (physical method). Among them, the physical method seems to be more attractive because most polymers and drugs do not have reactive groups for covalent bonding. Hence, formulations have been extensively investigated in recent decades for the physical entrapment of drugs within polymeric vehicles. Representative systems and their characteristics for the topical delivery are summarized in Table 1 for better understanding in perspective. As shown in Table 1, the formulations have been developed in terms of the minimal side effects, high bioavailability, and good patient compliance. This article was composed to give insight on the optimal design of polymeric vehicles for the topical drug delivery by surveying the previous studies concerned with the properties of polymeric materials affecting the permeation or penetration behavior of drugs. In this article, we excluded patch or film-type drug formulations and limited the polymeric materials to amphiphilic copolymers that can be used for the stabilization and delivery of drugs.

Polymeric materials for drug encapsulation Types of block polymers As listed in Table 1, various kinds of vehicles, such as liposomes, micelles, colloidal particles, have been developed in pharmaceutical and cosmetic industries, in which the duty of vehicles can be protection and delivery of drugs (Cho et al. 2010). In particular, well-defined structure of synthetic polymers is attractive in percutaneous treatments due to the ease of synthesis and the wide diversity of material selection. However, the polymers used in topical delivery should meet several stringent requirements, e.g., low toxicity or skin irritation, biocompatibility or biodegradability, appropriate molecular weights and amphiphilicity. Polymers used in the topical delivery can be classified into two classes, natural and synthetic polymers. Cellulose, chitosan, and alginate are natural polymers (Yang et al. 2008; Shim et al. 2004; Raghavachari and Fahl 2002). PEG, PGA, PLA, PCL, PPO, various kinds of vinyl and acryl polymers are synthetic polymers. Synthetic polymers, however, are more versatile than natural polymers because of the controllability of topology, composition, molecular weight, degradability, and so on. It is possible to enhance the stability and permeation efficiency of therapeutic drugs or control the release rate by manipulating the nature of

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Table 1 A variety of the formulations and characteristics for topical delivery system Formulations

Characteristics

Advantages

Refs.

Liposomes

Phospholipid as a wall material

Reduction of skin irritation and improvement of the solid-state stability of drugs

Date et al. (2006)

Polymersome

Polymer as a wall material

Higher stability and rigidness as compared to lipid

Rastogi et al. (2009)

Nanoparticles

Submicron (\1 lm) colloidal particles

High stability, high carrier capacity, and feasibility of the incorporation of both hydrophilic and hydrophobic drugs

Gelperina et al. (2005)

Microemulsion

Droplet size less than 150 nm, spontaneous formation, and stabilized by a surfactant

Thermodynamically stable colloidal dispersion, low viscous, high penetration rates into deeper skin layer

Santos et al. (2008) and Teichmann et al. (2007)

Vesicles

Water-filled colloidal particles

Honeywell-Nguyen and Bouwstra (2005)

Hydrogel

Three-dimensional network structure

Consisted with amphiphilic molecules, rapid entering into the SC, and remains in the deepest layer Facilitation of drug handling and high drug-loading capacity

Suhag et al. (2008) and Tsai et al. (1999)

Table 2 Typical examples of block copolymer vehicle system and their controlled properties Factor

Polymer

Model drug

Results/conclusion

Refs.

Particle size

PEG-b-PCL

Minoxidil

Smaller particle is more efficiently permeated through the hair follicles

Narrainen et al. (2002)

Hydrophobicity

PEG-b-PPS

CsA

Polymer having smaller PEG mass fraction can easily solubilize the CsA by mean of hydrophobic interactions, because of its high hydrophobicity

Velluto et al. (2008)

Hydrophobicity

PS-b-HEMA

NR

The incorporation of NR into a series of PS nanoparticles was sensitive to the percentage of the hydrophilic HEMA

Wu et al. (2009b)

Hydrophobicity

PEEP-b-PCL-b-PEEP

PTX

The drug loading efficiency of the PTX incorporating micelles increased with increasing the hydrophobic segment

