Cholesterol Modified Self-Assemblies and Their Application to

Review pubs.acs.org/Biomac

Cholesterol Modified Self-Assemblies and Their Application to Nanomedicine Francesca Ercole,† Michael R. Whittaker,† John F. Quinn,*,† and Thomas P. Davis*,†,‡ †

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia ‡ Department of Chemistry, University of Warwick, Coventry, ULCV4 7AL, United Kingdom ABSTRACT: Cholesterol is a ubiquitous molecule in biological systems, and in particular plays various important roles in mammalian cellular processes. The presence of cholesterol is integral to the structure and behavior of biological membranes, and profoundly influences membrane involvement in cellular mechanisms. This review focuses on the incorporation of cholesterol into synthetic nanomaterials and assemblies, focusing on LC phase behavior, morphology/selforganization and hydrophobic interactions, all important factors in the design of nanomedicines. We highlight cholesteryl conjugates, liposomes and polymeric micelles, focusing on self-assembly capabilities, drug encapsulation and intracellular delivery. An area of considerable interest identified in this review is the use of cholesteryl-functional vectors to deliver drugs or nucleic acids. Such applications depend on the ability of the nanoparticle carrier to associate with both the cellular and endosomal membrane.

1. INTRODUCTION Cholesterol is a lipid sterol that carries out many roles vital for the normal functioning of the human body. As a major component of eukaryotic cell membranes, a significant role involves modulation of membrane fluidity and permeability. In animals, cholesterol serves as a precursor to all the steroidal hormones,1 vitamin D, and bile acids2 essential for fat digestion. The brain contains 25% of the body’s membrane cholesterol, and up to 80% of brain cholesterol is located in myelin sheaths, where it facilitates the tight packing of the sheath membrane and thus enables normal brain development and neuron insulation. The blood-brain barrier prevents brain-cholesterol exchange with lipoprotein−cholesterol in the general circulation, and as such brain-cholesterol is recycled via an apolipoprotein-dependent mechanism (thereby minimizing loss to the circulation).3 Cholesterol metabolism, its elegant biosynthetic pathway, complex stereochemistry, and selfassembly behavior are aspects that have inspired Nobel winning scientists (Konrad Bloch and Feodor Lynen in 1964 and Michael S. Brown and Joseph L. Goldstein in 1985). Abnormal plasma cholesterol levels contribute to atherogenesisa fact that has attracted widespread attention, to dominate general perceptions of the effects of cholesterol on human health. However, cholesterol merits attention beyond the negative, as it performs a role essential for life, exhibiting and imbuing unique properties, and offering potential for many practical applications. Cholesterol is one of the most ubiquitous, natural materials that can be exploited in nanoscience, as it can induce self-assembly while eliciting specific © XXXX American Chemical Society

biological responses. The fundamental and essential role that cholesterol plays in biology, its interesting properties, and the growing synthetic techniques that are available for its conjugation could broaden our view of cholesterol as an important player in nanotechnology and disease treatment, particularly in areas such as imaging and drug delivery.4 This review will focus on how cholesterol has been applied, in a nanomedical context, for drug delivery and bioimaging, with a specific focus on polymeric constructs.

2. STRUCTURAL ASPECTS OF CHOLESTEROL AND EFFECT ON CELL MEMBRANES The cell membrane has many functions: it encloses and compartmentalizes the cell, regulates concentration gradients, plays a fundamental role in signal recognition, transduction, and amplification and generally provides an essential structural framework for metabolism. The cell membrane has a complex molecular architecture (Figure 1): at its most basic level it is composed of a phospholipid bilayer in which the hydrophilic head-groups are exposed to the aqueous environment and the hydrophobic chains are contained within its interior. A variety of integral proteins are buried in the membrane bilayer while others are bound to its surface as peripheral proteins. Many of these proteins possess oligosaccharides (sugars), which extend out into the aqueous medium. Glycolipids can also be Received: April 23, 2015 Revised: June 1, 2015

A

DOI: 10.1021/acs.biomac.5b00550 Biomacromolecules XXXX, XXX, XXX−XXX

Review

Biomacromolecules

Figure 1. Pictorial representation of the membrane of a living cell. (By courtesy of Encyclopaedia Britannica, Inc., copyright 2007; used with permission.)

Figure 2. (a) Chemical structure of sterol. (b) Chemical structure of cholesterol with the smooth α-face and rough β-face.

and specific membrane function. The ratio of cholesterol to phospholipids affects the stability, permeability, and protein mobility of the membrane. Therefore, membranes with high ratios have high stability and relatively low permeability, and their major function is often as a protective barrier (such as the epidermis layer of the skin). Membranes of intracellular organelles such as mitochondria have low cholesterol ratios and are consequently fluid and permeable, serving in metabolic reactions as well as in energy production. The brain and nervous system, which are rich in myelin sheaths (necessary for efficient nerve conduction) also contain high levels of cholesterol. The outer membranes of most cells have intermediate ratios indicative of both protective and metabolite-transport functions.9 The relative mobility and fluidity of the individual lipid molecules making up the membrane bilayer changes with temperature and composition, as studied using artificial membranes. For example, at low temperatures, the saturated hydrocarbon tails of phosphatidylcholines can pack together closely in a nearly solid gel state. On raising the temperature above 41 °C the regular order is lost as the tails mobilize. The membrane melts to change form at a temperature referred to as the melting temperature (Tm) (the gel to liquid-crystalline disordered phase transition). Biological membranes which are made up of a mixture of components including proteins have broader phase transitions compared to synthetic bilayers. Importantly, biological membrane composition is tightly regulated, partly to maintain fluidity at the body temperature of the organism. The influence that cholesterol exerts on the properties of a membrane lipid bilayer is complex and depends on the nature of the neighboring lipids, notably, the degree of chain unsaturation and length of their hydrophobic tail and the headgroup. It has been demonstrated that the addition of cholesterol to a pure phospholipid bilayer abolishes the normal

