Recent Advances in Drug Delivery of Polymeric Nano-Micelles

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1 Send rders for Reprints to Current Drug Metabolism, 2016, 17, REVIEW ARTICLE Recent Advances in Drug Delivery of Polymeric Nano-Micelles Zarith Asyikin binti Abdul Aziz 1, Akil Ahmad 1,3, Siti Hamidah Mohd-Setapar 1,2,*, Hashim Hassan 2, David Lokhat 3, Mohammad Amjad Kamal 4,5,6 and Ghulam Md Ashraf 4,* 1 Center of Lipids Engineering and Applied Research, Ibnu Sina Institute for Industrial and Scientific Research, Universiti Teknologi Malaysia, UTM Skudai, Johor, Malaysia; 2 Department of Chemical Engineering, Universiti Teknologi Malaysia, UTM Skudai, Johor, Malaysia; 3 Department of Chemical Engineering, College of Agriculture, Engineering and Science, University of KwaZulu- Natal, Durban-4041, South Africa; 4 King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia; 5 Enzymoic; 6 Novel Global Community Educational Foundation [Peterlee Place, Hebersham, NSW 2770, Australia] A R T I C L E H I S T R Y Received: March 20, 2016 Revised: July 21, 2016 Accepted: July 25, 2016 DI: / Abstract: In clinical studies, drugs with hydrophobic characteristic usually reflect low bioavailability, poor drug absorption, and inability to achieve the therapeutic concentration in blood. The production of poor solubility drugs, in abundance, by pharmaceutical industries calls for an urgent need to find the alternatives for resolving the above mentioned shortcomings. Poor water solubility drugs loaded with polymeric micelle seem to be the best alternative to enhance drugs solubility and bioavailability. Polymeric micelle, formed by self-assembled of amphiphilic block copolymers in aqueous environment, functioned as solubilizing agent for hydrophobic drug. This review discusses the fundamentals of polymeric micelle as drug carrier through representative literature, and demonstrates some applications in various clinical trials. The structure, characteristic, and formation of polymeric micelle have been discussed firstly. Next, this manuscript focuses on the potential of polymeric micelles as drug vehicle in oral, transdermal routes, and anti-cancer agent. Several results from previous studies have been reproduced in this review in order to prove the efficacy of the micelles in delivering hydrophobic drugs. Lastly, future strategies to broaden the application of polymeric micelles in pharmaceutical industries have been highlighted. Keywords: Nano-micelles, drug delivery, therapeutic effect, surfactants, block polymers. Please provide corresponding author(s) photograph size should be 4" x 4" inches Siti H. Mohd-Setapar 1. INTRDUCTIN The major problem being faced by modern pharmaceutical research is the hydrophobic characteristic of drugs, which is associated with ever increasing solubility problem for the past ten years [1, 2]. Currently, approximately 70% of new drug candidates are insoluble in water and organic media. Besides, 40% marketed oral drugs are considered as hydrophobic drugs [3, 4]. The limited drug dissolution rate and low bioavailability of drugs are among the bad effects of water insoluble drugs [1, 2]. In order to achieve drug therapeutic effect in blood, increased drug dosage needs to be considered [2]. However, enhanced drug dosage can lead to a variety of problems like high toxicity, high manufacturing cost, difficulties in drug formulation, etc [2, 5]. Numerous drug delivery systems have been introduced by researchers in pharmaceutical industries in order to solve the problems associated with poorly soluble drugs. According to the traditional methods, hydrophobic drugs were encapsulated in nanosized carriers like liposomes, niosomes, micelle, emulsion, etc [2]. Unfortunately, among these carriers (eg: liposome and niosome), there are some which are not able to hold the hydrophobic compounds *Address correspondence to these authors at the Center of Lipids Engineering and Applied Research, Ibnu Sina Institute for Industrial and Scientific Research, Universiti Teknologi Malaysia, UTM Skudai, Johor, Malaysia; Tel: ; Fax: ; sitihamidah@cheme.utm.my King Fahd Medical Research Center, King Abdulaziz University, P.. Box 80216, Jeddah 21589, Saudi Arabia; Tel.: ; s: ashraf.gm@gmail.com, gashraf@kau.edu.sa although they have the potential of improving hydrophobic drug solubility [6]. Thus, among multiple drug carriers that have been developed or undergoing development, micellar nanocarriers promise to resolve the drug solubility problem. Polymeric micelles are among the approaches, as multifunctional-based drug delivery system, for hydrophobic drugs and have gained a lot of attention in the last two decades [7]. They are nanosized compounds (>100 nm) which are formed by selfassembling amphiphilic block copolymers in water, above certain concentration called critical micelle concentration (CMC), and consist of hydrophilic and hydrophobic compartments [8]. In aqueous environment, the hydrophobic block co-polymer forms a semisolid core which act as the water insoluble drug reservoir, while the hydrophilic segment of the block co-polymer forms a coronal layer or acts as an outer shell to prevent the rapid clearance of the drug carrier [9]. Further explanation regarding formation of polymeric micelles is discussed in the following sub-sections. Fig. (1) represents spontaneous micelle formation with the hydrophobic drug being loaded inside the micelle core in aqueous media. Copolymers have two types of monomers with different solubility. The two types of monomers can be arranged into polymeric chain in different pattern to provide random, block and graft copolymer. Fig. (2) illustrates the arrangement of di-block and triblock copolymers which were prepared from the same hydrophilic and hydrophobic monomeric units. The major reasons to choose polymeric micelle as hydrophobic drug carrier are its biocompatibility and biodegradability characteristics [10]. Besides, polymeric micelles usually have much lower CMC values and are more stable compared to micelles developed from conventional detergents /16 $ Bentham Science Publishers

