Current Pharmaceutical Design, 2011, 17, 65-79 65 1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. Self Assembling Polymers as Polymersomes for Drug Delivery Jay Prakash Jain #1 , Wubeante Yenet Ayen #1 and Neeraj Kumar* 1 1 Department of Pharmaceutics, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar- 160062, India Abstract: Polymersomes are one of the most interesting and versatile architectures among various self assembled systems for drug deliv- ery. The stability and ability to load both hydrophilic and hydrophobic molecules make them excellent candidates to use as drug delivery systems. They demand for certain physicochemical parameters; especially hydrophilic to hydrophobic block ratio of copolymer to form vesicular morphologies. Different amphiphilic copolymers as well as their architectures show differences in the requirement of hydro- philic to hydrophobic block ratio to form polymersomes with various types of morphologies. This review focuses on basic aspects of po- lymersomes along with a series of copolymers employed for preparation of polymersomes and their potential applications as drug deliv- ery systems. Keywords: Self assembly, polymers, PEG-PLA, (PEG) 3 -PLA, polymersomes, polymeric vesicles, stimuli responsive, drug delivery. INTRODUCTION Cells contain, protect and transport biological substances as per physiological requirements. They can represent the basic model for any vesicular system and provide an understanding for the construc- tion of nature inspired vesicular systems from amphiphilic mole- cules that can be designed to carry, protect, target and release con- tents [1]. With basic understanding of cell morphology, phospholip- ids were first identified for preparation of vesicular systems, com- monly known as liposomes. Since their discovery, liposomes find diverse applications in the field of biology, chemistry, physics and as delivery systems for drugs and cosmetically active agents [2, 3]. They can be used as nontoxic, biodegradable and non-immunogenic delivery systems for drugs of different physicochemical properties (hydrophilic, hydrophobic or amphipathic) [1]. However, practical applications of liposomes have been continually hindered by lack of stability and rapid uptake by reticuloendothelial systems (RES). The former is presumably due to thin (3-5 nm) hydrophobic bilayer membrane. As a consequence, the vesicle properties like encapsu- lant retention and degradation are not well controlled. This issue was dealt by increasing the transition temperature of phospholipids and incorporation of cholesterol. The latter problem has been ad- dressed to an extent by PEGylation of lipids to form “stealth liposomes” [4, 5]. PEGylated-stealth liposomal doxorubicin (Doxil/Caelyx ® ) is the first liposomal anticancer drug formulation approved by Food and Drug Administration (FDA) in 1995. How- ever, phospholipids have limited flexibility for chemical modifica- tion by PEGylation as PEG-lipids at higher PEG densities have a tendency to form micelles due to disproportionately large PEG head groups. To counter this, the hydrophobic chain needs to be propor- tionally larger, which is not possible using phospholipids. A polymer approach to vesicle formation can broaden the range of the properties achievable due to plethora of possibilities to gen- erate such amphiphiles by controlling the polymer’s molecular weight, polydispersity and relative hydrophilic to hydrophobic block ratio [6-8]. The use of such amphiphilic block copolymers for generation of polymer vesicles, referred to as “polymersomes”, has now become an attractive strategy to create stable and biocompati- ble structures for drug delivery applications. Polymersomes are hollow, lamellar and spherical structures whose dimensions and *Address correspondence to this author at the Department of Pharmaceutics, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar-160062 INDIA; Tel: +91172-2292057; Fax: +91172-2214692; E-mail: [email protected] # Equal contribution in the manuscript. morphologies can be controlled by varying chemical constitution and size of copolymer, preparation methods, solution properties such as initial copolymer concentration, pH, temperature, solvent type, etc. [9-17]. The representative structures of copolymers used, types of encapsulants, model structure of polymersomes, and types of stimuli conditions responsible for degradation of polymersomes are demonstrated in Fig. (1). Due to their hollow and spherical morphology, polymersomes are capable of encapsulating various agents within the vesicle core or in the hydrophobic bilayer depending on the characteristics of encap- sulant as demonstrated in Fig. (1). In general, hydrophilic mole- cules are encapsulated within central aqueous core, whereas hydro- phobic molecules are entrapped into hydrophobic bilayer membrane [18]. ADVANTAGES OF POLYMERSOMES AS VESICULAR DELIVERY SYSTEMS Liposomes have been extensively studied over the years for various applications. However, poor stability of liposomal delivery systems limits their utility in drug delivery and resulted in a few marketed products regardless of extensive and long research in this area. The poor stability is due to chemical and physical properties of the liposomes. Chemical instability is caused by oxidation, acyla- tion and rapid hydrolysis of phospholipids whereas, physical insta- bility is mainly due to low molecular weights of phospholipids that lead to a thin (typically 3-4 nm) and leaky membrane [1, 19]. Such limitations with lipids have motivated development of po- lymersomes prepared using organic super amphiphiles such as di- block copolymers. Polymersomes have similar utility to their lipid counterparts but consist of two covalently linked distinct copolymer chains. Unlike liposomes (lipid vesicles), for polymersomes (poly- mer vesicles) synthetic control over amphiphilic block copolymer chemistry enables tunable design of polymersome material proper- ties through polymer synthesis [6, 8, 20, 21]. Importantly, copoly- mer molecular weights can be considerably larger (3-20 kDa) than those of natural lipid membranes (<1 kDa). For range of copoly- mers that self assemble into polymersomes, the thickness of hydro- phobic membrane varies from 8-22 nm depending on copolymer molecular weight and relative hydrophilic to hydrophobic block ratio [6]. Hence, polymersome membranes provide a novel experi- mental opportunity to study thickness dependence of membrane properties, such as stability, fluidity and permeability, in a system- atic way as represented in Fig. (2). In practical terms, many achievements with liposomes such as protein integration, fusion, DNA encapsulation, bio-compatibility,
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Current Pharmaceutical Design, 2011, 17, 65-79 65
1381-6128/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.
Self Assembling Polymers as Polymersomes for Drug Delivery
Jay Prakash Jain#1
, Wubeante Yenet Ayen#1
and Neeraj Kumar*1
1Department of Pharmaceutics, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar-
160062, India
Abstract: Polymersomes are one of the most interesting and versatile architectures among various self assembled systems for drug deliv-ery. The stability and ability to load both hydrophilic and hydrophobic molecules make them excellent candidates to use as drug delivery
systems. They demand for certain physicochemical parameters; especially hydrophilic to hydrophobic block ratio of copolymer to form vesicular morphologies. Different amphiphilic copolymers as well as their architectures show differences in the requirement of hydro-
philic to hydrophobic block ratio to form polymersomes with various types of morphologies. This review focuses on basic aspects of po-lymersomes along with a series of copolymers employed for preparation of polymersomes and their potential applications as drug deliv-
ery systems.
Keywords: Self assembly, polymers, PEG-PLA, (PEG)3-PLA, polymersomes, polymeric vesicles, stimuli responsive, drug delivery.