integrated into the structure of the membrane, with their lipid portions being part of the inner lipid membrane. The complex membrane structure also contains sterols of which cholesterol is most commonly represented. Sterols (steroidal alcohols), a subgroup of the steroids, have the basic ring structure shown as Figure 2a. Cholesterol (Figure 2b) has the basic ring structure of a sterol but also has a double bond in ring B: an aliphatic iso-octyl side-chain at position C17 and two axial methyl substituents, C18 and C19, attached at positions 10 and 13, in relative cis orientations. The four rings create a flat and rigid structure. The cholesterol ring system is asymmetric with one side planar, while the reverse is cissubstituted, characterized by the presence of the two methyl groups. The flat face of cholesterol is called the α-face, and all substituents located on this face (in trans conformation relative to C19) are called α, while the substituents located on the rough β-face (in cis conformation relative to C19) are called β. The short branched hydrocarbon tail makes the cholesterol molecule a largely hydrophobic structure, but the polar 3βhydroxyl group provides a weakly amphiphilic nature. The fused cyclohexane rings adopt a puckered and more stable chair conformation, predominating over the boat. This makes cholesterol a bulky, planar, and rigid structure compared to the other membrane components such as the fatty acid tails of phosopholipids and sphingolipids. Cholesterol is accommodated in the lipid membrane by orienting the steroidal ring parallel to the hydrocarbon chains of the lipids, and its hydroxyl headgroup is orientated in the aqueous phase. Cholesterol is an essential structural component of animal cell membranes, and serves to regulate several physical properties of the cell membrane. In particular, it reduces passive permeability of water, small molecules and gases5,6 and regulates fluidity and the phase behavior of membranes.7,8 The cholesterol content of a membrane varies according to tissue B


Review

Biomacromolecules

Figure 3. Main chemistries used to conjugate cholesterol.

scrutiny.20 Recently a new physical principle operating in biological membranes has been proposed that describes a “push−pull” mechanism in which cholesterol is pushed away from low-melting phospholipids and pulled toward highmelting lipids.21 Even though a definitive proof of lipid raft existence remains elusive, the cholesterol-dependent structures are surmised to behave as functional units supporting various cellular processes, including the regulation of membrane trafficking in both the exocytic and endocytic pathways, cell migration, and a variety of cell signaling cascades.22 A comprehensive description on cholesterol’s effects on lipid bilayers can be obtained by consulting reviews published in the area.11,23−26

sharp thermal transition between gel and liquid state, giving the membrane properties intermediate between the two phases, thus broadening the Tm of the membrane. Furthermore, cholesterol stiffens the membrane above the Tm by producing a condensing10 and ordering effect11 on neighboring lipids. In this case, the mobility of hydrocarbon tails of phospholipids is reduced due to their interaction with relatively rigid cholesterol. Cholesterol therefore enhances the rigidity of the membrane, making it less permeable to small water-soluble molecules. Below the Tm, in the gel state, the opposite effect occurs where cholesterol weakens the van der Waals interactions between the hydrocarbon chains on phospholipids, therefore inhibiting regularity and increasing the fluidity of the hydrocarbon chains. Since cholesterol is an essential component of eukaryotic cell membranes, its levels are tightly controlled in a healthy individual via several homeostatic processes including regulation of de novo synthesis, cellular uptake and deposition of cholesteryl esters into fat droplets within the cell.12 Disturbance of these tightly regulated processes leads to a variety of diseases of lipid metabolism. 13,14 Evidence has suggested that cholesterol is involved in the maintenance and function of “lipid rafts” in cellular membranes which are thought to function as platforms that concentrate and segregate certain protein receptors within the floating sea of the membrane bilayer.15−18 These highly ordered and tightly packaged membrane domains are enriched in cholesterol and also contain glycosphingolipids and phospholipids with a higher degree of saturated fatty acyl chains, compared to the rest of the membrane. Cholesterol is thought to serve as a spacer between the hydrocarbon chains of the sphingolipids and to function as a dynamic glue that keeps the raft assembly together.19 Removal of raft cholesterol leads to dissociation of most proteins from rafts and renders them nonfunctional. Lipid rafts have proved difficult to visualize so the lipid raft model, which has evolved from indirect evidence, has come under some

3. LIQUID CRYSTAL ORGANIZATION OF CHOLESTERYL DERIVATIVES Cholesterol therefore has a primary role in modulating the structural and dynamic properties of cellular membranes through its ability to interact and self-assemble with membrane lipids and proteins, thereby promoting the specific organization fundamental to cellular function and viability. The 3β-hydroxyl group on the cholesterol molecule provides a chemical handle for modification and conjugation. Once modified using the chemistries indicated in Figure 3, cholesterol becomes a cholesteryl group, which therefore denotes the conjugated molecule throughout this review. A common strategy for cholesterol conjugation, especially to polymers, makes use of the cholesterol chloroformate, a commercially available compound, giving access to carbamates and carbonates. The self-assembly of both molecules and macromolecules bearing cholesteryl groups into liquid crystal (LC) mesophases is a phenomenon that has proven useful in the field of nanotechnology and related industries such as electronics, personal care and pharmaceuticals. Cholesterol LC states have developed from a mere curiosity to a highly interdisciplinary C


Review

Biomacromolecules

Figure 4. (Left a−d) Examples of PEG-Chol anchors; (right) liposome with PEG-Chol anchor inserted into lipid bilayer.