Wang et al. (2008)

HLB

PEO-b-PPO-b-PEO

CBZ

The solubilization of CBZ in PEO–PPO–PEO of different HLB was monitored using UV-spectroscopy. The amount of solubilized CBZ was higher when the HLB polymer was lower

Kadam et al. (2009)

HLB

PEO-b-PCL

EPM

Drug-retaining capability is diminished when the PEO– PCL block copolymers have higher HLB value

Yoo et al. (1999)

Particle size, Hydration

PEG-b-oligo(DTOSA)-b-PEG

NR

An increase in the rate and extent of dyes penetration to deeper skin layer could be due to the small size and hydration properties of the nanospheres

Costache et al. (2009)

Morphological structure of vesicles

PEO-b-PLA, PEO-b-PEE

PKH2,PKH67

Worm-like micelles can be used as novel carriers for hydrophobic drugs into the hydrophobic core. The loading efficiency was shown to be in a range that does not give a toxic initial burst of drug

Kim et al. (2005)

Morphological structure of vesicles

PEG-b-D,L-PLA copolymer

Ovalbumin

Drug release from the microspheres was affected by the morphological structure of microspheres and properties of copolymer

Dorati et al. (2008)

polymers. Among the synthetic polymers, the most frequently studied material could be PEG. PEGylation what is called is to make a covalent bonding between PEG and other molecules, such as drug, polymer, DNA, protein, etc. It has been widely introduced for ‘stealth’ functionality against immunogenicity as well as long-term circulation.

Besides such a conjugation technique, therapeutic drugs can be stabilized or encapsulated by various physical or chemical methods. Particularly, the block copolymers of amphiphilic nature have been developed for the encapsulation of hydrophilic or hydrophobic drugs because it can easily form a self-

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assembled structure which can be used as a drug carrier. Representative amphiphilic block copolymers can be PluronicÒ (PEO-b-PPO-b-PEO or PPO-b-PEO-b-PPO) and PEG blocked biodegradable polyesters (PCL, PLA, PLGA, PGA, etc.), in which some polymers have already shown successful clinical results in transdermal therapy (Liggins and Burt 2002). To enhance the loading capacity and permeation efficiency, many researchers have been investigated corresponding factors in block copolymers, such as block length, HLB, topology, chemical composition, and so on. The typical examples of block copolymer vehicle systems are summarized in Table 2. Preparation methods for amphiphilic block copolymers Amphiphilic block polymers as a drug carrier have been intensively studied for pharmaceutical and medical fields so far. In general, hydrophilic blocks can improve the biocompatibility and hydrophobic blocks, the solubilization and stabilization of hydrophobic drugs. In synthesis, there are a few methods to prepare amphiphilic copolymers, such as free-radical polymerization (FRP) (Wu et al. 2009b), ring-opening polymerization (ROP) (Hong et al. 2005; Cho et al. 2009; Zha et al. 2006), living radical polymerization (LRP) (Narrainen et al. 2002), and living anionic polymerization (LAP). Henceforth, the characteristics of the most frequently used methods will be described. FRP is a conventional and wisely used process for polymerization. It has the major advantage that can be carried out in mild conditions than the other methods and a wide range of vinyl monomers can be used. However, this method exhibits very little control over the MW distribution and is difficult to prepare a variety of topologies, such as block copolymers, star polymers or comb polymers due to the termination reaction. LRP has been developed for the preparation of the welldefined amphiphilic block copolymers with narrow MW distribution, various HLB values, and a large variety of polymer structure. This technique is applicable to a wide range of vinyl monomers without any additional process like a protection reaction. There have been several LRP techniques developed; for example, atom transfer radical polymerization, nitroxide-mediated polymerization, reversible addition-fragmentation chain transfer polymerization, group transfer polymerization, and so on. In addition, several strategies for the linear block copolymers have been well known as follows: (a) sequential addition of monomers, (b) coupling of linear chains containing antagonist functions, (c) switching from one polymerization method to another, (d) use of dual (double-head) initiator consisting of two distinct initiating fragments. In the case of (a), the reaction is usually stopped before the complete conversion of the first monomer to avoid uncontrolled termination. The previously