2 2 Current Drug Metabolism, 2016, Vol. 17, No. 00 Aziz et al. which are as low as 10-6 M. [11]. In terms of thermodynamic stability, polymeric micelles, consisting of certain co-polymers, have high kinetic stability due to the presence of multiple sites that are capable of hydrophobic interaction between each polymer molecule [12]. High stability of polymeric micelle promises high efficacy in retention time of drugs (to be encapsulated) and prevention of the drug from enzymatic degradation and inactivation of bioactive molecules [10]. The advancement of the bioavailability of oral and topical drug, administered by using polymeric micelle, has been discussed by several researchers in achieving therapeutic effect of those drugs. In topical or transdermal drug delivery system, the application of biodegradable and biocompatible polymers has been widely studied as the skin (stratum corneum) limits the drugs penetration [13].The two types of polymers, which are used in topical drug delivery system, are natural and synthetic polymers. Natural polymers are comprised of cellulose, chitosan and alginate, while Polyethylene glycol (PEG), Poly(glycolic acid) (PGA), Poly (lactic acid) (PLA), Polycaprolactone (PCL), and Polyphenylene xide (PP) are the examples of synthetic polymers. However, compared to natural polymers, synthetic polymers are the most suitable for employing as drug vehicle, due to their topology controllability, composition, molecular weight, and degradability [14]. At present, more than 60% marketed pharmaceutical drugs are used as oral products. Drugs are preferred to be delivered by oral administration because of its simplicity, easiness and patient s acceptance especially in the case of repeated dosing in chronic therapy [16, 17]. Nimodipine [17], docetaxel [18], paclitaxel [19] and quercetin [20] are the examples of oral hydrophobic drugs that are encapsulated with polymeric micelles. Besides, in a few clinical trial studies, application of polymeric micelle as water insoluble drug vehicles have been conducted in a laboratory setting which will be further discussed in the following sub-sections. Fig. (1). Spontaneous micelle formation loaded hydrophobic drugs. Fig. (2). Main structure of block co-polymers. 2. CRE AND CRNA PLYMERIC MICELLES Hydrophilic corona of micelle-forming individual blocks usually consist of PEG blocks with molecular weight of 1 kda to 15 kda [10]. The merits of this polymer is economical, less toxic, high solubility and good steric protector for various macromolecules [1, 3]. However, there are several other hydrophilic polymers, used as an alternative to PEG, such as poly(n-vinyl-2-pyrrolidone) (PVP) [21], poly(vinyl alcolhol) (PVA) and poly(vinylalcohol-covinyloleate) [22]. PVP is often known as primary alternative for PEG [21] since both have similar characteristics including high biocompatibility that facilitates the formulation of several drug carriers [23]. Besides that, PVA and poly(vinylalcohol-covinyloleate) were used to prepare micelles enhancing transcutaneous permeation of retinyl palmitate [22]. Nevertheless, PEG is still the primary choice for hydrophilic micelle corona-forming block [6]. Several monomers, such as propylene oxide [24], L-lysine [25], aspartic acid [26], caprolactone [27], and some others, have been used to prepare hydrophobic core forming block. Additionally, palmitoyl, which is one of the example of chitosan grafted with hydrophobic groups, are currently becoming popular due to its high biocompatibility [28], while dendrimers micelles are also being recommended for developing hydrophobic core polymeric micelles. The common examples of co-polymeric blocks used to make polymeric micelles have been reproduced in Table PLYMERIC MICELLES FRMATIN Generally, polymeric micelles are formed by self-assembling of individual amphiphilic di-block or tri-block copolymers (unimers) which consist of hydrophilic and hydrophobic blocks [29]. The two main steps in the formation of polymeric micelles consist of the synthesis of the desirable amphiphilic polymeric molecules and its conversion to micelles at critical micellization concentration by using several techniques [29, 30] Critical Micelle Concentration Amphiphilic block copolymers are self-assembled through a thermodynamically driven and a reversible process. In order to characterize the self-assembling of amphiphilic molecules, CMC is the most important factor that needs to be discussed [29]. In aqueous solution at low concentration, copolymeric compounds exist as single molecules (unimers) and start to self-assembly when the copolymer concentration reaches the CMC [31]. At concentrations, below the CMC, the number of amphiphilic copolymers at air/water interface increases as concentration increases, but when they reach the threshold concentration (CMC point), the interface becomes saturated with the monomers and surface tension between air and water becomes constant [32, 33]. The schematic diagram of selfassembly of block copolymers at CMC is depicted in Fig. (3). The size of hydrophobic and hydrophilic chains of block copolymers influences the value of CMC. Large size of hydrophobic fragment results in lower CMC value while higher CMC value enhances hydrophilic domain area [34]. CMC values of surfactant micelles are not low as polymeric micelles and hence polymeric micelles are more stable and able to solubilize various hydrophobic entities efficiently using their hydrophobic core. Moreover, low CMC value of polymeric micelles possess high stability micelles structure with extreme dilution after intravenous administration to

3 Drug Delivery of Polymeric Nano-Micelles Current Drug Metabolism, 2016, Vol. 17, No Table 1. Commonly used co-polymeric blocks for formation of polymeric micelles [10]. Copolymers Chemical Structure of Repeating Units Abbreviations Hydrophilic Corona: Poly(ethylene glycol/oxide) H PEG, PE H 3 C Poly(N-isopropyl acrylamide) NH pnipam H 3 C Poly(acrylic acid) H PVP H 3 C Poly(2-oxazoline) H 3 C H N Polyethers Poly(propylene oxide) Hydrophobic Corona: PP H 3 C Poly(esters) Poly(L-lactide) H 3 C PLA