INTRODUCTION
Cells contain, protect and transport biological substances as per physiological requirements. They can represent the basic model for any vesicular system and provide an understanding for the construc-tion of nature inspired vesicular systems from amphiphilic mole-cules that can be designed to carry, protect, target and release con-tents [1]. With basic understanding of cell morphology, phospholip-ids were first identified for preparation of vesicular systems, com-monly known as liposomes. Since their discovery, liposomes find diverse applications in the field of biology, chemistry, physics and as delivery systems for drugs and cosmetically active agents [2, 3]. They can be used as nontoxic, biodegradable and non-immunogenic delivery systems for drugs of different physicochemical properties (hydrophilic, hydrophobic or amphipathic) [1]. However, practical applications of liposomes have been continually hindered by lack of stability and rapid uptake by reticuloendothelial systems (RES). The former is presumably due to thin (3-5 nm) hydrophobic bilayer membrane. As a consequence, the vesicle properties like encapsu-lant retention and degradation are not well controlled. This issue was dealt by increasing the transition temperature of phospholipids and incorporation of cholesterol. The latter problem has been ad-dressed to an extent by PEGylation of lipids to form “stealth liposomes” [4, 5]. PEGylated-stealth liposomal doxorubicin (Doxil/Caelyx
®) is the first liposomal anticancer drug formulation
approved by Food and Drug Administration (FDA) in 1995. How-ever, phospholipids have limited flexibility for chemical modifica-tion by PEGylation as PEG-lipids at higher PEG densities have a tendency to form micelles due to disproportionately large PEG head groups. To counter this, the hydrophobic chain needs to be propor-tionally larger, which is not possible using phospholipids.
A polymer approach to vesicle formation can broaden the range of the properties achievable due to plethora of possibilities to gen-erate such amphiphiles by controlling the polymer’s molecular weight, polydispersity and relative hydrophilic to hydrophobic block ratio [6-8]. The use of such amphiphilic block copolymers for generation of polymer vesicles, referred to as “polymersomes”, has now become an attractive strategy to create stable and biocompati-ble structures for drug delivery applications. Polymersomes are hollow, lamellar and spherical structures whose dimensions and
*Address correspondence to this author at the Department of Pharmaceutics,
National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S. Nagar-160062 INDIA; Tel: +91172-2292057;
Fax: +91172-2214692; E-mail: [email protected] #Equal contribution in the manuscript.
morphologies can be controlled by varying chemical constitution and size of copolymer, preparation methods, solution properties such as initial copolymer concentration, pH, temperature, solvent type, etc. [9-17]. The representative structures of copolymers used, types of encapsulants, model structure of polymersomes, and types of stimuli conditions responsible for degradation of polymersomes are demonstrated in Fig. (1).
Due to their hollow and spherical morphology, polymersomes are capable of encapsulating various agents within the vesicle core or in the hydrophobic bilayer depending on the characteristics of encap-sulant as demonstrated in Fig. (1). In general, hydrophilic mole-cules are encapsulated within central aqueous core, whereas hydro-phobic molecules are entrapped into hydrophobic bilayer membrane [18].
ADVANTAGES OF POLYMERSOMES AS VESICULAR
DELIVERY SYSTEMS
Liposomes have been extensively studied over the years for various applications. However, poor stability of liposomal delivery systems limits their utility in drug delivery and resulted in a few marketed products regardless of extensive and long research in this area. The poor stability is due to chemical and physical properties of the liposomes. Chemical instability is caused by oxidation, acyla-tion and rapid hydrolysis of phospholipids whereas, physical insta-bility is mainly due to low molecular weights of phospholipids that lead to a thin (typically 3-4 nm) and leaky membrane [1, 19].
Such limitations with lipids have motivated development of po-lymersomes prepared using organic super amphiphiles such as di-block copolymers. Polymersomes have similar utility to their lipid counterparts but consist of two covalently linked distinct copolymer chains. Unlike liposomes (lipid vesicles), for polymersomes (poly-mer vesicles) synthetic control over amphiphilic block copolymer chemistry enables tunable design of polymersome material proper-ties through polymer synthesis [6, 8, 20, 21]. Importantly, copoly-mer molecular weights can be considerably larger (3-20 kDa) than those of natural lipid membranes (<1 kDa). For range of copoly-mers that self assemble into polymersomes, the thickness of hydro-phobic membrane varies from 8-22 nm depending on copolymer molecular weight and relative hydrophilic to hydrophobic block ratio [6]. Hence, polymersome membranes provide a novel experi-mental opportunity to study thickness dependence of membrane properties, such as stability, fluidity and permeability, in a system-atic way as represented in Fig. (2).
In practical terms, many achievements with liposomes such as protein integration, fusion, DNA encapsulation, bio-compatibility,
66 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
etc. have been achieved with polymersomes too [6]. This broadens the range of polymersome properties through a wide choice of co-polymer molecular weight, polydispersity and relative hydrophilic to hydrophobic block ratio. Polymersomes are stable in biologically relevant temperatures and exhibit remarkable mechanical stability, for example, they can sustain >20% mechanical dilation (e.g. poly(ethylene oxide)-b-poly(butadiene) [PEO-b-PBD] vesicles) compared to phospholipid membranes that rupture at <5% stretch-ing. As discussed earlier, low stability of liposomes is attributed to their thin hydrophobic membrane, which makes them susceptible to
fluctuations and defects. However, polymersome membranes have a lower lateral diffusivity and a higher viscosity, with a fluidity that decreases with increasing molecular weight and most dramatically with chain entanglement [15, 22]. These properties can be further enhanced when the shell is cross-linked by methods such as UV irradiation of cross linkable functional groups like methacrylate and butadiene [21, 23-25]. The enhanced stability (both in vitro and in vivo) and ability to tune their size and incorporate responsive or functional species are additional advantages [26-28].
Fig. (1). A systematic demonstration of various types of amphiphilic block copolymers which can self assemble into polymersomes for encapsulation and
controlled release of bioactive moieties in passive way or in response to stimuli.
Fig. (2). Schematic diagram showing changes in properties of bilayer membrane with increase in the molecular weights of macromolecular amphiphiles.
Liposomes have phospholipids which have molecular weight of <1kDa whereas copolymers in polymersomes can have one order magnitude higher molecular
weight and thus can affect physical properties accordingly.
Self Assembling Polymers as Polymersomes for Drug Delivery Current Pharmaceutical Design, 2011, Vol. 17, No. 1 67
REQUIREMENTS OF POLYMER COMPOSITION FOR
SELF ASSEMBLY INTO POLYMERSOMES
The basic requirement for self assembly structures is the am-phiphilic nature of macromolecules and in case of polymers, the presence of two blocks having different solubility in the aqueous environment (viz. hydrophilic and hydrophobic). Amphiphilic block copolymers can self assemble in water into various ordered mesophases, typically lamellar structures. Conceptual diagram of polymersomes with commonly used hydrophilic and hydrophobic block copolymers is illustrated in Fig. (3).
The ratio of these blocks controls the architecture of these as-semblies [29, 30]. A delicate balance of hydrophilic to hydrophobic segment is required to design copolymers, which preferably form vesicular structures to other structures [15, 31]. Diblock copolymers with small hydrophilic fractions (fEO) (fEO<20%) and large molecu-lar weight hydrophobic blocks exhibit a strong propensity for se-questering their immobile hydrophobic blocks into solid-like parti-cles [32, 33]. Increasing fEO to ~20–42%, generally shifts the as-sembly towards more fluid-like vesicles or other ‘‘loose’’ micellar architectures. For fEO >42%, however, generally both worm mi-celles (up to ~50% fEO) and spherical micelles are formed [31, 34, 35]. For example, typical non-biodegradable amphiphilic block copolymer composed of PBD-b-PEO is reported to form polymer-somes when hydrophilic fraction is in the range of 25-42% [6] and copolymers based on poly(ethylene oxide)-b-poly(caprolactone) (PEO-b-PCL) formed polymersomes when hydrophilic fraction is 11.8-18.8% [36]. A recent report from our group [37, 38], using branched copolymer composed of three poly(ethylene glycol) chains linked to a PLA chain via citric acid [(PEG)3-PLA] obtained polymersomes for 10-30% hydrophilic fraction [37, 38]. Molecular weight plays a major role at low hydrophilic fraction but very minor role in case of mesophases and the fact has been proven by Jain and Bates [31]. In case of degradable polymer chains, for example, poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA), the phase transitions become very important due to hydrolytic degradation of hydrophobic block which is reflected by an increase of hydrophilic fraction thereby changing polymersomes into micellar structures. This phenomenon plays an important role to deliver drugs in acidic environment like tumor region where acidic environment facilitates the faster degradation of hydrophobic block and increase in hydro-philic fraction which leads the micellar (macromolecular surfac-tants) formation. It is anticipated that the micelles have membrane disruptive surfactant activity and destabilize endolysosomal mem-
branes and help in delivery of drugs to their targets within cells [39].