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polymerized chain is isolated by precipitation and used as a macroinitiator for another block extension. If the additional monomer is added before the complete conversion of the first block, a gradient copolymer is formed. The synthesis of gradient copolymers simplifies the process with some loss of control over the polymer structure. In the case of (c), macroinitiators are prepared and used for the chain extension with PEG, PPG, poly(butadiene), poly(siloxanes), and so forth (Narrainen et al. 2002). LAP may be used for the preparation of nearly monodispersed block copolymer with predetermined molecular characteristics because it has the fast initiation step as compared with the propagation step. To avoid side reactions, however, functional groups should be protected during polymerization. PEG-b-PPS block copolymers were synthesized by LAP of propylene sulfide upon a PEG macroinitiator, formed by deprotection of a PEG thioacetate (Velluto et al. 2008). Well-characterized block copolymers based on the biocompatible PEG as soluble sequence and the biodegradable PMM as hydrophobic one have been synthesized by LAP (Larras et al. 2000). ROP is often used to introduce the hydrophobic, cyclic monomers to the hydrophilic blocks. The hydrophobic and cyclic monomers including PLA (Drumond et al. 2008), PGA (Gautier et al. 2009), PCL (Ge et al. 2002; Haw and Kim 1997; Cho et al. 2008), PHB have been found to be good materials because of their biocompatibility, low toxicity, and biodegradability. Sn(Oct)2 has been the most often used catalyst for ROP because of its high catalytic activity as well as US FDA approval as a food additive, although its potential toxicity in biomedical application is controversial.

Important factors of block copolymers for topical delivery Hydrophobicity and hydrophile–lipophile balance Hydrophobic nature is an important factor for the solubilization capacity of drugs. In usual, the solubilization capacity of a specific drug can be quantified by UV–Vis spectrophotometry with varying the concentration of polymeric vehicle. To prove the effect of hydrophobicity on the drug uptake, NR was encapsulated with three kinds of polymers and it was found that the skin uptake of NR increased with decreasing hydrophobicity (PS [ PCL [ CAB) (Wu et al. 2009a). The HLB value of amphiphilic block copolymers is another critical factor because the formation of micelle, as a drug vehicle, is mainly driven by hydrophobic interaction (Signori et al. 2005). Wu et al. reported the results that the increase of HEMA block length in the PS-b-HEMA block copolymer reduced hydrophobicity, and which led to low

Topical delivery and related analytical methods

NR loading in the nanoparticles of PS-b-HEMA. They concluded that the uptake of drug into the skin was less efficient as the hydrophobicity of carrier (i.e., the nanoparticle) decreased (Wu et al. 2009b; Thevenin et al. 1996). The importance of HLB in surfactant micelle system has been already noticed in several literatures (Wu et al. 2001; Thevenin et al. 1996). In SC layer, the layered lamella structure composed of ceramide, fatty acid, and cholesterol is strongly affected by pH, and which implies that its ‘normal’ layered morphology can be maintained in the presence of water (Friberg 1990). Like other chemical enhancers as well as surfactants, the block copolymers with appropriate HLB can effectively disturb the layered lamella structure. To solubilize CBZ, PEO-b-PPO-b-PEO triblock copolymers were investigated (Kadam et al. 2009). Especially, the experiments were focused on the effect of different MW and HLB. The comparisons of the solubilization of CBZ in HLB (9) and HLB (14) demonstrated that the hydrophobic interaction between polymer and drug was a critical factor for the solubilization of CBZ. PEO-b-PCL copolymers were also studied to encapsulate the water-soluble drug, EPM (Yoo et al. 1999). They prepared three kinds of polymers having different HLB and they reported that the block copolymer nanoparticles with low (\9) and middle (9–11) HLB values showed the highest drug retaining capacity. In addition, as a result of the daily EPM release from the PEO-b-PCL nanoparticles, the nanoparticles with low HLB showed the most prolonged release. This sustained-release behavior may support the concept of an onion-like micelle because the drugs entrapped into the concentric layered region would be liberated by the diffusion through a tortuous pathway. Cho et al. (2009) studied the permeation of the low MW PEO-b-PCL-b-PEO copolymers having different HLB values in the SC layer of hairless mouse by using CLSM and FITC-dye labeling technique. They reported that most of the copolymers of low HLB (7.4) localized at the SC layer and a few those of high HLB (11.2) localized at both SC and VE layer. This implied that the partition of block copolymers would be different with HLB value. The HLB value of drug itself affects the drug release rate and partitioning in SC. Watanabe et al. measured the HLB values of several model drugs, i.e., derivatives of vitamin C, E, and coenzyme Q10 by using IR attenuated total reflection and near-IR diffusive reflection methods and demonstrated the effect of HLB values on iontophoresis efficiency (Watanabe et al. 2006). Morphology and vehicle size Block copolymers can form a self-assembled structure due to non-covalent interactions (hydrogen bonding or van der