4 4 Current Drug Metabolism, 2016, Vol. 17, No. 00 Aziz et al. Table (1) contd. Copolymers Chemical Structure of Repeating Units Abbreviations Poly(lactide-coglycolide) H PDLLA H Poly( -amino ester) CH 2 PCL H 2 C Polyamides Poly(L-Histidine) phis H 3 C NH Fig. (3). Schematic representation of the theory of critical micellization concentration (CMC) for solutions of block copolymers. patients [35, 36]. Polymeric micelles having the diameter >100nm and this unique characteristic favors the assembling of the micelles for easy penetration and accumulation at the site of illness and is still a good alternative for poorly soluble drug carriers, as applied by several researchers Polymeric Micelles Drug Encapsulation Techniques Hydrophobic drugs encapsulation techniques using polymeric micelles can be done by chemical conjugation [37] or physical entrapment [38]. The compatibility of Drug and hydrophobic block (also known as drug loaded efficiency) can be tested by using Flory-Huggins interaction parameter [39]. The interaction between hydrophobic block copolymer and drugs is the main factor for the solubilization and stabilization of drugs loaded in polymeric micelle [40]. Thus, the stability of hydrophobic drugs, loaded inside micelles core, can be enhanced with proper choice of the hydrophobic block copolymer [33]. In the encapsulation technique, chemical conjugation is about the formation of covalent bond between particular groups of drugs and hydrophobic core of polymeric micelle [41]. However, chemical conjugation possesses some disadvantages in hydrophobic drug encapsulation and hence the water insoluble drug incorporation by using physical procedure needs to be considered. The examples of physical entrapment methods are discussed in the next sub-sections below Dialysis Method This method starts with the step of bringing the drug and copolymer from a solvent, like ethanol in which both are soluble, into a solvent (such as water) which is selective for the hydrophilic part of the polymer. Thus, with the initial solvent being replaced by the selected one, the hydrophobic part of the polymer starts to form micellar core which incorporates the water insoluble drug. The organic solvent can be fully removed by extending the dialysis over several days [41]. This technique has been applied in previous study concerning the preparation of PE-b-PBLA micelles from various solvents. In this experiment, PE-b-PBLA was dissolve in 20 ml of each N,N-Dimethylformamide (DMF), Acetonitrile (CAN), tetrahydrofuran (THF), N,N-dimethylacetamide (DMAc), ethyl alcohol and Dimethyl sulfoxide (DMS) respectively and the solutions were stirred for overnight at room temperature [42]. Then, water was used to dialyze the solutions through molecular porous dialysis tubing, followed by lyophilisation. The highest PE-b-PBLA micelles yield was 87% w/w from dialysis against water using DMAc. In addition, as compared to other solutions, PE-b-PBLA micelles formation by using DMAc as an initial solvent had a more narrow size distribution [38] Mechanical Dispersion Method In mechanical dispersion technique, organic solvent or a blend of solvents is used to dissolve hydrophobic drug and copolymer. This step is followed by solvent evaporation using rotary evaporator for small-scale preparation, while for large-scale preparation, thin film method or explosion proof-spray dryer was used. Then, once the micelle is formed, the drug-copolymer complex can be transferred out by agitating with aqueous solutions. This method is the most suitable for large hydrophilic segment of block copolymers and is usually prepared by ultrasonification to form the micelles [43]. This technique was used to prepare paclitaxel incorporated with diblock copolymer [44]. Throughout the preparation, acetonitrile was used to dissolve paclitaxel and copolymer followed by solvent evaporation under nitrogen stream at temperature of 60 C

5 Drug Delivery of Polymeric Nano-Micelles Current Drug Metabolism, 2016, Vol. 17, No within 2 hours to form a solid paclitaxel/pdlla-b-mepeg matrix. Then, the matrix solution was separated out by preheating the matrix in warm water bath to form a transparent gel-like sample. Finally, water was added into the sample and stirred with glass rod or vortex mixer to form a clear micellar solution [43] Co-Solvent Evaporation Method In this method, volatile water-miscible organic solvents, such as acetonitrile, tetrahydrofuran, acetone and methanol, are used to dissolve the drug and copolymer. In order to push the self-assembly of hydrophobic blocks to micelles, water is added into the solution. Evaporation method is used to remove the organic solvent [45]. Self-assembling of PEG-b-PCL micelles by addition of water has been investigated in a study, in which acetonitrile was used to dissolve drug and the copolymer. The micelles formation was observed by Dynamic Light Scattering. In between 10% and 30% critical water content (CWC), depending on the Point Cloud Library block size, initial micelle structures were observed at diameter of 200nm to 800nm. Hence, as the water fraction increased to ca. 40%, the diameter of the micelles undergo sharp collapse to 20nm to 60nm, forming monodisperse micelles [45] il in Water Emulsion Method This method is concerned about the preparation of copolymer aqueous solution by adding drug, which dissolves in waterinsoluble volatile solvent (like chloroform), to form emulsion of oil in water [41]. A study investigates the comparison of the efficacy of dialysis and oil in water techniques. Based on the investigation, the emulsification method show more efficiency in Doxorubicin encapsulation with PE-b-PBLA micelles having DX loading of 12% (w/w) as compared to dialysis method which had only 8% (w/w) DX loading with the block copolymer [46]. Another study discussed about oil in water technique to prepare indomethacin (IMC)- incorporated micelles. The experiment was carried out by dissolving of 60mg of PE-PBLA (without drug) in 120mL of distilled water and homogenized by sonication for 30 seconds. A chloroform solution of IMC (6mg in 1.8mL) was added drop wise into PE-b- PBLA-distilled water solution under vigorous stirring at room temperature. The chloroform evaporates through the open system. Amicon YM-30 ultrafiltration membrane was used to filter the solution in order to remove unbound IMC and contaminants of low molecular weight and the lyophilized one [38]. 