SELF ASSEMBLING AMPHIPHILIC COPOLYMERS
Linear block copolymers have been most widely studied for polymersomes preparation. However, few recent publications have also reported branch, graft and dendritic block copolymers can form the polymersomes structure (Table 1). Initial work on the polymer-somes was focused on creating these superstructures and studying their behaviour using non-biodegradable copolymers. Polystyrene was the first and most widely used non-biodegradable hydrophobic block polymer. Eisenberg’s group [9, 10, 12, 17, 40, 41] studied polystyrene based block copolymers and reported various vesicular, tubular and micellar structures. Formation of various kinds of mor-phologies were studied by varying copolymer composition, initial copolymer concentration, polydispersity of the corona chain, nature of the common solvent, amount of water present in the solvent mix-ture, temperature, presence of additives such as ions, ho-mopolymers, or surfactants [9-12, 16, 17, 41]. These morphologies include small uniform vesicles, large polydisperse vesicles, en-trapped vesicles, hollow concentric vesicles, onions, and vesicles with tubes in the wall. However, these polymersomes were not utilized for drug delivery until recently when boronic acid deriva-tives of the polystyrene (PS) were used for the preparation of sugar responsive polymersomes based on PEG-b-PS block copolymers for controlled delivery of antidiabetic drugs. In this type of po-lymersomes, mixture of amphiphilic and stimuli-responsive boronic acid-containing block copolymers were used. The boronic acid containing block copolymers formed phase-separated domains in the polymersomes, which could be dissolved by increasing pH of the medium or by introducing sugar molecules (monosaccharides) such as D-glucose [42].
Later on, other non-biodegradable hydrophobic blocks such as poly(2-cinnamoylethyl methacrylate) (PCEMA) [43], poly(di-methylsiloxane) (PDMS) [24], poly(propylene oxide) (PPO) [44], poly(ethyl ethylene) (PEE) [6] and poly(butadiene) (PBD) [45, 46] were also used to synthesize diblock copolymers for preparation of polymersomes. Amongst these, PBD is the most widely explored for various biomedical applications. PBD based copolymers have also been used for artificial leukocyte to deliver therapeutic agents [47]. These copolymers have also been blended with biodegradable polymers such as PCL and PLA for reducing the drug release rate from polymersomes [39, 48-50].
Fig. (3). Schematic presentation of the vesicle of block copolymers and a section removed to reveal the structure through membrane with some examples of
hydrophobic and hydrophilic blocks of amphiphilic copolymers.
68 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
Table 1. Amphiphilic Copolymers Used to Prepare Polymersomes.
A) Linear
A-1. Non-Biodegradable Polymers as Hydrophobic Block
Copolymers Applications Ref.
Hemoglobin delivery [26, 69, 70]
Magnetic Resonance Image (MRI)-guided delivery [71]
PR_b-targeted delivery of tumor necrosis factor- [72]
Targeted drug delivery to inflammatory sites [47]
As oxygen carrier [69]
In vivo diagnostic and drug delivery applications [73, 74]
Poly(ethylene oxide)-b-poly(butadiene) (PEO-b-PBD)
Drug delivery [20, 26]
Poly(ethylene oxide)-b-poly(butadiene)-Cross linked (PEO-
b-PBO)
Delivery system for chelated Gadolinium
(MR contrast agent)
[75]
Biomedical applications [76] Poly(ethylene oxide)- b-poly(butylene oxide) (PEO-b-PBO)
Incorporated with PEO as excipient,
Cell entrapment for transport and delivery,
Three dimensional (3D) scaffold for tissue engineering application
[77]
Poly(ethylene oxide)-b-poly(isobutylene) (PEO-b-PIB) Drug delivery [78]
pH and ionic strength responsive polymersomes [79] Poly( -l-glutamic acid -b- poly(butadiene)) (PGA-b-PBD)
Drug delivery [45]
Dextran-b-polystyrene (Dex-b-PS) Drug delivery [80]
Poly(ethylene glycol)-b-polystyrene boronic acid (PEG-b-
PSBA)
Sugar responsive drug delivery [81, 82]
Polystyrene-b-poly(isocyanoalanine(2-thiophene-3-yl-
ethyl)amide (PS-b-PIAT)
Drug delivery [83]
A-2. Biodegradable (Polyesters) Polymer as Hydrophobic Block
Copolymers Applications Ref.
Doxorubicin delivery [39, 48]
Drug delivery (Fluorescein isothiocyanate-dextran as model hydrophilic drug) [84]
Poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-
PCL); Poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-
PLA)
Delivery system for peptide brain delivery [polymersomes conjugated with
mouse-anti-rat monoclonal antibody OX26 (OX26-PO)]
[85]
Poly(ethylene glycol)-b-(2-nitrophenylalanine)-b-
poly(caprolactone) (PEG-b-NPAL-b-PCL)
Photo(UV) responsive vesicles [86]
Doxorubicin delivery (PCL is acrylated and photo-cross linked to stabilize mem-
brane)
[87, 88] Poly(ethylene oxide)-b-poly(caprolactone) (PEO-b-PCL)
As therapeutic oxygen delivery system [50]
Poly(ethylene oxide)-b-poly( -methyl-caprolactone)
(PEO-b-PMCL)
For deep tissue in vivo imaging [89]
Delivery system for imaging agent in MRI [90] Poly(ethylene oxide)-b-poly(caprolactone) blend
Poly(ethylene oxide)-b-poly(butadiene)
(PEO-b-PCL)/(PEO-b-PBD) Delivery of siRNA and AON [52]
Self Assembling Polymers as Polymersomes for Drug Delivery Current Pharmaceutical Design, 2011, Vol. 17, No. 1 69
(Table 1) Contd….
Copolymers Applications Ref.
Delivery of siRNA and AON [52] Poly(ethylene oxide)-b-poly(lactic acid) blend
Poly(ethylene oxide)-b-poly(butadiene)
(PEO-b-PLA)/(PEO-b-PBD) Simultaneous delivery of paclitaxel and doxorubicin [18, 91,
92]
Delivery system for therapeutic oxygen [50] Poly(ethylene oxide)-b-poly(lactic acid)
(PEO-b-PLA) Incorporated with poly(N-isopropylacrylamide) for sustained delivery of large
and small molecules
[49]
Poly(ethylene glycol)-b-poly(lactic acid)-b-Poly(l-
glutamic acid) or poly(ethylene-glycol)-b-
Poly(caprolactone)-b-poly(l-glutamic acid)
(PEG-b-PLA-b-PGA) or (PEG-b-PCL-b-PGA)
Oral delivery of insulin [93]
Poly(2-hydroxyethyl aspartamide) grafted with lactic acid
oligomers
Delivery systems in biotechnology, pharmaceutical and cosmetic fieldss [94]
A-3. Polycarbonates as Hydrophobic Block
Copolymers Applications Ref.