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Waals interaction) and the morphology of block copolymer aggregates depends on block length and the chemical nature of repeating unit (Signori et al. 2005). A variety of morphologies have been reported, such as sphere, vesicle, rod, lamella, tubular, and so on. Some of these structures are potential feasibility for drug delivery (Discher Dennis and Eisenberg 2002). The controlled aggregate architecture can be achieved by adjusting the solution conditions. To form a drug vehicle, amphiphilic block polymers are dissolved in a solvent suited to both blocks, and then a poor solvent for one block (e.g. ‘‘A’’) should be added. The poor solvent derives the A-block association and increases the interfacial tension. As the concentration of poor solvent increases, the shape of the aggregate is changed from spheres to rod and vesicles. Although many researchers have been tried to elucidate the effects of the morphology on the drug release behavior, it has not been demonstrated clearly. Among the efforts, it is noticeable that polymeric vehicles having amphiphilic domain are suitable for drug delivery because they can encapsulate both hydrophobic and hydrophilic drugs as compared with other morphologies (Harrington et al. 2000). Xiong et al. investigated the release kinetics of MAMA (hydrophobic) and procaine hydrochloride (hydrophilic) as a model drug. They prepared the nanoparticles containing the drug from the PLAb-F127-b-PLA block copolymers having different ratio of PLA to F127 (Xiong et al. 2005). Unlike the other previous publications, however, the authors reported the reverse results that the loading capacity of MAMA in the PLA-bF127-b-PLA (PLAF127-23) block copolymer having shorter hydrophobic PLA was slightly higher than that of the polymer (PLAF127-29) having longer hydrophobic PLA chain. It was suggested that the morphology of PLAF127-29 nanoparticles consists of normal bi-layer vesicles, while PLAF127-23 nanoparticles forms onionlike vesicles with three layers where the inner most layer of PLAF127-23 nanoparticles represents the hydrophobic PLA core. The size of vehicle is also an important factor. Although the polymeric microparticles with a diameter ranging from 3 to 10 lm selectively penetrate through either hair follicles or sweat ducts, the particles larger than 10 lm remain on the skin surface (Lauer 1999). It has been known that ultra fine (\30 nm) nanoparticles are more suitable for the penetration via intercellular route. Even if the penetration mechanism of nanoparticle vehicle systems has not been demonstrated clearly, a dramatic increase in the intercellular uptake of particles was achieved by reducing the size of the vehicles (Panyam and Labhasetwar 2003; Bencini et al. 2008). Desai et al. (1996) reported that the penetration efficiency of the nanoparticles (*100 nm) was 15 to 250-folds greater than microparticles.