4. PLYMERIC MICELLES AS RAL DRUG DELIVERY ral route is the most preferable route in drug administration which possesses several advantages. From patients points of view, oral drug administration is the most convenient and painless selfmedication, especially for chronic disease remedies [47]. However, even though it is broadly used in pharmaceutical industries, there is problem regarding the drugs low bioavailability which affects the formulation of drug in oral delivery [2]. Water insoluble drug may slow down the drug dissolution rate or the saturation solubility which assemble incomplete drug release, resulting in poor bioavailability of drugs [2]. Drug intrinsic properties and physiological conditions are the two variables that need to be considered as they may affect drug bioavailability [7]. ral drug administration must successfully pass through the chemical and enzymatic gastrointestinal (GI) liquid, cross the mucus layer and epithelium before being absorbed [48]. Low drug absorption is caused by the drug intrinsic properties which involve poor stability in gastrointestinal (GI) tract, low mucosal permeability, and low solubility in mucosal fluid [31, 32]. Besides, drug absorption is also influenced by physiological factors, like colonic microflora, surface area, enzymatic activity, gastrointestinal transit time, and manipulating ph [51]. Thus, in this regard, encapsulation of hydrophobic drug with polymeric micelles can give high impact in improving stability, solubility and bioavailability of drugs. In addition, polymeric micelle core may protect the drugs from rapid clearance from circulation and increase the amount of drug available for absorption ral Drug Absorption in Gastrointestinal Tract Gastrointestinal tract is a part of the human organ system, where oral drug administration should survive during its transition through it [7]. It is responsible for building the organ and glands extract nutrients from the ingested food [15]. Moreover, the presence of salivary amylase and gastric protease cause the absorption process to occur partially in the oral cavity and the stomach, however the largest part of drug absorption occurs in the small intestine [52]. In the small intestine, there are many ways for nutrients to be absorbed, however for oral drugs absorption there are only two pathways involved namely transcellular pathways (Fig. 4a) and paracellular pathways (Fig. 4e) [15]. Normally, transcellular pathway absorbs low molecular weight hydrophobic drugs, which diffuses through the membrane, and the rate of absorption is measured by the concentration gradient across the intestinal membrane (Fig. 4c) [7]. Compared with hydrophilic entities, they are not able to diffuse through the intestinal membrane because of their low affinity for lipidic constituents [53]. Therefore, paracellular pathway is the only route which is available to be used for their absorption process since there is no appropriate transporter for membrane [7]. Moreover, several anti-cancer drugs like paclitaxel, which is the most effective anti-cancer drug, suffers from poor absorption in the gastrointestinal tract [54]. This is due to the harsh environment in GI tract which affects the drug absorption process. Hence, development of polymeric micelles loaded hydrophobic drug can be the protective vehicles to avoid destruction in GI tract and enhancing the drug absorption [51]. Fig. (4). Schematic representation of the mechanisms involved in the absorption of exogenous drugs in the small intestine. (a) transcellular transport; (b) active transport; (c) facilitated diffusion; (d) receptor-mediated endocytosis; (e) paracellular transport; (f) pinocytosis [2]. The good drugs dilution and stability in gastrointestinal environment depend on the lower polymeric micelles CMC values [55]. A Low CMC value is normally caused by the large amount of hydrophobic regions in the micelle core. In order to achieve lower CMC, the chain length at polymer shell should be controlled, while increasing the chain length in the polymer core [2]. The significance of hydrophobic chain in micellar core, with respect to the CMC values, has been proved by several researchers. Kang et al proved that on increasing the hydrophobic chain of triblock copolymer, polyvinylpyrrolidone-block-poly(d,l-lactide)-block- polyvinylpyrrolidone (PVP-b-PDLLA-b-PVP) the CMC values get reduced [56]. Table 2 represents the result of the Critical Aggregation Concentration values (CAC) of various triblock co-polymers that have been tested in the study. The result shows that the values

6 6 Current Drug Metabolism, 2016, Vol. 17, No. 00 Aziz et al. Table 2. Characterization Data for Triblock co-polymer and Star Block Copolymer [56]. No Block Co-Polymer (VP)/DLLA CAC (mg/l) 1 PVP-b-PDLLA-b-PVP (5:1) 4.47 : PVP-b-PDLLA-b-PVP (4:1) 3.15 : PVP-b-PDLLA-b-PVP (2.5:1) 2.41 : Star-PDLLA-b-PVP (7:1) 5.72 : Star-PDLLA-b-PVP (4:1) 3.56 : Star-PDLLA-b-PVP (2:1) 1.8 : of CAC vary according to the different hydrophilic chains. The CAC values of A-B-A block copolymer reduced as hydrophilic chain ratio decreased. The values were reported in the range of 5 to 24 mg/l. Unfortunately, in order to ensure the micelle stability in the GI tract, not only the manipulation of the polymeric micelles CMC value is important, but another parameter that needs to be considered, so as to achieve good absorption of polymeric micelle, is the alteration in the range of ph values [2]. Assorted studies have been carried out to investigate the consequence of ph sensitive polymeric micelles towards drug release and oral bioavailability of hydrophobic drug. Satturwar et al manipulated ph sensitivity of PEG-bpoly(alkyl(meth)acrylate-co-methacrylic acid) loaded with