Poly(ethylene glycol)-b-poly(trimethylene carbonate) (PEG-b-PTMC) Drug delivery (Fluorescein isothiocyanate-dextran as model hydro-
philic drug)
[84]
Stimuli responsive (pH, ionic strength) [28] Poly(l-glutamic acid)–b-poly(trimethylene carbonate) (PGA-b-PTMC)
Temperature and pH sensitive drug delivery [53, 64]
Poly(ethylene glycol)-b-poly(2,4,6-
trimethoxybenzylidenepentaerythritol carbonate) (PEG-b-PTMBPEC)
pH sensitive polymersomes for simultaneous delivery of paclitaxel
and doxorubicin
[95]
A-4. Protein/peptide based Polymersomes (Peptosomes)
Copolymers Applications Ref.
Doxorubicin delivery [60, 96] Poly( -benzyl l-glutamate)-b-hyaluronan
(PBLG-b-HYA) Docetaxel delivery [68]
Poly( -benzyl l-glutamate)-b-coiled-coil Peptide (PBLG-b-E) Drug delivery [59]
A-5. Miscellaneous Linear Block Copolymers
Copolymers Applications Ref.
Transdermal drug delivery, siRNA delivery [97, 98]
Intraepithelial and/or transepithelial delivery of
drugs
[66]
Poly(2-methacryloyloxy-ethyl phosphorylcholine)-b-poly(2-diisopropylamino ethyl
methacrylate) (PMPC-b-PDPA)
Intracellular delivery of nucleic acids [99]
Poly(ethylene oxide)-b-poly(2-diisopropylamino ethyl methacrylate) (PEO- b-PDPA) Intraepithelial and/or transepithelial delivery of
drugs
[66]
Cholesterol-end-capped poly(2-methacryloyloxyethyl phosphorylcholine)
(Chol.-b-PMPC)
Doxorubicin delivery [67]
Poly(dimethylsiloxane) (PMDS) with 2-(dimethylamino)ethyl methacrylate
(DMAEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) (DEAEMA)
(PDMS-b-PDMAEMA) and (PDMS-b-PDEAEMA)
In vivo diagnostic applications [100]
70 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
(Table 1) Contd….
Copolymers Applications Ref.
Poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline)
(PMOXA-b-PDMS-b-PMOXA)
Drug delivery [101]
Poly(ethylene oxide)-b-poly(acrylic acid)-b-poly(N-isopropylacrylamide)
(PEO-b-PAA-b-PNIPAM)
Temperature sensitive triggered delivery of pDNA,
siRNA, pharmaceutical proteins and peptides
[56]
Cytoplasmic delivery of large molecules, such as
peptides, proteins, AON and DNA
[102] Poly(ethylene glycol)-SS-poly(propylene sulfide) (PEG-SS-PPS)
In vivo diagnostic applications [103]
Poly(ethylene oxide)-b-poly(propylene sulfide)-b-poly(ethylene oxide) (PEO- b-PPS-
b-PEO)
Glucose Oxidase delivery [54, 55]
Poly(ethylene glycol)-b-poly(fumaric/sebacic acids) AB and ABA type Drug delivery (Calcein as model hydrophilic drugs) [57]
B) Non-linear Amphiphilic Block Copolymers
B-1. Dendritic Block Copolymers
Poly(ethylene glycol)-b-2,2-bis(hydroxymethyl)propionic acid (bis-MPA) (PEG-b-AZO) Photo(UV) responsive vesicles [104]
Poly(amindoamine) Dendron-b-poly(l-lysine) (PAMAM-b-PLL) Drug delivery [105]
B-2. Tripodal Copolymers
Cyclotriphosphazenes grafted poly(ethylene glycol) and a hydrophobic oligopeptide Delivery of anticancer agents and other bio-
medical applications
[106]
B-3. Type 3-Miktoarm Star Copolymers
Poly(ethylene glycol)-poly(lactic acid)2 (PEG-(PLA)2) Doxorubicin delivery [107]
B-4. Branched Copolymers
methoxy-poly(ethylene glycol)3-citrate-poly(lactic acid) ((mPEG)3-PLA) Amphotericin B and Doxorubicin delivery [37, 38,
61, 62]
B-5. Grafted Copolymers
methoxypoly(ethylene glycol)-ethyl-p-aminobenzoate (polyphosphazenes) (PEG/EAB–PPPs) Doxorubicin delivery [58]
Use of biodegradable polymers, which are clinically more use-ful as hydrophobic block in polymersomes appeared lately in the literature. Polymersomes based on biodegradable polyesters, for example, PEO-b-PLA, have been used for delivery of small mole-cules such as doxorubicin [18, 36], paclitaxel [18], etc. and large molecules such as small interfering ribonucleic acid (siRNA) or antisense oligonucleotides (AON). This was focused on factors affecting drug loading and functional delivery of siRNA and AON with non-ionic, nano-transforming polymersomes. It has been shown that these biodegradable delivery systems are taken up pas-sively by cultured cells after which they undergo degradation and transform into micelles thus allowing endolysosomal escape and delivery of either siRNA into cytosol for mRNA knockdown or else AON into the nucleus for exon skipping within pre-mRNA. It was demonstrated that these vesicular structures may be used as the replacement of Lenti-virus due to polymersome mediated knock-down which appears as efficient as common cationic lipid transfec-tion and about half as effective as Lenti-virus after sustained selec-tion [51, 52].
Polycarbonates such as poly(trimethylene carbonate) (PTMC) are another category of biodegradable polymers which have been used as hydrophobic blocks to prepare polymersomes. These poly-mers are stimuli sensitive to pH and temperature, and were initially
used for delivery of anticancer drugs [28, 53]. Some other hydro-phobic polymers which have been used as hydrophobic blocks in-clude poly(propylene sulphate) (PPS) [54, 55], poly(N-isopro-pylacrylamide) (PNIPAM) [56], poly(fumaric/sebacic acids) [57], poly(phosphazenes) [58] to name a few. Protein based polymer-somes (peptosomes) have also been prepared and used for delivery of various bioactive agents [45, 59, 60]. Details are described else-where in this review. Recently efforts in our group [37, 38, 61, 62] have focused on the development of polymersomes from biode-gradable synthetic branched amphiphilic copolymer consisting of two previously FDA approved building blocks, PEG and PLA. Unlike other polymersomes reported in the literature, which are formed from linear block copolymers, this amphiphilic copolymer is synthesized from three PEG chains (with the aim of increasing PEG surface density of polymersomes) linked to PLA via citric acid. A controlled release of amphoteric drugs such as, ampho-tericin B was achieved [62]. Further, pharmacokinetics and phar-macodynamics evaluation of amphotericin B loaded polymersomes has been performed in murine models and results demonstrated better efficacy and tolerability of polymersome loaded amphotericin B compared to Fungizone (marketed micellar formulation of am-photericin B using deoxycholate) [61].