research field, being one of the most studied applications in materials science. Chemical linkage of cholesterol’s rigid planar steroidal ring system to a highly mobile and flexible hydrocarbon tail results in cholesteryl esters and ethers that display mesomorphic properties. This results from a tendency for the molecules to aggregate into large three-dimensional structures in which the position and orientation of the molecule is organized to endow LC properties.27 Cholesteryl benzoate was the first LC compound discovered by Reinitzer in 1888,28 and gives its name to one of the earliest classes of LC subgroups to be defined, i.e., the cholesteric (or chiral) nematic phase. The cholesteric phase appears in organic compounds that consist of elongated (nematogenic) molecules without mirror symmetry, i.e, chiral molecules. Due to cholesterol’s rigid structure with eight chiral centers, typical representatives of these compounds are cholesteryl derivatives.29,30 Cholesteric LCs organize into a helical (twisted) supramolecular structure, which affords particular optical properties, such as selective reflection and transmission of light, thermochromism, and circular dichroism. The possibility of changing their optical properties with an applied electrical field results in several types of electro-optic effects. Altogether this makes cholesteryl derivatives very interesting candidates for applications in modern LC technology.31,32 Since the discovery of cholesteryl benzoate, literally thousands of cholesteryl-based LCs involving monomers, oligomers, and dimers have been reported.29,33,34 Along with many industrial developments in the field of LC technology, basic research has continued to be carried out into polymeric LCs. One research area involves the incorporation of rod-shaped mesogenic groups, also known as low molecularweight LCs, into polymers. This incorporation can be carried out by connecting the mesogenic groups via flexible spacers, either as side groups to the main chain or directly within the main chain itself.35,36 Neat or melt-state ordering of polymers functionalized with cholesteryl groups has been used to investigate fundamental phase behavior of LC states.37 The LC properties of cholesteryl polymers can be tailored by the judicious selection of backbone, comonomer, dopants, and degree of cross-linking. Cholesteryl polymers can form smectic, nematic, cholesteric or blue mesophases, which can be utilized

for specific applications, e.g., cholesteric and blue mesophases are used in optoelectronic, color information technology, and laser applications.34 In the case of side chain cholesteric LC polymers, homopolymers containing only cholesteryl mesogenic units normally fail to exhibit cholesteric properties, with smectic structures dominating, even though their corresponding cholesteryl monomers readily form cholesteric mesophases. Various strategies have been explored as routes to generating cholesteric LC polymers: the copolymerization of two cholesteryl monomers having widely varying and flexible spacer length;38 the incorporation of nonmesogenic chiral sidechains;39 the addition of photochromic nonmesogenic groups40 as well as the incorporation of nonchiral nematic monomers.41 Polymers with cholesteryl side-chains that can self-assemble to form layered smectic mesophases are common, e.g., polynorbornenes, polysiloxanes, polymethacrylates, and polyacrylates.42,43 Investigation into the neat state LC phases of many LC cholesteryl polymers mainly relates to their application as optical-electrical devices. However, some neat state LC phases have been studied with a view to biomedical applications. For example, the LC phases of cholesteryl oligo(Llactic acid) were explored for their interactions with cells.44 Incorporation of cholesteryl functionality into a polymer is normally implemented via a cholesteryl-based monomer; however, cholesterol initiators have also been reported. For example, cholesterol has been used to initiate the ring opening polymerization of lactic acid to form cholesteryl-(L-lactic acid) oligomers, thermotropic LCs, for self-assembly into lamellar structures consisting of interdigitated bilayers.45 Cholesteryl end-capped polycarbonates exhibiting LC characteristics, synthesized by the ring-opening polymerization of 2,2dimethyltrimethylene carbonate and initiated by cholesterol, have also been reported.46 Many other examples of LC polymers based on the cholesteryl group acting as a mesogen exist in literature; however, this is beyond the scope of this Review.47,48 Lyotropic LC phases, having long-range orientational order, induced by the addition of a solvent, are abundant in living systems.49 In fact, research into the physical properties of biologically important cholesteryl esters has revealed a D


Review

Biomacromolecules participation of cholesteryl LC states in disease processes.50 Droplets of cholesteryl esters appear in a variety of normal and pathological cellular processes. For example, they have been found in neural tissue prior to nerve myelination;51,52 the adrenal gland accrues cholesteryl esters for the synthesis of steroid hormones;53 lipoprotein particles responsible for cholesterol transport in the blood are known to have a core rich in cholesteryl esters54 and atherosclerosis involves cholesteryl ester accumulation with formation of both intraand extracellular deposits forming a major part of the lesion.55 In the latter two systems, the cholesteryl esters are primarily cholesteryl linoleate and cholesteryl oleate which have been found to undergo a phase transition from a smectic LC to disordered liquid phase, close to or above body temperature. In isolated pure systems, these cholesteryl esters not only have slightly higher transition temperatures but also exhibit an additional intermediate cholesteric LC phase. Recent evidence has also shown altered cholesterol distribution near amyloid deposits in Alzheimer’s affected brains.56 Cholesterol in bile can also crystallize to form gall stones, which can block the bile ducts.57 Investigations into the LC phase behavior and transition temperatures of pure cholesteryl esters, and factors that influence their behavior have therefore been important to understand the phase behavior of cholesteryl esters in biological systems.

stabilizing the structures and reducing the leakage of contents.83,84 As mentioned previously, the effect of cholesterol on a lipid bilayer is to increase the degree of orientation of lipid tails, and reduce the rate of exchange of lipids in the LC phase, leading to a laterally condensed membrane with increased packing density, higher mechanical strength and lower permeability. Therefore, cholesterol is often incorporated into preclinical and clinical liposomal drug carrier formulations to decrease membrane fluidity and provide favorable drug retention properties. Table 1 lists liposomal drug vectors approved for clinical application that contain cholesterol in the formulations. Table 1. Examples of Approved Liposomal Products Containing Cholesterol name DaunoXome Myocet DepoCyt Marqibo Doxil STEALTH

liposomal excipientsa

drug

DSPC; cholesterol egg phosphatidylcholine; cholesterol DOPC; DPPG; cholesterol

daunorubicin citrate doxorubicin-citrate

Egg sphingomyelin; cholesterol MPEG-DSPE; HSPC; cholesterol

cytarabine (cytosine arabinoside) vincristine sulfate (vinca alkaloid) doxorubicin hydrochloride

a

Distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dipalmitoylphosphatidylglycerol (DPPG); methoxyPEG(2000)-distearoylglycerol phosphoethanolamine (MPEG-DSPE); fully hydrogenated soy phosphatidylcholine (HSPC).