hydrophobic drug, named candesartan cilexetil in amorphous form [57]. In-vitro analysis was conducted to monitor the release of candesartan cilexetil by changing the ph values. As reported in this study, the drug release from micelles was triggered as ph increased from 1.2 to 7.2. In another study, scientists investigated the effect of in vitro drug release from micelle formed by encapsulating hydrophobic drug, namely progesterone (PRG), with PEG-b-poly(alkyl acrylateco-methacrylic acid). The PRG was encapsulated by several types of A-B block co-polymer which include PEG-b-P(EA 17 -co- MAA 17 ), PEG-b-P(EA 20 -co-maa 12 ), and PEG-b-P(nBA 12 -co- MAA 21 ). The PRG entrapment efficiency in all polymers was found to be more than 90% indicating drug loading of >9% w/w. PEG-b- P(EA 17 -co-maa 17 ) and PEG-b-P(EA 20 -co-maa 12 ) polymers with EA group exhibited the release of more than 50% of the hydrophobic drug after 2 hours in a medium of ph 1.2. Besides, the fastest PRG release (in 2 hours), resulting in the release of 55% drug is observed in the case of PEG-b-P(nBA 12 -co-maa 21 ). In this study, it was observed that there was no change in the release profile at ph above 7.0 [16]. Hence, it may be agreed that the increase in drug bioavailability is very important to ensure good drug stability, in the GI tract, as well as reducing drug leakage and precipitation in the stomach. These two studies indicate that the micelle stability profile can be altered by manipulating block copolymers, which are ph sensitive under different environment. This fact may be utilized for controlling drug release and as a possible way to increase the bioavailability of poorly water-soluble drugs Clinical Trials in ral Drug Delivery The investigations of the application and efficacy of polymeric micelle, in enhancing the solubility of oral drug administration, including the encapsulation of hydrophobic oral compounds by polymeric micelles have been carried out by several pharmaceutical researches. Shamma et al. studied the incorporation of pluronic/ phosphatidylcholine/polysorbate 80 mixed micelles (PPPMM) with poor oral bioavailability drug, Nimodipine [17]. Nimodipine (NM) is used in oral drug administration for treating stroke and migraine [51, 52]. Besides, this drug is characterized as Class II drug by the Biopharmaceutical Classification System (BCS) with high permeability and poor solubility characteristic [60]. The study was done to intensify nimodipine aqueous solubility, so as to improve its bioavailability, increase drug retention time and assist in the delivery of the drug into brain tissue. NM-loaded with PPPMM was prepared by using thin film hydration technique and subjected to several analyses such as drug payload, solubilization efficiency (SE), micellar size, zeta potential, and transmission electron microscopy (TEM). The study reported drug payload of all NMloaded PPPMM ranging from 0.95 to 1.1 mg/ml, while their solubilization efficiency for samples stored at 5±3 C for 24 hours ranged from 92.5±1.48% to 99.2±2.01%. Such high value of SE indicates that the accommodation of nimodipine in the micelle core was very stable and efficient. n the other hand, the mean particle size of the drug loaded polymeric micelle ranged from ±9.08 nm to ±15.89 nm while the particle size distribution values ranged from 0.35 to The zeta potential was found at a range -22.3±0.005 to -31.2±0.00g mv. The value of more than ±20.0 results in good stability of the sample [61]. In another study, the investigation of the efficacy of diblock copolymers micelles, PEG-p(CL-co-TMC) [methoxypoly(ethylene glycol)-poly(caprolactone/trimethylene carbonate)] as oral drug delivery system, by using risperidone as the model drug, was carried out [62]. Risperidone is an antipsychotic medicine which is characterized as Class II drug by biopharmaceutical classification system (BCS) with good permeability but poor solubility in water. ne of the objectives of this study was to estimate the enhancement of risperidone oral bioavailability after encapsulation with the diblock copolymers, in an in-vivo experiment. During the oral administration experiment, male Wistar rats were used and risperidone solubilized at 2.5 mg/ml in 0.625% w/v tartaric acid, or in 10% w/v PEGp(CL-co-TMC) polymeric micelles. Pharmacokinetic-pharmacodynamic study was performed in order to evaluate the polymeric micelles for oral drug delivery. Two main therapeutic targets for antipsychotic drug which are dopamine D2 and serotonin 5-HT2A [63] was used to measure their central occupancy so as to study the ability of PEG-p(CL-co-TMC) solution to deliver risperidone into brain after oral treatment. Risperidone solution with the diblock copolymers was compared with risperidone aqueous solution containing tartaric acid. The result of kinetics dopamine D2 and serotonin 5-HT2A receptor occupancy in the brain after oral application of 2.5 mg/kg risperidone in 0.625% tartaric acid or in 10% micellar solution [62]. According to reported data, it can be seen that there is no huge difference between both formulations. However, after 10 minutes of administration, the level of 5-HT2A receptor was low when formulating the drug with copolymer, 27.7±12% compared to occupancy for risperidone with tartaric acid solution (62±4%). From the above result, determination of plasma level between the two formulations was performed for pharmacokinetic study. Even through in this study, there is no significant difference be-