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Topology of copolymers Topology of copolymers is the structure of a single copolymer molecule. Although the influence of polymer topology on drug release has not been clarified, it is a critical factor affecting its applicability in drug delivery (Gaucher et al. 2005). The simplest structure can be linear polymers of homopolymer, AB-type diblock, ABA- or BAB-type triblock, and multi-block copolymers (Qiu and Bae 2006). Their typical applications center on polymer– drug conjugation. The most important aspect of the conjugation lies in the localization of the conjugates by efficient cleavage mechanism. The other important factors in the copolymer design should include non-toxicity, non-immunogenicity, biodegradability, and sterilization (Elvira et al. 2005). Compared with linear polymers, graft copolymers may provide considerable functionalities onto polymer backbone by chemical modification. It is believed that the self-assembly from the graft copolymers easily undergo inter-molecular entanglement among the longer main chains or intra-molecular association so that they tend to form spherical micelles. Most graft polymers can be called comb-shaped or brush-shaped copolymers; longer main chain as backbone (Mn * 104 g/mol) and shorter side chains as graft segments (Wang et al. 2005). Gref et al. prepared amphiphilic dextran by coupling between PCL monocarboxylic acid and the hydroxyl groups on dextran. By using the comb-like copolymers (dextran–PCL), they prepared the nanoparticles containing BSA of which surface was enriched with lectin. The surface-bounded lectin conserved its hemagglutinating activity, suggesting the possible application for targeted drug delivery (Gref et al. 2002). Star polymers have a three-dimensional hyperbranched structure. As compared with linear or graft copolymers, star polymer as drug carrier seems rather limited so far. It has been shown that star-shaped polymers exhibit a smaller hydrodynamic radius and lower solution viscosity as compared with the linear polymers of the same molecular weight and composition. However, the most significant feature of the star polymer-based self-assembly is superior structure stability to the others. Djordjevic et al. evaluated amphiphilic star-like macromolecules (ASMs) as a drug vehicle. Indomethacin, piroxicam, and ketoprofen as model drugs were individually encapsulated with ASMs. The effects of the ASMs on the percutaneous permeation of NSAIDs across hairless mouse skins were evaluated in in vitro test. The results indicated that the ASMs delayed the drug release rates, whereas the solubility measurements showed that the ASMs increased the aqueous solubility of NSAIDs (Djordjevic et al. 2003). Dendrimers, in particular, have attracted attention for drug-delivery applications because of the high density of functional group, well-defined shapes and sizes

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(monodispersity), and low viscosity. The interior of dendrimer has a void space and functional groups, so they are wellsuited for the carrier molecules in drug delivery. With dendrimers as drug-loading core, it was demonstrated that the micelles prepared with larger dendrimer core showed higher encapsulation capability than those with smaller cores (Tolia and Choi 2008; Liu et al. 2000). Cheng et al. (2007) have studied PAMAM dendrimers for the transdermal delivery of NSAIDs (ketoprofen and diflunisal) that have limited the clinical applications because of side effects caused by oral administration.

Analytical methods for topical delivery Investigation of in vitro or in vivo drug release behavior is often a difficult, time- and cost-consuming process. However, adequate analytical methods for the characterization of vehicles and resulting efficacy would be a prerequisite procedure for successful topical delivery. In fact, numerous parameters should be characterized; for example, molecular weights, chemical composition, HLB, Nagg, CMC (or CAC), HDV, f-potential, morphology, degradability, encapsulation (or loading) efficiency, skin permeation (or penetration) rate, skin irritation or toxicity, partition between SC and VE, and so on (Harrington et al. 2000). None of them should be omitted for the successful topical delivery. In addition, it is impossible to adopt a single test system which could be used to study the drug release behavior of each formulation, since there are extra factors governing the drug release properties, such as type of drug (Nii and Ishii 2005), the dosage form, the excipients, penetration enhancers (Furuishi et al. 2007), and other formulation variables. In the following sections, several major techniques to assess the polymeric vehicle system in topical delivery are discussed. Encapsulation efficiency Encapsulation efficiency is a basis for the calculation of drug permeation and which is sensitive to a number of factors associated with physico-chemical properties of polymers and corresponding vehicles. In general, the drug loading content and encapsulation efficiency can be calculated as follows; drug recovered in formulations nanospheres or vesicles recovered  100 (% ) ð1Þ