4. LIPOSOMES 4.1. Role of Cholesterol. Cell biologists are well acquainted with the bilayer arrangement of molecules in a lamellar LC phase, since this forms the fundamental structure of most cellular compartmental membranes. A liposome is an artificially prepared spherical vesicle composed of one or more lamellar phase lipid bilayers enclosing an interior aqueous space (as in Figure 4). Since the first report that phospholipids in aqueous systems can form closed bilayered structures (by Bangham et al. in the early 1960s), liposomes have evolved from being model membrane systems into sophisticated carriers for a wide variety of therapeutic agents.58,59 To date they represent the most extensively studied drug delivery vehicles for pharmaceutical drugs such as anticancer agents.60−67 The lipid molecules that are most often selected to prepare liposomes are glycerophospholipids, sphingolipids, and cholesterol itself. This is largely due to their abundance in natural cell membranes, their ability to mimic the behavior of biological cell membranes, and their biocompatibility and degradability. Lipid-based vesicles have become very sophisticated in terms of design, composition, size, and capacity to encapsulate materials. The structure of liposomes allows for delivery of hydrophilic cargo loaded in the aqueous compartment, or hydrophobic cargo embedded in the lipid bilayer. The therapeutic agents that can be carried within the vesicles range from conventional drugs, pro-drugs, anticancer agents,68−71 antibiotics,72,73 gene-active substances (including plasmids, antisense oligonucleotides and small interfering RNA (siRNA)), through to contrast agents for imaging and diagnosis.74−79 Liposome applications have also extended into lesser-known aspects of bionanotechnology, such as nanofactories and catalysis.80 Early work on the in vivo activity of liposome-entrapped drugs in animal models showed that the first-generation liposomes often had difficulty in retaining some types of entrapped molecules in their interior due to a tendency to leak through the lipid boundary.65,81,82 Cholesterol addition was found to change the content of the liposome bilayer, thus

4.2. Poly(ethylene glycol)−Cholesteryl Conjugates: Anchors for Liposomes. One of the primary goals driving the use of self-assembled vectors such as liposomes for chemotherapy is to favorably mediate the biodistribution and pharmacokinetics of drugs, thus broadening the so-called therapeutic window. Alterations to biodistribution in tumoraffected tissue can occur through a mechanism known as the enhanced permeability and retention (EPR) effect (also referred to as passive targeting).85 Normal tissues possess vasculature with tight junctions between endothelial cells (∼5 nm), thus preventing the extravasation of small particles. Diseased tissue (e.g., tumors), possesses vasculature that is porous and leaky due to enhanced interstices between endothelial cells, allowing vesicles with diameters up to 400 nm to extravagate through the gaps into the surrounding tissue, where they can passively accumulate.86 The EPR effect is also accentuated by the reduced lymphatic drainage of capillaries surrounding the tumors.85 In order to lengthen circulation time and reduce clearance before the drug arrives at the diseased tissue, most vesicles currently in development normally consist of an outside coating of polyethylene glycol (PEG). This acts as an impermeable, hydrophilic layer on the outer surface, minimizing opsonization87 and imbuing stealth properties to avoid recognition by the monocytic phagocyte system (MPS). PEGylation also minimizes the aggregation of vesicles and improves long-term stability of the formulation. This combination of effects means that PEGylated-liposomes, commonly referred to as “stealth liposomes”, have greater potential to accumulate in tumor tissue and thus carry out their chemotherapeutic action locally, reducing “off-target” effects. The potential benefits of this approach have led to a wealth of biomedical research, in vitro and in vivo, leading to changes in clinical practice and new clinical trials.88−91 One noteworthy E


Review

Biomacromolecules

low with accumulation being limited to the periphery of the tumor.105,106 To overcome this issue, surface-tethered ligands may be introduced to the nanoparticle surface to facilitate binding to cells, leading to internalization via receptor-mediated endocytosis; this has become the subject of intense research.66,107−109 The search for new ligands for targeting has been focused on specific receptors that are overexpressed on target cells, such as cancer cells. Targeting ligands can be simple peptides, proteins (including antibodies) or protein fragments, carbohydrates, vitamins, or small molecules.110 For example, antibodies against transferrin, or transferrin itself, are popular ligands for liposome targeting to tumors.111 Other ligands include those that target receptors for folate,112 epidermal growth factor (EGF),113 or antibodies against cell surface antigens, which can be overexpressed in different tumor types.114,115 In order to provide a biologically active ligand on the outside of the nanoparticle capable of interacting with a specific target, a number of approaches can be used, as reviewed by Nobs et al.116 For instance, cholesteryl-based anchors can be used for the construction of receptor targeted PEGylated liposomes which have been shown to be an effective alternative to lipidbased anchors such as those based on phospholipids. As an anchor for liposomal formulations, a cholesteryl derivative has the advantage of being electrically neutral, compared to a DSPE phospholipid anchor, which introduces a negative charge to the liposome surface (which may result in plasma protein binding).117 Moreover, cholesteryl derivatives are less susceptible to degradation during storage (depending on the conjugation chemistry used), and are also cheaper to make.118 Cholesteryl-based anchors are normally made up of three structural elements: the hydrophobic cholesteryl portion that inserts into the hydrophobic part of the membrane, a hydrophilic linker or spacer, and a functional end group. Anchors and tethers with a wide range of different functional end groups have been created and applied in nanomedicine.119 One example is maleimido-PEG-cholesteryl (Mal-PEG-Chol), which has been used to anchor cetuximab monoclonal antibody and Fab fragments, to produce immuno-liposomes, which were internalized by EGF receptor targeting cells.120 Folate moieties have also been introduced to liposomes using folate-PEG-Chol anchors.121 Folate receptor targeted liposomes carrying doxorubicin were shown to be 38 times more toxic to cancerous cells than nontargeted control liposomes (Figure 4d).122 Others who have studied the anchor with similar results noted that the colloidal stability of the folate-PEG-Cholanchored liposomes was superior to non-PEGylated liposomes, thus demonstrating that PEG is important not only as a spacer but also for its protective and stabilizing properties.123,124 A liposomal formulation of docetaxel targeting the folate receptor was synthesized using the anchor folate-PEG-cholesteryl hemisuccinate (Fol-PEG-CHEMS), which showed promising tumor cell-selectivity.125 Studies from the same group indicated that, compared to Fol-PEG-Chol (which contains a carbamate linkage), Fol-PEG-CHEMS is superior at retaining its folatereceptor targeting activity during prolonged storage. Therefore, the chemistry used for construction of the anchors is of considerable importance.126 Many other examples of using PEG-Chol-based anchors for receptor-targeting have been published. Targeting to glucose transporters on the blood brain barrier was investigated for delivering drugs to the brain using a glycosyl derivative of cholesterol (Glucose-PEG-Chol), which anchors glucose