7 Drug Delivery of Polymeric Nano-Micelles Current Drug Metabolism, 2016, Vol. 17, No Table 3. Polymeric Micelles Application in Hydrophobic ral Drug Delivery. Polymers CMC (mg/l) Incorporated Drug References PEG-DSPE/TPGS a Paclitaxel [70] PEG-b-P(VBDENA) b Paclitaxel [71] PEG-b-PLA Griseofulvin [72] PEG-b-P(CL-co-TMC) c 30 Risperidone [62] Ketoconazole Indomethacine PEG/MG/SA d Risperidone [73] Hydrocortisone Cyclosporin PEG-b-PLA/PLA-CH NA Itraconazole [74] PE-b-PP-b-PE 1.6 x10-4 to 6.9x10-5 Camptothecin [75] tween risperidone tartaric acid solution and risperidone-loaded polymeric micelle solution. It means that, the two solutions have same bioavailability. Hence, in this study, it was concluded that this novel copolymer has high opportunities in enhancing the solubility for poor oral drug bioavailability. Another study aimed to encapsulate hydrophobic drug, named Genistein, with tri-block copolymers (Pluronics F127) so as to see the ability of polymeric micelles in enhancing the drug bioavailability [64]. Genistein is a phytoestrogen compound, found in soy product, which is efficient in treating cancer [57, 58], cardiovascular diseases [67], osteoporosis and postmenopausal symptoms [68]. This drug is hydrophobic and its aqueous solubility is 0.81μg/mL [69] and also considered as low oral bioavailability. In the investigation, solid dispersion method was used to incorporate Genistein into Pluronics F127 and analyzed by its in-vitro characteristics like drug loading amount, drug loading efficiency, and genistein release profile. Drug loading amount and efficiency of incorporated genistein with the polymeric micelles was discussed by Kwon et al. [64]. Genistein-loaded pluronics f127 was prepared by manipulating genistein/tri-block copolymer ratios. The drug loading amount was increased from 6.51 to % with the increment of genistein/pluronics f127 ratios, from 10/135 mg to 17.5/135 mg. The increase in drug loading amount indicates the ability of polymeric micelle to incorporate the poor solubility drug in the micelle core. n the other hand, the maximum drug loading efficiency was achieved with the genistein/pluronic F127 copolymer ratio of 17.5/135 mg. According to these three examples, concerning the application of polymeric micelles in drug delivery system, it can be seen that the efficacy of polymeric micelles are an excellent carrier for hydrophobic drugs. Therapeutic effect has been investigated and evaluated by various researcher s since 1990 s. Thus relating with this topic, the other investigations of various polymeric micelle in enhancing solubility oral drug administration have been summarized and listed in Table PLYMERIC MICELLE AS ANTI-CANCER DRUG DE- LIVERY AGENT In 2012, World and Health rganization (WH) updated cancer as a chronic disease which attacks people almost every single year and causes to increasing morbidity and mortality [51, 52]. Several types of cancer, suffered by men and women, namely lung [78], lymphoma [79], stomach [80], breast cancer [81], etc., are the primary causes of deaths each year. Nowadays, major cancer therapies like chemotherapy, radiation, and surgery are applicable, depending on the stage of each type of cancer [82]. Chemotherapy is the most preferable treatment in cancer therapy. Unfortunately, it possesses poor water solubility and pharmacokinetics, and the therapeutic effect is compromised by short circulation time and toxicity. The poor characteristic of anticancer drug may cause various problem such as short drug circulation time, inappropriate biodistribution and possibility to attack healthy tissue (leading to toxicity and side effect) [83]. Besides, the crucial causes for the failure of chemotherapeutic agent as cancer therapy is multidrug resistance (MDR) in cancer [84]. The MDR causes cytotoxic molecules to be out of the target cells which include antibiotics, antimalarials, herbicides, and chemotherapeutic agent for cancer [85]. Since 100 years ago, enormous effort and work have been carried out so as to improve these limitations and to achieve excellence in the anti-cancer drug therapeutics. As the examples, Paul Ehrlich the recipient of Nobel Prize for Physiology and Medicine in 1908, proposed the concept of magic bullet which promises the advantageous of drug delivery to target cells. Besides, nanocarriers have gained more attention in research field as anti-cancer drug delivery which include liposomes, nanoparticle, and micelles [86]. Encapsulating anti-cancer drug with polymeric micelle is one of the promising drug delivery system which is used in pharmaceutical study in order to enhance the drugs solubility [9] The micelle core is able to solubilize and minimize loss and degradation of the anti-cancer drug. Small size of polymeric micelles allows the drug to escape from the target area, once the micelles are in the tumor vicinity, and extended the drug retention time due to the lack of lymphatic drainage in tumor sites [2]. Hence, these sub-sections focus on research in the use of polymeric micelles as delivery chemotherapeutic agent Polymeric Micelles Targeting Strategies in Cancer Therapy Traditional or standard chemotherapeutic drugs face various challenges in targeting tumor site which include non-systematic drug distribution into tumor site, insufficient drug concentration reaching the tumor, lack of efficiency to avoid toxicity and side effects to normal tissues and cells [29]. However, the combination of anti tumor agent with polymeric micelles can efficiently defeat these limitations [87, 88] by passively and actively targeting strategies EPR Effect Targeting Strategies of Polymeric Micelle Solid tumors can be considered as cancer and non-cancer depending on the types of cells formation. Tumors are caused by dis-

8 8 Current Drug Metabolism, 2016, Vol. 17, No. 00 Aziz et al. tinct pathophysiological characteristic that does not exist in normal tissues, resulting into hypervasculator, lymphatic drainage system, and increasing vascular permeability [89]. Inside solid tumors, leaky blood vessel and poor lymphatic drainage system resulting the enhanced permeation and retention (EPR) effect and it allows polymeric micelles to escape from leaky capillary bed [29] and passive targeting tumor site so as to effectively deliver the antitumor drugs (Fig. 5). utstanding drug accumulation in tumor site can be achieved by using polymeric micelles as the drug vehicle [87]. This strategy can be known as passively nano-carriers tumor targeting in anti-cancer drug delivery. In polymeric micelles passive targeting technique, the small size of the micelles (<100 nm) are able to avoid the action of reticulo-endothelial system so as to enhance the drug