Self Assembling Polymers as Polymersomes for Drug Delivery Current Pharmaceutical Design, 2011, Vol. 17, No. 1 71
With respect to hydrophilic block of amphiphilic copolymer, poly(acrylic acid) (PAA) was the first hydrophilic polymer utilized for preparation of amphiphilic copolymer PS-b-PAA and further used for preparation of polymersomes to study their morphologies [12, 17]. Some other hydrophilic block polymers include poly(2-methyloxazoline) (PMOXA) for synthesis of amphiphilic copoly-mer poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMOXA-b-PDMS-b-PMOXA) [24], poly(iso-prene) (PI) in poly(isoprene)-b-poly(2-cinnamoylethyl methacry-late) (PI-b-PCEMA) [43], poly(4-vinyl pyridine) (P4VP) [11], poly(4-vinylpyridinum methyl iodide) (P4VPMeI) [63], PEG (or PEO) [18, 36], etc. Among all hydrophilic polymers, PEG (or PEO) is the most explored hydrophilic block for preparation of polymer-somes due to its following properties:
• Water-soluble, nonvolatile, unctuous and flexible in nature
• Nontoxic (FDA approved for internal consumption) and poorly immunogenic
• Successfully used for PEGylation of various small and large molecule drugs and delivery systems
• Forms well defined bilayers when used in proper proportion with hydrophobic counterparts
• Renders proteins and delivery systems nonimmunogenic and tolerogenic
• Prolongs the clearance time of PEGylated delivery systems and drugs in vivo by overriding RES
• Disposed off from body by glomerular filtration
Other than PEG, poly(l-glutamic acid) (PGA), a polypeptide and biodegradable polymer, is also used as hydrophilic block for polymersomes preparation, for example PTMC-b-PGA [28, 64], poly(dimethyl aminoethyl methacrylate)-b-poly(l-glutamic acid) (PDMA-b-PGA) [65], PBD-b-PGA [45]. Due to instability of PGA at low pH, polymersomes containing PGA have been used for de-livery of drugs to the biological environment at low pH such as tumor site. Some other less frequently explored hydrophilic blocks include phosphorylcholines [66, 67], hyaluronan for poly( -benzyl l-glutamate)-b-hyaluronan (PBLG-b-HYA) [60, 68] and poly-peptides for poly( -benzyl l-glutamate)-b-coiled-coil peptide (PBLG-b-E) copolymer [59].
CHARACTERIZATION OF POLYMERSOMES
The polymersomes can be characterized for their physical, me-chanical and chemical properties. The techniques used are same as applicable to colloidal particles (solid or vesicular particles) and mostly involve light scattering and various types of microscopic techniques.
Scattering Methods
Scattering of laser light due to sample particulate obstruction depends on the size of particle and hence can be used for size measurement in 1-1000 nm range. Two variations i.e. dynamic and static light scattering have been used for size measurement of parti-cles [108].
Visual Techniques
Direct visualization by various types of microscopy can reveal the size, morphology, surface properties, etc. of polymersomes. Microscopy is the most powerful, fast and easy tool for polymer-somes characterization. Resolution with light microscopy is very less compared to electron microscopy which is widely used for polymersome analysis.
Light Microscopy
Various types of light microscopy techniques are described in the literature with different resolution and contrast. Transmission light microscopy [57], phase contrast microscopy [109, 110] and differential interference contrast (DIC) microscopy [37] are some of
light microscopic techniques used for characterization of polymer-somes. Basic principles of each microscopic technique are found in the respective references and interested readers are referred to original papers for the details of techniques. The microscopic im-ages using all three techniques are depicted in Fig. (4A-C).
Light Microscopy Utilizing Fluorescence:
Fluorescence gives unique advantages over simple light mi-croscopy, due to very low limit of detection or high sensitivity and possibility of attaching single or multiple fluorotags for visualizing particular target molecule or structure [111-113]. Polymersomes can be fluorescent by encapsulating a fluorescent dye (Fig. (5A)) or the membrane can be simply stained by lipophilic probe which aggregates in the hydrophobic part. Alternatively, fluorophore can be covalently attached to the membrane molecules. A common problem with conventional fluorescence microscopy is that when fluorochromes are excited and detected, the resulting fluorescence from those fluorophores bound to the surface is overwhelmed by the background due to the population of non-bound or non-encapsulated molecules. The problem has been addressed by a technique called total internal reflectance fluorescence microscopy (TIRFM). A TIRFM elicits an evanescent wave that selectively illuminates and excites fluorophore in a restricted region of the specimen immediately adjacent to the interface of two different density media. The evanescent waves are generated only when the incident light is totally reflected from the interface. The wave de-cays exponentially with distance from the interface thus the TIRFM enables a selective visualization of surface region such as the mem-brane of a vesicular systems [114, 115].
Confocal Laser Scanning Microscopy (CLSM)
It is one of the most significant advances in optical microscopy that affords the ability to collect sharply defined optical sections from which 3D renderings can be created. CLSM offers several advantages over conventional wide field microscopy including, the ability to control depth of field, elimination or reduction of back-ground information away from the focal plane and capability to collect serial optical sections from thick specimens (Fig. (5B-C)). CLSM enables visualization of steady state structure as well as dynamic processes. Hence, the technique is very useful for vesicu-lar systems but the major limitation to its application as a standard tool is relatively high equipment costs [7, 74, 116].
Electron Microscopy
Electron microscopes [(e.g. transmission electron microscope (TEM) or scanning electron microscope (SEM)] use a focused beam of electrons instead of photons and developed to overcome the limitation of magnification and resolution in the optical micros-copy. TEM is routinely used to analyze the structure of polymer-somes and is reported by a number of researchers working in the area of polymersomes [54, 69, 92, 117]. To improve phase contrast, negative staining is used where it permits very high resolution im-aging of surface and sub-surface details (Fig. (6A)). A mixture of stain and sample of interest is allowed to dry, and then the stain accumulates on the vesicles and appears darker than the surround-ings. The extreme conditions (e.g. temperature and high vacuum) for specimens in the operation of TEM resulted in development of cryo-TEM which enables investigation in natural environment [34]. SEM is also a useful tool for studying the surface properties of polymersomes. It requires sample surface to be conductive or semi-conductive for study and hence, a thin layer of conducting material like carbon or gold is coated. SEM image is generated by secondary electrons, produced by impact of primary electron beam on conduc-tive surface and gives 3D appearance of surface. Example image of polymersomes prepared from (PEG)3-PLA is presented in (Fig. 6 (B)) [37].
Atomic Force Microscopy (AFM)
It utilizes a sharp probe moving over the surface of a sample in a scan. The force between tip and the sample is very small usually
72 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
less than 10-9
N and mostly measured by optical beam reflected from the mirror surface on the backside of cantilever onto a position sensitive photo detector. The technique has significantly contrib-uted in investigation of surface topography of various systems and can be equally important in case of polymersomes [46, 80]. AFM image of polymersomes from (PEG)3-PLA are represented in Fig. (6C).
Mechanical Characterization
The elastic behaviour of polymersomes is measured by mi-cropipette aspiration technique. It is employed to obtain elastic bending and stretching moduli which are deduced from the observa-tion of vesicle membrane extension into pipettes with diameters of several micrometers as a function of suction pressure [20]. Mi-cropipette aspiration makes use of video microscopy to follow the vesicle shape with the applied suction pressure. Further, this tech-nique can be used to directly measure area compressibility modulus, bending moduli, lysis tension, lysis strain, and area ex-pansion of membrane fluid phase [6, 118]. However, it is limited to giant vesicles (micrometer size) due to the necessity of optical mi-croscopy visualization.
Other Techniques
Some other techniques like UV spectrophotometry, Fourier transform infra red (FTIR) [119] and differential scanning calo-rimetry (DSC) [120] have been used for characterization of po-lymersomes. UV and FTIR spectroscopies have been used to char-acterize the cross linking of polymersomes in the PI-b-PCEMA copolymer based on the fact that absorption from CEMA disappears
during UV irradiation of a vesicle solution. From DSC analysis, thermal changes in the system, for example, due to transitions in morphologies can be studied. In general, use of particular technique depends on application/intended use, the characteristic of the po-lymersomes and specific properties of block copolymers used.