clinical product is PEGylated liposomal doxorubicin (Doxil/ Caelyx), a stable and long-circulating liposome formulation that has become widely used in chemotherapy (Table 1). To form a hydrated PEG layer on a liposome surface, PEG− lipid polymer conjugates are commonly used. Cholesteryl− PEG (PEG-Chol) can be inserted into liposomes as part of the formulation with the cholesteryl group of the conjugate conferring cohesion to the lipid bilayer.92,93 Different molecular weight PEG can be routinely coupled to cholesterol by formation of ether, ester, carbonate or carbamate bonds. PEG can also be linked to cholesterol through a spacer arm (PEGLinker-Chol) such as diaminobutane (Figure 4b).94 Investigations have been carried out into the effects of PEG-Chol and cholesterol in liposomal formulations, both on drug loading and pharmacokinetics and permeability.95 Several drug release experiments revealed that PEG-Chol can in fact induce membrane defects in the liposome, resulting in concomitant release of cargo, especially at high PEG-Chol densities.96 Another potential problem is that a PEG coating on a liposome can act as a steric barrier for drug release and cellular uptake. One approach to solve this problem is the use of liposomes coated with a pH-sensitive PEG coating. The protective coating is detached by the acidic environment of a tumor to allow internalization of the nanocarriers and concurrent release of the loaded drug. A hydrazone-based acid-cleavable PEG-Chol conjugate (Figure 4c) has been applied for this purpose.97 Further, when PEG and cholesteryl groups are attached through an ester linkage, this provides a system for releasing contents by a process described as esterase-controlled dePEGylation (Figure 4a).98 PEG-Chol conjugates have been traditionally applied as stabilizing anchors for liposomes to prolong their circulation time, and also behave as solubilizing surfactants which serve to prevent aggregation of vesicles.99 However, PEG-Chol conjugates are themselves amphipilic molecules and are thus able to self-assemble into micelles. Such micelles have been shown to have promise as delivery vectors for poorly watersoluble anticancer agents such as quercetin,100 doxorubicin,101 and docetaxel.102 Interestingly, PEGylated liposomes have been shown to induce significant immune responses when repeatedly injected into the same animal. This means that a previously injected dose of PEGylated liposomes can cause a reduction in the circulation time of a subsequent dose that accumulates in the liver and spleen. This phenomenon, referred to as accelerated blood clearance (ABC), is understood to be induced by antiPEG IgM antibodies.103 In order to prevent this occurrence, Xu et al.104 made cleavable PEG−lipid derivatives, i.e., liposomes modified with methoxyPEG(2000)−cholesteryl hemisuccinate (PEG-CHEMS) or methoxyPEG(2000)−cholesteryl methyl carbonate (PEG-CHMC). It was demonstrated that repeated injection of PEG-CHEMS liposomes did not induce an ABC phenomenon or cause an increase in liver accumulation. By comparison, repeated injection of conventional methoxyPEG(2000)−distearoylphosphatidylethanolamine (PEGDSPE) liposomes in rats induced the ABC phenomenon, as well as causing increased uptake in the liver. Only a slight ABC phenomenon was induced by repeated injection of PEGCHMC liposomes; however, in this case, increased uptake in the liver was still observed. While PEGylation has been shown to increase circulation time of the carrier and improve arrival at the tumor site, the actual fraction of nanoparticles entering the tumor cells is often F


Review

Biomacromolecules

Figure 5. Synthesis of polymer-caged liposomes (PCLs). Reproduced from ref 136. Copyright 2007 American Chemical Society.

residues to the outside of liposomes.127 TAT (Transactivatingtransduction) peptide (which has well-known cell penetrating properties) was incorporated into carriers using TAT-PEG-bChol, with the aim of targeted delivery of antibiotics to the brain.128 The use of PEG-Chol conjugates in drug delivery has been comprehensively reviewed by Qian et al., and the reader is directed there for a more complete consideration of the topic.129 More general reviews on the use of liposomes as drug carrier systems may also be of some interest.61,66,130 4.3. Polymer-Cholesteryl Conjugates as Anchors for Liposomes. PEG-Chol linear anchors have been applied to enhance the properties of liposomes, e.g, to increase stability and circulation time, and to introduce surface targeting capability. However, in the event of dissociation from the surface, liposome stability can be compromised. While surface cross-linked liposomes have been shown to have higher stability, such liposomes do not necessarily allow for controlled release of the payload.131 The incorporation of synthetic polymeric cholesteryl-based anchors in liposomes is one approach to increase the stability of the liposome. In one study, a synthetic cyanoacrylate polymer was made as the anchor containing pendant folate, cholesteryl, and PEG groups which was inserted into bilayers of liposomes. The folatereceptor targeted, docetaxel-loaded liposomes showed promising results for enhancing chemotherapeutic action in tumor tissue.132 Polymeric anchors have also been used to prepare liposomes with both enhanced stability and stimuli-responsive properties. Application of thermosensitive liposomes in combination with regional hyperthermia represents a novel strategy for tumorspecific drug delivery based on the idea that the permeability of tumor vasculature can be enhanced further by heat, causing more liposomes to accumulate intratumorally.133 Further, when the liposomes reach the heated tumor tissue area, the drug can be released due to changes in the permeability of the lipid membrane. Modification of liposomes with temperatureresponsive poly(N-isopropylacrylamide) (PNIPAm) results in thermosensitive liposomes, which can be applied for the delivery of drugs to tumors.134 Due to the sharp coil-to-globule