Drug loading content =

drug fed  drug loss drug fed  100 (% )

Encapsulation efficiency =

ð2Þ

Topical delivery and related analytical methods

The above equations can be used in both micellar and particular vehicle systems. The encapsulation efficiency varies with the solubilization capacity of vehicle against drug, and which can be usually manipulated by HLB, molecular weights, affinity or interaction between drug and polymers, polymer concentration, etc. (Shim et al. 2004; Kandavilli et al. 2002; Yeo and Park 2004). Sometimes vehicle size or HDV strongly determines the encapsulation efficiency (Frauke et al. 2001). As a measuring technique, HPLC is the most frequently used method for the quantitative analysis of the encapsulation and release rate of drugs. In this method, the vehicles are previously disrupted in a relevant solvent and the drug has to be isolated in pure solvent. In liposome system, the encapsulation efficiency of the hydrophilic, lipophilic, and amphiphilic drugs were investigated by using a HPLC with UV detection (Nii and Ishii 2005). Sun and Chiu investigated a new method to measure the encapsulation efficiency of individual lipid vesicles (Sun and Chiu 2005). After trapping the single vesicle optically, they irradiated a single 3-ns UV laser pulse to release the molecules encapsulated within the vesicle and they could measure the number of released molecules by the confocal detection with a laser excitation at 488 nm (CW Ar? laser). Although this method has a drawback in which only one vesicle can be analyzed at a time, it gives detailed information on the encapsulation efficiency. The drug loading content can be determined by measuring the amount of drug loss. Yu et al. (2008) fabricated the nanospheres and vesicles containing Ca2? and CO32as a drug. They used alginate as a drug carrier. After the fabrication, the aqueous phase was collected and the drug content in the aqueous phase was measured to determine the drug loss. The drug concentration was determined by an UV–Vis spectrophotometer. Zhang et al. (2004) measured the encapsulation efficiency of a marker (homocarnosine) by using proton NMR technique without separation of the encapsulated marker. Although numerous techniques including chromatography, centrifugation or dialysis have been used to remove the free (unencapsulated) drug, it may lead to leakage of vesicles. Therefore, quantitative methods requiring physical separations would not be desirable. Skin tape stripping Tape stripping in its standard form is a well-suited method to investigate the penetration depth or rate in the SC layer by collecting a thin SC layer of 0.5–1 lm thickness (Coderch et al. 1996). After the treatment of therapeutic drugs onto skin, an adhesive tape is put onto the treated skin area and is then taken off again after a certain application time. The tape contains corneocytes as well as the corresponding amount of applied formulation. To quantify the amount of

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drug in an individual tape, a number of methods were proposed in the literature: weighing (Kalia et al. 2000); absorbance in the visible range (Marttin et al. 1996); determination of the area of stained corneocytes (Lademann et al. 2009). A disadvantage of this method is that the amount of the corneocytes taken changes per stripping on the patients. Furthermore, the type of substance applied and the penetration time have an influence on the amount of corneocytes on the individual tape. Therefore, it is not easy to determine the penetration kinetics from the amount of drug permeated in SC layer. A schematic cut through the SC demonstrating the position of areas used for the tape stripping is shown in Fig. 1a. Figure 1b is a representative penetration profile of clobetasol propionate measured by the tape stripping method. This profile can be calculated by adding the single amounts of the SC removed during the procedure. Clobetasol propionate is a corticosteroid used to treat various skin disorders including eczema and psoriasis (Shah et al. 2002; Weigmann et al. 1999). The transdermal delivery of low molecular weight heparin (LMWH) was investigated with skin tape stripping to estimate the enhancement strategies combined with micro-needle, ultrasound, or iontophoresis (Lanke et al. 2009). The flux of LMWH was significantly enhanced as compared with each self-supported method. The increase in LMWH flux is most likely because the SC layer is removed during tape stripping. As the SC layer becomes thinner, the primary barrier for LMWH transport can be ignored. The increase in TEWL levels following the tape stripping also indicates the removal of the SC layer. Tsai et al. used the tape stripping to disrupt the permeability barrier of skin. They examined the MW cutoff of PEG penetration through the tape stripped or SDStreated skin with various TEWL levels. They demonstrated that the higher amounts and larger molecules of PEG could be penetrated into the two disrupted skins than normal skin (Tsai et al. 2003). The tape stripping technique can provide a predictive assessment of drug penetrated into the skin as well as better understanding of the drug permeation mechanism. Diffusion cell analyses In vitro skin permeation study is carried out for the measurement of diffusive drug release rate and optimization of promising formulations, in which a dissolution test has been widely employed for evaluating a variety of dosage forms, such as suspension, transdermal patches, semi-solid formulations, etc. The representative apparatuses and type of dosage forms for the dissolution test are summarized in Table 3. There are several kinds of diffusion cell techniques including horizontal-type skin permeation system (Reimus et al. 2007) in which solute diffuses horizontally; FDC (Maffei et al. 2004; Prasad et al. 2007; Bosman et al. 1998)