POLYMERSOMES FOR DRUG DELIVERY APPLICA-TIONS
Polymersomes have emerged as versatile platforms in drug delivery applications and have received special attention in last decade due to their stability, decreased leakage of the encapsulant, site specificity, longer blood circulation and overall potential thera-peutic and diagnostic applications. Though there is a huge potential for application of polymersomes in different disease conditions, currently, the research work is mainly focussed on using these structures in cancer therapeutics. Because of the complexity of can-cer, polymersomes are of special interest in delivering therapeutic agents, as discussed in the following sections.
The Challenges of Cancer Therapy
Cytotoxic anticancer therapy has relied primarily on the en-hanced proliferative rate of cancer cells, using drugs that act on DNA, enzymes such as the topoisomerases that are important in DNA replication (e.g. doxorubicin), and tubulin (e.g. paclitaxel). However, in patients with appreciable tumor burdens, there is a re-growth and spread of often more malignant and multidrug-resistant tumors following chemotherapy [121]. This is due to the fact that hypoxic cells in the center of tumors can be essentially dormant and much less susceptible to conventional cancer chemotherapy and
Fig. (4). Light microscopy images of polymersomes prepared from (PEG)3-PLA copolymer A) Transmission light microscopy; B) Phase contrast microscopy;
and C) Differential interference contrast microscopy. Each technique shows different contrast for inner lumen and outer wall of the vesicles.
Fig. (5). Microscopic images of polymersomes prepared from (PEG)3-PLA copolymer A) Fluorescence microscopy in the presence of calcein as a hydrophilic
fluorescent probe and cross sections of polymersomes by confocal laser scanning microscopy using B) Fluorescein (hydrophobic dye) C) Propidium iodide
(hydrophilic dye) as fluorescent probes.
Self Assembling Polymers as Polymersomes for Drug Delivery Current Pharmaceutical Design, 2011, Vol. 17, No. 1 73
there is also limited drug penetration [122]. The acquisition of drug resistance may be due to high hydrostatic pressure and/or presence of over-expressed receptors, such as plasma membrane P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP). These proteins actively pump out a large spectrum of struc-turally unrelated drugs, leading to sub-therapeutic intracellular drug concentrations [123, 124].
The toxicity associated with cancer chemotherapy is frequently the rate limiting factor in the clinical use of best available single or combination chemotherapeutic agents. Recent gains in cancer che-motherapy include the use of drug delivery systems, preferably polymer based delivery systems being investigated to enhance the clinical utility of established anticancer agents. Thus, the develop-ment of a polymeric drug delivery systems, which can encapsulate and release highly toxic therapeutic agents at desired site of action is getting considerable attention.
The Rationale for use of Polymersomes as Drug Delivery Sys-tems
While the search for new antineoplastic agents is in progress, optimization of delivery for existing drugs will remarkably improve the current scenario in management of cancer. Chemotherapy is the major therapeutic approach for treatment of cancers with high me-tastatic potential. However, the clinical application of conventional anticancer drugs involves high patient risks because the drugs are not specific to cancer cells as most of anticancer drugs are antipro-liferative thereby non-selectively attacking rapidly proliferative healthy cells. The inefficiency and toxicity of chemotherapy have been primarily associated with the formulation and biodistribution of the drug, and the acquisition of the drug resistance by cancer
cells. For instance, paclitaxel (Taxol®) is a representative example
of a drug that suffers from formulation issues because of its poor permeability and solubility [125], while doxorubicin is well known for its high volume of distribution because of its low molecular weight and amphipathic nature. Delivery systems of anticancer drugs must show sustained, controlled, and targeted release to de-liver a large portion of the administered drug to the desired site [126]. An ideal drug delivery system which would have a high drug loading capacity and encapsulation efficiency, long circulation time and adequate stability in the blood stream, and selective accumula-tion at the site of action together with suitable drug release profile and good biocompatibility, is a current demand of optimal cancer therapy. In this section, we highlight application of polymersomes in cancer therapy. Polymersomes possess a number of attractive biomaterial properties such as good stability and encapsulant reten-tion ability. As a result, they have received special attention during the past one decade with a higher potential to use as delivery sys-tems for anticancer drugs over existing lipid vesicles. The morphol-ogy and architecture of polymersomes with thick hydrophobic membrane and hydrophilic reservoir can be exploited for delivery of combination of anticancer drugs in clinical use.
Photos et al. injected various polymersomes prepared from PEO-b-PEE and PEO-b-PBD copolymers into rats and showed that their in vivo circulation times were approximately two times longer than stealth liposomes (coated with PEG) [27]. Studies carried out by different groups of authors have also proven that polymersomes are prime candidates as delivery systems for highly toxic therapeu-tic agents such as doxorubicin or hydrophobic agents such as pacli-taxel and docetaxel. In general, polymersomes as delivery systems
Fig. (6). Images of polymersomes prepared from (PEG)3-PLA copolymer A) Transmission electron microscopy (negatively stained); B) Scanning electron
microscopy; C) Atomic force microscopy.
74 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
can augment the pharmacokinetic and pharmacodynamic profiles of drug molecules [18, 39, 60, 64, 68, 91, 95, 96, 127-129].
Therapeutic potential of polymersomes encapsulating antican-cer drugs such as paclitaxel and doxorubicin have been demon-strated in vitro and in vivo in animal models by Discher’s group [18, 39, 91]. Blends of polymersomes prepared from non-biodegradable PEO-PBD and biodegradable PEO-PLA amphiphilic block copolymers were used. Studies in animal tumor models with doxorubicin and paclitaxel encapsulated in the interior aqueous lumen and thick hydrophobic wall, respectively have shown prom-ising pharmacologic results. In tumor bearing animal models, selec-tive accumulation of polymersomes in tumor, sustained peak tumor concentration and increased tumor drug exposure was achieved, which, in turn showed enhanced antitumor activity when compared to free drugs. Polymersomes with combination of both drugs clearly induced a 2.5 fold higher apoptosis in tumor after 2 days and results demonstrated a higher maximum tolerated dose, as well as in-creased tumor shrinkage and maintenance compared to free drugs [18]. Further, interesting approaches were introduced using biode-gradable polymersomes constructed from PEG-b-PCL amphiphilic copolymer for targeted delivery of doxorubicin into tumor cells [74].
In another study, self-assembled PEO-PBD polymersomes were evaluated as paclitaxel delivery systems and in vitro cytotoxicity study to inhibit proliferation of MCF-7 cell lines was demonstrated. The results showed that paclitaxel-loaded polymersomes have de-sirable inhibitory and sustained release profile as compared to pacli-taxel alone [46]. Recently, Lecommandoux’s group [60, 68, 96] conducted in vitro and in vivo evaluation of docetaxel and doxoru-bicin-loaded biodegradable polymersomes prepared from PBLG-b-HYA. Docetaxel-loaded polymersomes were stable either in solu-tion or in lyophilized form, which demonstrated controlled release behaviour over several days and showed high in vitro toxicity after 24 h in MCF-7 and U87 cell lines, compared to free docetaxel. In vivo studies in tumor bearing mice showed improved pharmacoki-netic, biodistribution and efficacy profile of docetaxel [68]. The same group [68, 96] has investigated the intracellular delivery of doxorubicin-loaded PBLG-b-HYA based polymersomes in high (MCF-7) and low (U87) CD44 expressing cell lines. The flow cy-tometry data demonstrated successful uptake and high accumula-tions in MCF-7 compared to U87 cell lines. Cytotoxicity of the drug was concentration and exposure time dependent. The pharma-cokinetic and pharmacodynamic profiles of the formulation demon-strated reduced cardiotoxicity compared to free doxorubicin. The results suggested that intracellular delivery of doxorubicin-loaded PBLG-b-HYA based polymersomes was dependent on the CD44 expression level in cells due to presence of hyaluronic acid on the surface of polymersomes. The cytotoxicity and internalization mechanism of doxorubicin-loaded polymersomes have also been evaluated in C6 glioma tumor cell lines [60]. In addition, C6 glioma cell lines are expressing CD44 receptors at their surface, which should help the hyaluronan based polymersomes internalization through endocytosis. Hence, this system could be used as self tar-geting drug delivery system in over expressed CD44 glycoprotein cells of breast cancer [60, 68, 96].