transition of the polymer chain at the lower critical solution temperature (LCST), PNIPAm anchored into liposome bilayers can lead to lateral phase separation of the bilayer, consequently allowing cargo release through membrane defects. Additionally, a PNIPAm coating also provides stabilizing properties. Recently, cholesteryl-modified NIPAm oligomers (NOs) with two polymer architectures were used to prepare NIPAm-anchored liposomes.135 These were main chain NIPAm oligomers (MCNOs, where a cholesteryl group was positioned at the end of the main chain) and side chain oligomers, (SCNOs, where the cholesteryl groups were pendant from the main chain). A biotinylated cholesteryl anchor was also inserted into the liposomes to supply a cancertargeting property and enhance cellular uptake. Both NO modified liposomes tended to release cargo faster at 37 °C than pristine liposomes, due to the lateral phase separation that occurs when the temperature is raised above the LCST of the NIPAm chains. Further, SCNO liposomes lead to faster release (compared to MCNO liposomes) due to “comb” aggregation effects originating from the interaction of the oligomers on the lipid membrane upon heating. The inclusion of pH-responsive moieties is another promising approach by which liposomal drug release can be triggered in the tumor environment. An acid-cleavable PEG lipid (cholesteryl-vinyl ether-PEG) was synthesized and dispersed as a minor component with dioleoylphosphatidylethanolamine (DOPE) to produce stable liposomes. Vinyl ether protonation and hydration, followed by hemiacetal cleavage at acidic pH, results in dePEGylation of the stabilizing lipid. In turn, this allows DOPE to adopt the nonlamellar, inverse hexagonal phase, causing the bilayer arrangement to become destabilized.42 Lee et al. demonstrated that cholesterylterminated poly(acrylic acid) (Chol-PAA) can be readily inserted into a known liposome system and then cross-linked to stabilize the bilayer membrane.136 Cross-linking of the carboxylic acid moieties on the surface of polymer-incorporated-liposomes (PILs) was achieved via amide bond formation using 2,2-(ethylenedioxy)-bis(ethylamine), yielding polymercovered (caged)-liposomes (PCLs) (Figure 5). The PCLs were found to be highly stable and could be lyophilized into powder G


Review

Biomacromolecules

Figure 6. TEM images of self-assemblies of block copolymers in water (samples stained by uranyl acetate): (a) PEG5000-b-PAChol (14/86); (b) PEG2000-b-PAChol (28/72). (c) Chemical structure and characteristics of amphiphilic LC block copolymers containing a cholesteryl-based mesogen; scale bar 200 nm. Reproduced from ref 148. Copyright 2007 American Chemical Society.

ization (ATRP).140 These techniques facilitate the synthesis of low polydispersity polymers with well-defined architectures, compositions, end groups and block lengths. Living radical polymerization strategies are particularly relevant for the synthesis of amphiphilic block and random copolymers for self-assembly in water. Various morphologies are possible, such as ordered monolayer or bilayer structures (e.g., polymerosomes) and core−shell nanostructures (e.g., micelles).141 In the latter case, the inner core is comprised of hydrophobic segments, while the surrounding corona is provided by the hydrophilic component. The hydrophobic core serves as a reservoir for poorly water-soluble and amphiphilic drugs, while the hydrophilic shell (corona) stabilizes the core. A corona composed of PEG, or another nonfouling polymer, prolongs circulation time in blood to increase accumulation in tumor tissues by the EPR effect. Amphiphilic polymers containing cholesteryl functionality commonly form micelles, nanogels, and hydrogels, which all have the ability to encapsulate and release drugs. While the self-assembly of amphiphilic block copolymers in water to form nanostructures such as micelles is a well-known phenomenon, a wide variety of self-association phenomena may also be anticipated for amphiphilic random copolymers incorporating hydrophobic groups. The self-association is dependent on the type of hydrophobic groups, their content in the copolymer, their sequence distribution, and the type of hydrophilic monomer units. The self-assembly behavior of amphiphilic random copolymers containing cholesteryl groups was reviewed by Yusa.142 Several phenomena regarding the association of polymer-bound hydrophobes in water are explained. Association can occur either within a single polymer chain or between different polymer chains; an intrapolymer association will result in a chain loop while interpolymer associations can form physical cross-links. The extreme case of interpolymer association can lead to network formation or bulk phase separation. Intrapolymer hydrophobic associations can instead lead to the formation of single molecular self-assemblies or “flower-like” unimolecular micelles. These can collapse further into a highly compact assembly. When intrapolymer associations dominate, but a portion of hydrophobes still undergo interpolymer association, intermolecularly bridged flower-like micelles can be formed. One of the drivers for using cholesteryl-based building blocks as part of the polymeric structure is their ability to undergo hydrophobic interactions, and thus promote self-assembly in

and redispersed without loss of structure. Furthermore, the vehicles could be induced to release a model payload (calcein) under acidic conditions (pH 4 and 5.5), demonstrating enhanced stability as well as responsiveness. Low pH is thought to induce a random coil-to-globular phase change for polymers in the PCL membrane due to increased hydrophobic interactions between polymer chains. Moreover, protonated carboxylic acid groups can hydrogen bond to the phosphodiester head groups of the lipid molecules in the membrane, thereby decreasing the lipid−lipid interactions responsible for membrane stabilization. Both these effects are thought to perturb the membrane structure, inducing the formation of pores sufficiently large to allow leakage of contents. Another system that has been investigated is cholesterylterminated poly(N,N-dimethylaminoethyl methacrylate), CholPDMAEMA, incorporated into lecithin-based liposomes. In this case, the liposomes were stabilized by incorporation of CholPDMAEMA at neutral pH and destabilized with slight changes of pH, most likely due to interaction of the protonated tertiary amino groups with the liposomal membrane at low pH (5.5).137