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Fig. 1 a A schematic cut through the SC demonstrating the position of the areas used for tape stripping in relation to the treated skin area. b The penetration profile of clobetasol propionate (Shah et al. 2002; Weigmann et al. 1999)

Table 3 Representative dosage formulations and apparatus used for drug release behavior (Rastogi et al. 2009) Type of dosage form

Release method

Refs.

Topicals-semisolids

Diffusion cell

Pygall et al. (2007), Tallury et al. (2009) and Teichmann et al. (2007)

Transdermal-patches

Paddle over disk

Microparticles

Flow through cell

Pongjanyakul et al. (2003) and Aqil et al. (2008) Raffin et al. (2007) and Conti et al. (1995)

Suspensions

Paddle

Simon et al. (1994) and Sjoeqvist et al. (1993)

and the flow-through diffusion cells (Bajpai et al. 2006; Kirino et al. 2009) are vertical type methods to investigate the drug release behavior. Among them, FDC has been frequently used to estimate the in vitro permeation of drugs in ordinary topical delivery systems because the horizontal type diffusion cells cannot be used for gel or cream-type formulation. This technique evaluates the drug release patterns (time-lag, steady-state release rate of penetration or permeation, etc.) from the formulations. In this system, the cell is composed of two compartments: donor and receptor. The donor compartment is a place where the drug is applied uniformly. From this compartment, drug vehicle or drug passes through a model membrane (e.g., hairless mouse skin) that is located between the donor and receptor compartments. The receptor compartment contains the receptor solution of which temperature is usually controlled with a thermostat. The conditions of the receptor solution, such as temperature and buffer composition, can have a significant influence on the drug permeation through the membrane. In general, physiological saline or phosphate-buffered solution maintained at 37 °C is used for the receptor solution (ElKattan et al. 2000; Bosman et al. 1998). In the case of

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dosage formulations containing non-fluorescent materials, the contents of drug can be determined by HPLC or UV absorption analysis (Shim et al. 2004; Carafa et al. 2004). The permeation mechanism of the dosage formulations containing fluorescent materials can be ascertained by CLSM after the diffusion cell analysis (Rastogi et al. 2009). Confocal laser scanning microscopy For the desirable administration of drugs, various polymeric vehicles have been used as well as the other kinds of vehicles, surfactants, solvents, fatty acids, since the penetration depth is strongly affected by the formulation. There have been several articles published; however, the penetration of polymeric vehicles through the SC layer has been argued until now. Some researchers insist that the MW of materials must be less than 500 Da to allow penetration into the normal skin and larger molecules may penetrate through the disordered skin of disrupted barrier function (Bos and Meinardi 2000). The others suggested that microparticles with a diameter under 10 lm selectively penetrated through the hair follicles and sweat ducts (Lauer 1999). In short, the sweat ducts, hair follicles, and the intercellular lipid layer might be the possible penetration routes for the polymeric vehicles containing drug; however, their chemical composition, size, and molecular weight seem to be very limited depending on the penetration pathway. In some cases, the penetration of polymeric vehicles (like other chemical enhancers) might be needless due to their potential toxicity. Nevertheless, tracing polymeric vehicles in the SC and VE layers is important to elucidate the governing factors that effect on the drug permeation behavior in the polymeric vehicle system. For this purpose, CLSM can be a good method, which has been a well-established technique with high-resolution images in biological fields (Alvarez-Roman et al. 2004). However, most pharmaceutical substances and polymeric