In addition to small molecules, peptides, proteins, and nucleic acids have been encapsulated in block copolymer assemblies by different groups of researchers [26, 50, 69, 130, 131]. The ability to encapsulate proteins and macromolecules within polymersomes provides a promise for future protein and gene therapies, which are currently facing delivery obstacles. Lee et al. [26] successfully encapsulated myoglobin, hemoglobin, and albumin in PEO-b-PBD based polymer vesicles but with variable efficiency. Rameez et al. [50] developed hemoglobin-loaded polymersomes using biodegrad-able and biocompatible amphiphilic diblock copolymers composed of PEG, PCL, and PLA. O’Nell et al. [131] demonstrated encapsu-lation of proteins with high encapsulation efficiency within po-
lymersomes prepared from block copolymers of PEG-b-PPS. Battaglia’s group [99, 130] have encapsulated DNA in polymer-somes prepared from PMPC-b-PDPA copolymer, which are pH sensitive and capable of releasing their contents upon exposure to the low pH media in endolysosomes, while the polymersomes pro-vide protection to DNA during storage. Few examples showing polymersomes as macromolecule delivery systems are represented in Table 2.
Release Mechanisms
Once polymersomes are reached to target site, such as tumor tissues, they have to release their encapsulated drug contents. The most common mechanism for release of encapsulant from polymer-somes is via hydrolytic degradation of hydrophobic block of am-phiphilic copolymers, for instance, polyester blocks such as PLA or PCL. This release mechanism takes advantage of the molecular shape dictated morphology of block copolymer aggregates. As the chain end of hydrophobic block hydrolyzes, the hydrophobic block systematically changes the molecular shape by increasing hydro-philic fraction and changing ultimately into micellar morphologies. This hydrolytic degradation is modulated by pH and accelerated at acidic pH, for example, in tumor microenvironment and endolysosomal compartments. Macromolecular surfactants (micel-lar structures) formed because of hydrophobic block degradation further porate endolysosomal membrane and help encapsulant to escape into cytoplasm [39]. In contrast to gradual mechanism of release caused by hydrolytic degradation of hydrophobic block of copolymer, due to synthetic nature of polymersomes, various re-lease mechanisms can be designed through response to external stimuli such as pH [91, 135], temperature [135-137], and oxidation-reduction conditions [54, 55, 102, 138]. This external stimuli re-sponsive approach has been created to exploit the sensitivity of specific hydrophobic blocks to external stimuli.
Temperature Responsive Polymersomes
Vesicles composed of copolymers of PEG and the temperature sensitive PNIPAM undergo phase transitions in response to tem-perature. A unique property of PNIPAM is its ability to undergo a reversible coil-to-aggregate transition in response to changes in temperature in aqueous solution. Below the lower critical solution temperature (LCST), the polymer is in an extended, water soluble conformation; above this temperature, it forms insoluble, hydro-phobic aggregates. When incorporated into a diblock with PEG, vesicles are formed at temperatures above the LCST. When the temperature is decreased below the LCST, the PNIPAM block be-comes solubilised and the vesicle disassembles. As a result, en-trapped molecule is released from the vesicle when the temperature decreases below the LCST [136, 137]. Reversibly cross-linked temperature responsive nano-sized polymersomes were prepared for protein delivery using triblock copolymers, PEO-b-PAA-b-PNIPAM. FITC-dextran was encapsulated as a model protein with a high encapsulation efficiency (>85 wt%) making these nanop-olymersomes a promising smart delivery systems for triggered in-tracellular delivery of small molecules, pDNA, siRNA, and phar-maceutical proteins and peptides [56].
Oxidation-Reduction Responsive Polymersomes
Hubell’s group [54, 55, 102] developed polymersomes, which dem-onstrate either oxidation [54, 55, 138] or reduction [102] responsive release mechanism that can be cued in vivo especially at the sites of inflammation and endolysosomal compartments. For oxidation responsive approach, a triblock copolymer consisting of two hydro-philic blocks of PEG surrounding a hydrophobic block of PPS, PEG-PPS-PEG, which self assembles to form polymersomes has been established. Under reducing conditions, the PPS block con-tains thio-ether moieties that are hydrophobic and stabilize the ve-sicular bilayer. When subjected to an oxidant, such as hydrogen peroxide, these functionalities become oxidized, affording rela-tively hydrophilic sulphoxide and sulphone functionalities. This
Self Assembling Polymers as Polymersomes for Drug Delivery Current Pharmaceutical Design, 2011, Vol. 17, No. 1 75
change in hydrophobic content destabilizes the lamellar bilayer and the vesicle ruptures [54, 55, 138].
Unlike oxidation responsive systems, reduction stimuli respon-sive systems can be developed by incorporating disulfide linkage between PEG and PPS [PEG-SS-PPS]. Such polymersomes can be made to remain intact in oxidizing environments (i.e. blood plasma) and to disassemble in reducing environment such as cytosol. This reducing environment results in a bursting rupture of polymersomes due to the cleavage of the disulfide bond between PEG and PPS blocks. This delivery system was evaluated using a hydrophilic fluorescent molecule (calcein) to demonstrate the reductive sensi-tivity in the presence of cysteine as the reducing agent [102].
pH Sensitive Polymersomes
In tumor microenvironment, hyper proliferative cancer cells adapt to use anaerobic glycolysis (hypoxic metabolism) to obtain extra energy that result in acidic microenvironment. Therefore, contrary to the normal blood pH of 7.4, extracellular pH values in numerous cancer tissues has been determined to be around 6.8 to 7.0 [91]. Moreover, cellular compartments, such as endolysosomes, exhibit even lower pH levels of approximately 4 to 6. Accordingly, using pH sensitive block copolymers will allow development of polymer-somes, which are capable of dissociating in response to decreased pH levels, for intracellular delivery. The acidic pH found in tumor tissues and in endolysosomal compartments of cells provides poten-tial localized trigger for the release of payloads from such a pH sensitive polymersomes. This approach has been designed and ex-ploited to free payloads of polymersomes incorporated either on hydrophobic thick wall (e.g. paclitaxel) and/or interior aqueous lumen (e.g. doxorubicin) upon accumulation at the tumor site and/or entry into the cytoplasm [18, 39, 91, 139].