circulation time efficiency in targeting the pathological tissues. Hydrophobic core as the drug reservoir efficiently against plasma protein adsorption and also because of hydrophilic polymer coating or branch copolymer used [90 92]. Besides the potency of polymeric micelles in EPR effect in delivery various anti-cancer drugs have been evaluated in pre-clinical and clinical studies. Fig. (5). Schematic representation of enhanced permeability and retention effect (EPR effect). Passive tissue targeting is achieved by extravasation of PMs through increased permeability of the tumor vasculature and ineffective lymphatic drainage. Matsumuka research group constructed self-assembly SN-38- loaded polymeric micelles (PEG-PGlu), formulation named NK012. In pre-clinical studies, the formulation was accumulated selectively and stay for a long time in solid tumor tissues by employing EPR effect [93, 94], while in clinical showed the NK012 formulation promised anti-tumor activity characteristic. Unfortunately, there are several challenges involve in EPR effect polymeric micelles targeting strategies. In EPR effects case, there was high osmotic pressure in tumor space after the drugs had escaped from poor lymphatic drainage and accumulated. This phenomenon possess to drug redistribution across several cancer tissues [95]. Moreover, tumor porosity and pore size are not fixed and varies according to the types and status of the tumor. Hence, the passive targeting system may not distribute into all tumors [96] Active Targeting Strategies Polymeric Micelles As the alternative to passive targeting, active targeting promises several advantageous compared to EPR effect. This strategies for drug targeting performed various method to enlarge amount of tumor cells selectively and enhance intracellular drug delivery [97]. The techniques involved in active strategies system consist of ligand-receptor targeting, antibody-antigen targeting, aptamers and stimuli-responsive targeting. Ligand receptor targeting method is based on receptor mediated endocytosis [98]. The bonding between ligand and polymeric micelles as targeting molecules bind with their specific receptors inside cell membrane, express or overexpressed on tumor cells surface and expected to efficiently deliver drug into tumor cells [99, 100]. Liu and co-workers developed paclitaxel, PTX-loaded polymeric micelles self assembled with ligand HA-C18 folic acid modified and bind with CD44 receptor to target MCF-7 and A549 cells. The study reported the compound expressed on the both cells [101, 102]. Additionally, incorporation of paclitaxel with polymeric micelles and modified by cyclic RGD peptide [c(rgdyk), cyclic arginine-glycine-aspartic acid-d-tyrosine-lysine] and bind together with integrin v 3 resulting into overexpressed on tumor vasculator and U87MG glioblastoma cells. As the result, in-vitro physicochemical characterization of the formulation showed sufficient loading capacity, size distribution and encapsulated efficiency. In addition, for in-vitro cytotoxicity studies demonstrated in enhanced the anti glioblastoma cell cytotoxic efficacy by 2.5 folds [103, 104] In antibody-antigen targeting method, antibodies are first introduced as one of the technique to cure cancer [105]. This targeting system has been extensively used after the discovery of hybridoma technology. The formulation of micelles containing Pluronics and murine polyclonal antibodies as actively targeted micelle-based drug delivery system has been first developed by Kabanov and his co-workers. The study aimed to efficiently deliver the antipsychotic drug into brain [106]. Moreover in Torchilin s study, hydrophobic drug, photodynamic loaded with PEG-PE polymeric micelle modified by monoclonal antibody 2C5 was formulated and indicated greatly enhanced drug targeted onto tumor cells compared to nontargeted drug loaded micelles [107]. Aptamer mechanism consist of single-stranded DNA or RNA oligonucleotides which usually compared with antibodies due to their effectiveness in specificity and affinity in targeting molecules [108]. More than 10 years back, aptamers received more attention in cancer therapeutics field because of their enormous benefits over other targeted therapeutics (Table 4). In addition, aptamers are able to be easily chemically modified depending on the diagnostic and therapeutic purposes [109]. Additionally, aptamers size is much smaller than other targeted therapeutic like monoclonal antibodies, thus more easily to be penetrated onto tumor cell [110]. The application of aptamers in tumor targeting, polyethyleneiminepolyethyleneglycol (PEI-PEG) modified with anti-psma aptamer (APT) was developed as a carrier for co-delivery of small hairpin RNA (shrna) against the anti-apoptotic gene Bcl-xL, and the anticancer drug DX. The study reported APT-mediated co-delivery of an anti-cancer drug and shrna against Bcl-xL may broaden the healing window and allow for the selective demolition of cancer cells [111] Clinical Trials Enormous efforts in developing hydrophobic anti-cancer drug incorporated with polymeric micelles to enhance drug therapeutics in cancer therapy are the reason on why many polymeric micelles are under clinical research. So far, there are various formulations have been evaluated in clinical trials and will be discussed in more detail SP1049C Several block co-polymers, Pluronics L61 and F127 have been used in formulating SP1049C and it was formulated by physically encapsulated with Doxorubicin (anti cancer agent). SP1049C was shown high efficacy anti-tumor activity achieved by MTD at 70mg/m 2 in phase I study which same dose with conventional doxorubicin, but SP1049C was much better in drug performance and safety [112]. In pharmacokinetic result reported by Alakhov s research team, in vitro study shown the formulation revealed excellent efficacy against variety tumor cells compared to doxorubicin alone [113]. Elsewhere, the cytotoxic activity of SP1049C was greatly effective against multidrug resistant cells which usually affect the potency of doxorubicin and other cytotoxic drugs [114].