Topical delivery and related analytical methods (a) (a)

431

(b) (b)

50 μm

50 μm

(d) (d)

(c) (c)

nanoparticles containing hydrophobic OMC could enhance the penetration of the molecule into the SC layer, compared to a non-particulate formulation at the same concentration (Mitriaikina and Mueller-Goymann 2007). To prove the penetration mechanism, a surrogate hydrophobic NR was encapsulated and the cutaneous distribution of NR was visualized using CLSM. Although the CLSM images could not show the evidence that the fluorescence was from the NR encapsulated with the nanoparticle, the images clearly demonstrated the enhanced distribution of NR. For a certainty, CLSM is a critical method to visualize the localization of fluorescent probes. It can represent the preferred penetration pathways; nevertheless, the selection of fluorophore is limited and no calibration methods of fluorescence intensities has been demonstrated (Alvarez-Roman et al. 2004).

Conclusions 50 μm

50 μm

Fig. 2 The CLSM images of the W/O/W multiple emulsions containing b, d FITC-labeled PEO-b-PCL-b-PEO copolymers and a, c unlabeled copolymers with different molar ratio of [CL] to [EO]: (a) [CL]/[EO] = 0.16 with a trace of FITC, b [CL]/[EO] = 0.18, c [CL]/[EO] = 0.51 with a trace of FITC, and d [CL]/[EO] = 0.53 (scale bar = 100 lm). The original images were modified from Cho et al. (2009)

materials are unfortunately non-fluorescent. Therefore, the usage of appropriate fluorescent compounds is reasonable whether it is applied chemically or physically (Stracke et al. 2006). For example, protein drugs and polymeric vehicles can be covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs can be labeled by simple blending with the fluorescent dye having similar solubility (Pygall et al. 2007). Although there are a number of fluorescent dyes, FITC isomers are the most commonly used and which can be easily modified with other molecules. Tallury et al. (2009) synthesized the FITC-labeled chitosan polymer to prepare ultra-small (\30 nm), stable, and water-dispersible nanoparticles which could be safely used for biomedical imaging applications. Cho et al. synthesized the FITClabeled and unlabeled PEO–PCL–PEO triblock copolymers to encapsulate water-soluble drug in multiple emulsion system. The representative CLSM images of W/O/W emulsions are shown in Fig. 2. The CLSM images of FITC-labeled copolymers clearly exhibit that the inner W droplets in green color are well dispersed in the oil phase (stained with NR) (Cho et al. 2009). In topical delivery system, in vivo test is very important to elucidate not only the mechanism and reliable depth profile but also the pathways of the drug release within the SC (Teichmann et al. 2007). It was reported that the polymeric

There have been considerable advances in the polymeric vehicles for the topical/transdermal delivery systems. In particular, significant efforts have been focused on understanding of the relationship between the characteristics of polymeric materials and drug permeation behavior. Encapsulation is a useful method to modify the physicochemical properties of drugs and it provides a facilitative mean to deliver the drugs. However, the feasible mechanism of drug permeation has not yet been investigated clearly and sufficiently, when the drug is applied on the skin. Therefore, further systematic researches should be needed to demonstrate the release kinetics and drug partitioning in each skin layer by using the well-defined and tailor-made polymeric materials. Acknowledgments This study was supported by Kyungpook National University Research Fund, 2011. Conflict of interest

The authors declare no conflict of interest.

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