5. CHOLESTERYL AMPHIPHILIC POLYMERS 5.1. Self-Assembling Nanoparticles. Like self-assembling liposome systems, polymeric nanoparticulate drug delivery vehicles have become a promising option for clinical development. Colloidal polymer based systems with a size range of 10− 1000 nm have attracted attention due to the flexibility offered by the increasing array of macromolecular synthetic methods, the diversity of polymers that can be synthesized, and the ability to readily conjugate drugs to the resulting structures. Polymers used with this approach include biodegradable ring-opening polymers such as polylactide, polycaprolactones, and polycarbonates, chain growth polymers such as polyacrylates, and natural polymers, such as polysaccharides, gelatin, alginate, collagen, and chitosan. Importantly, many of these materials can be easily modified using well-documented conjugation techniques.107,108 The ability to engineer stimuli-responsive properties into polymer systems has greatly increased the number of possible applications for self-assembled polymer particles, particularly for drug release.138 One of the advantages of a polymer based system is the level of sophistication and design that can be achieved by taking advantage of the synthetic tools that are now available in polymer chemistry. Notable examples are reversible addition−fragmentation chain transfer (RAFT)139 polymerization and atom transfer radical polymerH


Review

Biomacromolecules aqueous media.142 Because of the strong tendency for selfassociation of cholesteryl (Chol) groups, as well as their structural rigidity, a water-soluble polymer may be modified into a strongly associative polymer by covalently incorporating even a small amount of Chol moieties into the polymer chain.143−147 Morishima et al. studied the self-assembly behavior of cholesteryl modified polyanion, sodium 2(acrylamido)-2-methylpropanesulfonate (AMPS).146 Two copolymers of AMPS were investigated, one with cholesteryl methacrylate (CholMA), and the other with cholesteryl 6methacryloyloxyhexanoate (Chol-C5-MA). In the former, Chol is directly attached to the main chain by an ester bond, whereas in the latter, Chol is linked to the main chain via a pentamethylene spacer. With a Chol content in the polymers as low as 0.5 mol %, a bridged “flower-type” micelle was the proposed structure for the Chol-C5-MA copolymers. These showed a much stronger tendency for Chol units to preferentially associate between polymer chains (interpolymer associations) compared to the CholMA copolymers. The self-assembly of amphiphilic block copolymers in which the hydrophobic block contains cholesteryl groups (which act as mesogens) and the hydrophilic block is a linear PEG chain have previously been described. As expected, these materials showed interesting LC morphologies when dispersed in water.148 Nanofibers with lamellar fine structure were formed using PEG5000-b-PChol (14/86% by mass), and polymersomes were formed using PEG2000-b-PChol (28/72) (Figure 6). Smectic organization in the LC hydrophobic block was found to be essential for self-assembly into nanofibers for the former block copolymer. The synthesis and morphology of nanoassemblies (vesicles or short cylindrical micelles) formed in water from amphiphilic diblock copolymers containing a cholesteryl-based smectic LC hydrophobic block have also been reported.149 5.2. End-Functional, Cholesteryl Polymers That SelfAssemble. Amphiphilic diblock copolymers incorporating a block composed entirely of pendant cholesteryl monomer units, such as poly(cholesteryl methacrylate-block-2-hydroxyethyl methacrylate (PCMA-b-PHEMA), have been synthesized.150 However, a cholesteryl group located at the end of the hydrophilic polymer chain is often sufficient for inducing selfassembly in water, with many such systems reported. Welldefined surfactants were synthesized via ATRP of various oligo(ethylene glycol)methacrylates using cholesteryl-2-bromoisobutyrate as the initiator. Depending on the hydrophilic−hydrophobic balance, these surfactants give micellar or aggregated structures in water.151 A combined micellar system was prepared using cholesteryl-end functionalized poly(Nisopropylacrylamide) (Chol-PNIPAm) mixed with another amphiphilic polymer, methoxy poly(ethylene glycol) monostearate (mPEG-SA).152 The self-assembly behavior in aqueous media has also been investigated for α,ω-functional hydrophilic polymers (telechelics) in which cholesteryl groups are incorporated at both ends of the polymer chain.143,153,154 There is also the possibility of installing two cholesteryl groups at a single-chain end. Specifically, this approach was applied using homopolymers of N,N-dimethylacrylamide (DMA) and N-(2-hydroxypropyl)methacrylamide (HPMA), as well as statistical copolymers with N-acryloxysuccinimide (NAS). The presence of two spacially close and rigid cholesteryl groups located at the α-chain terminus was observed to drive vesicle formation, even at extremely low levels of incorporation relative to the hydrophilic components in the system.155,156