Armes’s group [130, 140] demonstrated a new class of diblock copolymer vesicles based on a biocompatible monomer, MPC, and a second monomer, DPA, which confers pH sensitivity to the mem-brane wall. These PMPC-b-PDPA diblock polymersomes were prepared by self assembly of a biocompatible diblock copolymer in purely aqueous solution simply by controlling the solution pH and were found to be colloidally stable at physiological pH. At physio-logical pH (pH 7.4), the tertiary amines of the PDPA block are neu-tral, making this portion of the diblock hydrophobic. However, decreasing the pH protonates the amines and then destabilizes the lamellar bilayer ultimately resulting in vesicle disruption. Moreo-ver, vesicle dissociation occurs completely below pH 6 and PDPA homopolymer dissolves in water below pH 6 as a weak cationic
polyelectrolyte, but becomes insoluble above pH 6 due to deproto-nation of its tertiary amine groups. The polymersomes were evalu-ated for doxorubicin encapsulation and release profile showed sig-nificantly sustained release of the drug at pH 7.5, due to entrapment inside vesicles. In vitro cell experiments showed efficient transfec-tion in both CHO and HDF cell lines for DNA delivery. However, studies concerning their biodegradability and cytotoxicity are not yet reported [130, 140].
Chen et al. [95] explored use of pH sensitive biodegradable polymersomes which were prepared based on diblock copolymer of PEG and an acid labile polycarbonate, PTMBPEC. Polymersomes prepared from PEG(1.9k)-b-PTMBPEC(6k) revealed average sizes of 100–200 nm. The acetals of polymersomes, though stable at pH 7.4, were prone to fast hydrolysis at acidic pH of 4.0 and 5.0, with half lives of 0.5 and 3 days, respectively. Drug encapsulation stud-ies revealed that these polymersomes were able to simultaneously load paclitaxel and doxorubicin hydrochloride.
Adams’s group [141] prepared pH sensitive vesicles using PEO-b-PDEAEMA. Initially, the PDEAEMA block is fully proto-nated and forms a cationic polyelectrolyte in aqueous solution and at low pH the polymer is unimerically dissolved. As pH is raised the charge is removed gradually until PDEAEMA block is com-pletely deprotonated and becomes hydrophobic at high pH, induc-ing self assembly. To investigate encapsulation efficiency of these delivery systems, polymersomes were prepared by switching pH of the solution in the presence of aqueous fluorophores, as a proof of concept.
CONCLUSIONS AND FUTURE PERSPECTIVES OF PO-
LYMERSOMES
Self assembling amphiphilic copolymers as constructs of po-lymersomes for drug delivery systems were described here. Po-lymersomes have attracted considerable attention and become one of the most attractive platforms as drug delivery systems and in-creased application of these systems is expected. In this review, the applications of polymersomes with special attention to cancer che-motherapy have been described. Polymersomes have been explored for co-delivery of synergistic drugs using large hydrophilic reser-voir for hydrophilic molecules and thick hydrophobic wall for hy-drophobic molecules, thus enabling exploitation of synergistic ef-fects of anticancer therapy. The most important approach will be targeting polymersomes to the specific cell surface receptors, which is potential approach of utilizing the hydrophilic surface of po-
Table 2. List of Copolymers and Corresponding Types of Macromolecules Encapsulated.
Copolymer Macromolecule Release Mechanism Ref.
PEO-b-PCL-b-PLA Bovine serum albumin (EE~10-13%) pH and temperature [132]
siRNA, AON [52] PEO-b-PLA; PEO-b-PCL
Hemoglobin (EE~20%)
pH and temperature
[50]
PMPC-b-PDPA pDNA (EE~25%) pH [99, 130]
PMOXA-b-PDMS-b-PMOXA Trypsin, Nucleoside hydrolase - [133, 134]
PEG-b-PPS-b-PEG Glucose-oxidase Oxidation [54]
PEO-b-PBD Myoglobin (EE~55%), Hemoglobin (EE~4.5%)
Albumin (EE~5%)
- [26, 69, 70]
PEG-b-PPS Ovalbumin (EE~37%),
Bovine serum albumin (EE~19%), Bovine -globulin (EE~15%)
Oxidation [131]
76 Current Pharmaceutical Design, 2011, Vol. 17, No. 1 Jain et al.
lymersomes that would carry a molecule, which can bind to tumor cells. Clinical trials of polymersomes are still several years away, but the researchers are now focusing on targeting and imaging spe-cific cell types with polymersomes using animal models. In general, from application perspective, development of polymersomes that would carry, protect, target and release chemotherapeutic agents in spatial and temporal controlled manner is actively ongoing and the design would provide a versatile application in drug delivery appli-cations. Besides drug delivery applications, polymersomes are found to be useful for imaging of cancer tissues tagged with a suit-able contrast agent or fluorescent agent which can allow taking the images of deep seated tumors [74, 142].
ABBREVIATIONS
AFM = Atomic force microscopy
AON = Antisense oligonucleotides
CLSM = Confocal lasser scanning microscopy
DEAEMA = 2(diethylaminoethyl methacrylate)
Dex = Dextran
DIC = Differential interference microscopy
DMAEMA = 2(dimethylaminoethyl methacrylate)
DNA = Deoxyribonucleic acid
DSC = Differential scanning calorimeter
FDA = Food and drug administration of USA
fEO = Hydrophilic fraction
FTIR = Fourier transform infra red
HYA = Hyaluronan
LCST = Lower critical solution temperature
MRI = Magnetic resonance imaging
MRP = Multidrug resistance protein
NPAL = 2-Nitrophenylalanine
P4VPMeI = Poly(4-vinylpyridinum methyl iodide)
PAA = Poly(acrylic acid)
PBD = Poly(butadiene)
PBLG = Poly(�-benzyl-l-glutamic acid)
PBO = Poly(butylenes oxide)
PCEMA = Poly(2-cinnamoylethyl methacrylate)
PCL = Poly(caprolactone)
PDMA = Poly(dimethylaminoethyl methacrylate)
PTMBPEC = Poly(2,4,6-trimethoxybenzylidenepentaerythritol carbonate
PIAT = Poly((isocyanoalanine(2-thiophene-3-yl-ethyl)amide
PMPC = Poly(2-methacryloyloxy ethyl phosphorylcho-line)
PR_b = Fibronectin mimetic alpha(5)beta(1) specific peptide
TIRFM = Total internal reflection fluorescence micros-copy
PDPA = Poly(2-diisopropylamino ethylmethacrylate)
PEE = Poly(ethyl ethylene)
PEG = Poly(ethylene glycol)
PEO = Poly(ethylene oxide)
PGA = Poly(l-glutamic acid)
P-gp = P-glycoprotein
PI = Polyisoprene
kDa = Kilo Dalton
PIB = Poly(isobutylene)
PLA = Poly(lactic acid)
PMCL = Poly(�-methyl caprolactone)
PMOXA = Poly(methyloxazoline)
PDMS = Poly(dimethylsiloxane)
PNIPAM = Poly(N-isopropylacrylamide)
PPO = Poly(propylene oxide)
PPS = Poly(propylene sulphate)
PS = Polystyrene
PSBA = Polystyrene boronic acid
UV = Ultra violet
PTMC = Poly(trimethylene carbonate)
RES = Reticuloendothelial systems
SEM = Scanning electron microscopy
siRNA = Small interfering ribonucleic acid
TEM = Transmission electron microscopy
ACKNOWLEDGEMENTS
Jay Prakash Jain is thankful to Director, NIPER for the award of PhD fellowship and Wubeante Yenet Ayen is thankful to Jimma University, Ministry of Education, Federal Democratic Republic of Ethiopia for full funding to carry out research work toward PhD program. Financial support from the Ministry Grant for Nanotech-nology Drug Delivery (C-11) is duly acknowledged.
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Received: November 22, 2010 Accepted: February 8, 2011
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