9 Drug Delivery of Polymeric Nano-Micelles Current Drug Metabolism, 2016, Vol. 17, No Table 4. Points to consider for successful cancer therapeutics. Properties Requirements Candidates Target specificity and Binding affinity Low nm - pm Antibodies, Aptamers, Peptides Screening and Production Efforts Screening : In vitro Fast and Low ZCost Peptides, Aptamers Immunogenicity Low Humanized Antibodies, Aptamers Modification Easy to Conjugation Small Molecules, Peptides, Aptamers NC-6004 (Nanoplatin) This drug-loaded polymeric micelle formulation involved chemotherapeutic key drug named cisplatin (cis-dichlorodiammineplatinum [II]: CDDP] incorporated with micellar nanoparticle for the therapies of lung, gastrointestinal, and genitourinary cancers. However, the applications of this drug are limited due to critical side effects like chronic neurotoxicity and kidney failure [94]. Hence, NC-6004 was developed so as to overcome the problems and enhance drug efficacy. It was formulated by the complex incorporation of CDDP and di-block copolymers poly-(ethylene glycol)- poly-(glutamic acid), PEG-pGlu. This formulation was reported to high accumulation in solid tumors and resulted in increasing drug retention time in blood circulation. The results were according to passive targeting mechanism (20 fold higher than free cisplatin). Additionally, 4mg/kg of the polymeric micelle drug administration to four mice resulted with complete tumor regression and without any weight loss [115] NK012 This formulation was developed by physical entrapment of block copolymer poly (ethylene glycol)-polyglutamate (pglu) and 7-ethyl-10-campothecin (SN-38). This drug is a biologically active metabolite of irinotecan hydrochloride (CPT-11) and having the anti-tumor activity but has not been used in clinical study due to its hydrophobic characteristic [116]. SN38-loaded PEG-pGlu was formulated by covalently bonded of drug and PGlu segment by condensation reaction between phenol of SN-38 and carboxylic acid of PGlu, and the reaction was boosted by 1,3-diisopropylcarbodiimide and N,N-dimethylaminopyridine as coupling agent and catalyst respectively [116, 117]. Koizumi et al reported that CPT-11 had poor efficacy in cytotoxic effect against cancers compared to NK012 exhibited much higher cytotoxic effect against cancers like lung and colon cancer. Plus, NK012 also had significant antitumor activity against bulky SBC-3/Neo (1,533.1 F 1, mm3) and SBC-3/VEGF tumors (1,620.7 F mm3) compared with CPT-11. In drug distribution analysis, there was excellent in drug accumulation performance in SBC-3/VEGF tumors compared to SBC-3/Neo tumors. Thus, it can be seen that this formulation could be used as promising anti-tumor agent [116] NK105 Paclitaxel is one of the chemotherapeutic agents which usually used in treatment of lung, ovarian and breast cancer [118]. Unfortunately, patients who had been treated with paclitaxel suffered with serious adverse effects like neutropenia and peripheral sensory neuropathy. In addition, compounds like Cremophor EL and ethanol are used as solubilizing agent for paclitaxel, however there was reported in hypertensive reaction by 2-4% patients treated with this drug [119]. Thus, new paclitaxel formulation such as NK105 was formulated and evaluated. The micellar solution was made from the drug conjugated with PEG and modified polyaspartate as hydrophobic chain. The paclitaxel was physically incorporated with the polymeric micelle by hydrophobic interaction between drug and polymer core [98]. In preclinical in vivo studies with colon 26- bearing CDF1 mice, the AUC of NK105 was 50 times higher, while the maximum plasma concentration (Cmax) in the tumors was three times higher as compared to PTX [93]. In addition, significant reduction of side effects, caused by Cremophor EL and ethanol after systemic PTX administration, occurred with NK105 [120]. In addition, phase I study with NK105, less hypersensitivity reactions occurred in patients suffering from pancreatic, bile duct, gastric, and colonic cancers compared to systemic PTX treatment [121]. Above discussions are the example of hydrophobic anti-cancer drug loaded with various types of polymeric micelles and almost all results show the bioavailability of the drug can be enhanced by polymeric micelles as drug vehicles in targeting cancer cells. 6. PLYMERIC MICELLE AS TPICAL DRUG DELIVERY Topical or transdermal drug delivery system involves a technique where the drugs are delivered topically onto human skin to achieve specific therapeutic effects and this therapy has been applied for a long time to treat local ailments. Cosmetics, pharmaceutics, and biotechnology industries are consider this drug delivery system since it possess efficient and safe in delivering drugs such as enhancing bioavailability, painless and non-invasive drug dosage, adverse effects reduction and many mores [14]. However, there are several limitations in transdermal route even though it seems to be the best alternative for oral and hypodermic administration. Limited drugs are available to administered topically onto human skin due to stratum corneum which act as the major barrier for the penetration of drug [122]. Stratum corneum (SC) located at outermost first human skin layer and highly organized crystalline lipid lamellae. Various physical mechanisms have been applied in order to disrupt and weaken the SC layer such as iontophoresis [123], electroporation [124], and sonopheresis [125]. Unfortunately, these techniques are more suitable to be used for water soluble drug since skin layer under SC layer, named VC layer has hydrophilic characteristic [126]. For hydrophobic drugs, encapsulation technique is the best way to deliver drug safely inside drug reservoir (core) to the target side. The classes of the drug vehicles used to encapsulate the drugs are lipids, surfactant, polymers, and so on. Among them, polymeric nanoparticle received good attention during past decades due to their benefits and versatilities as drug delivery [127, 128] Types of Block Polymers In transdermal route, polymers can be classified into two categories namely natural and synthetic polymers. Natural polymers are consist of chitosan, cellulose, and alginate [129, 130], while the lists for synthetic polymers are PEG, PGA, PLA, PCL, PP and acryl polymers [14]. However, synthetic polymers are more beneficial rather than natural due to their controllability in topology, molecular weight, degradability, composition, etc. This occurs because among natural polymers, there are less effective method to enhance their stability, drugs permeation efficiency, and drug control release rate. Poly(ethylene glycol) is the vast studied synthetic polymer and able to make covalent bond with other compounds like polymer, drugs, DNA and many more. Apart from that, it has also been broadly used against immunogenicity besides long-term circulation