In another interesting study, a partially cholesterylsubstituted 8-arm poly(ethylene glycol)-block-poly(L-lactide) star polymer (8-arm PEG-b-PLLA-cholesteryl) exhibited temperature-induced gelation (34 °C) in water, whereas unmodifed 8-arm PEG-b-PLLA failed to gel irrespective of concentration.157 Further, copolymers based on adenine and thymine methacrylate have also been synthesized with cholesteryl groups located on either one or both ends of the polymer chain. These molecules were shown to self-assemble and associate with the anticancer drug cis-dichlorodiammine platinum(II), which is known to bind irreversibly to the bases in DNA.158 Pluronic F68, is a commercially available nonionic surfactant consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide units, approved by the U.S. Food and Drug Administration for intravenous injection. However, it has severe drawbacks as a building block for drug delivery vectors as it has a high critical micelle concentration (CMC, 4.8 × 10−4 M) originating from its short propylene oxide segment, restricting drug loading and inducing poor dilution stability.159 To overcome the instability issue, a novel cholesteryl end-coupled F68 derivative was reported (F68-CHMC). This material was shown to have a CMC 400 times lower than that of F68, indicating much improved self-assembly capability.160 In vitro release studies demonstrated sustained-release from cabazitaxelloaded F68-CHMC micelles. Moreover, an in vivo antitumor activity test using a mouse S180 xenograft model showed superior inhibition for the micelles (79.2%) compared to Tween 80-cabazitaxel (56.2%) vehicle. 5.3. β-Cyclodextrin-Cholesteryl Inclusion Complexes. β-Cyclodextrin (β-CD), composed of sugar molecules bound together in a ring and displaying hydrophobicity on the inside and hydrophilicity on the exterior, is well-known for its involvement in host−guest interactions.161 The ability of β-CD to form an inclusion complex with various hydrophobic molecules in water has led to its use in a wide variety of biomedical applications, ranging from drug solubilization and delivery, through to building vectors for the delivery of siRNA and pDNA.162 β-Cyclodextrin is known to form an inclusion complex with the cholesteryl group, and this has been studied in different polymeric systems. Self-assemblies of cholesterylbearing pullulan can dissociate upon the addition of β-CD due to capping of the hydrophobic cholesteryl groups by complexation with β-CD.163 Reassociation of the cholesteryl groups can be induced by adding 1-adamantanecarboxylic acid, which preferentially complexes with β-CD.164 Cholesteryl-functionalized poly(L-lysine), which forms a helical secondary structure, was also found to undergo changes in secondary structure (αhelicity) induced by host−guest interaction with β-CD.165 Cholesteryl-end functional and biodegradable star polymers166 and gold nanoparticles grafted with cholesteryl endfunctional linear polymers have been synthesized and studied for their ability to form inclusion complexes with β-CD.167 A self-assembling hydrogel system has also been reported based on 8-arm, star PEG end-modified with either β-CD or cholesteryl functionalities (via a hydrolytically cleavable succinate linker). After dissolving both these polymeric components in an aqueous environment, a physical gel was formed due to the formation of β-CD/cholesteryl inclusion complexes.168,169 Poly(aspartic acid) bearing β-CD side groups was recently reported to form micelles in water with cholesteryl-end functionalized poly(D,L-lactide) by host−guest I


Review

Biomacromolecules

Figure 7. (a): Chemical structures of HPMA copolymers incorporating acid-cleavable Dox and cholesteryl groups; arrow indicates specifically where R group was attached. Adapted from ref 178. Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. With permission from John Wiley and Sons. (b) Chemical structure and proposed pearl necklace morphology of HPMA copolymer with pendant Dox and cholesteryl moieties. Reproduced from ref 180. Copyright 2012 American Chemical Society.

value for both nanoparticle systems in PBS (pH 7.4) was determined to be 33.4 and 38.3 °C, respectively. The cholesteryl grafted polymer yielded a higher encapsulation efficiency for drugs and a more appropriate LCST.175 Nanocarriers (such as polymeric micelles) that exhibit a transformation in their structure due to a pH change can be used to selectively deliver various anticancer drugs to tumor sites by passive targeting. The tumor extracellular environment is more acidic than normal tissues and the bloodstream because of the high rate of aerobic and anaerobic glycolysis in cancer cells.176 Moreover, after endocytosis of the carrier, the endosomes or lysosomes present an even more acidic environment. The intravesicular pH drops along the endocytic pathway, from pH 6.0−6.5 in early endosomes to pH 4.5−5.5 in late endosomes and lysosomes. pH-triggered drug release can therefore be envisaged using drugs that are covalently bound to the carriers by pH-cleavable bonds that are relatively stable at neutral pH (corresponding to the blood environment), but degrade to release the active drug under the acidic conditions of the endosome. Once cleaved, the drug is able to diffuse through the membrane and access intracellular targets.177 Another effective strategy to drive intracellular drug delivery is to use the acidic conditions within the late endosome to drive the transition from an intact vehicle encapsulating the drug outside the cell, to disassembly and cargo release inside the cell. Self-assembled drug delivery systems based on linear HPMA copolymer have been studied in which doxorubicin (Dox) and cholesteryl derivatives were bound by pH-sensitive hydrazone bonds to the same polymer backbone (Figure 7a). Defined amounts of HPMA, cholesteryl and Dox in the polymer structure led to self-assembly into micelles with cholesteryl groups located in the core and Dox in the corona. The pH sensitivity of the hydrazone bonds lead to fast release of Dox from the micelles at pH 5.0, these conditions mimicking those in the late endosomes of tumor cells. The pH-sensitive conjugates therefore can take advantage of this acidic environment to release the cargo. Cholesteryl groups were released at a slower rate, leading to slower disintegration of the micelles, thus delaying removal of the carrier system from the body by the kidneys.178 In an earlier study by the same authors, various HPMA copolymers were synthesized with only Dox bound to the polymer backbone by hydrazone bonds. The structure of the conjugates differed in the type and content of hydrophobic substituent introduced into the polymer structure (dodecyl, oleic acid, and cholesteryl moieties). Experiments in

inclusion complexation, thereby allowing the encapsulation of protein.170

6. CHOLESTERYL POLYMERS AS DRUG DELIVERY PLATFORMS For drug delivery applications, strongly associative amphiphilic copolymers incorporating different amounts of pendant cholesteryl groups in their hydrophobic domains are more commonly represented in the literature than systems that incorporate cholesteryl groups only as end groups. The encapsulation of hydrophobic drugs is well reported. The entrapment efficiency and loading capacity of polymeric micelles bearing a cholesteryl side-functional hydrophobic block, investigated using Nile Red and Ibuprofen, was found to be dependent on cholesteryl content.171 Polymeric micelles made from poly(ethylene oxide)-b-poly(α-cholesteryl carboxylate-ω-caprolactone) were investigated for their ability to encapsulate the hydrophobic drug, cucurbitacin I.172 Further, a micellar delivery system for the hydrophobic drug indomethacin, prepared from cholesteryl-functionalized carboxymethylcellulose, showed high levels of loading and steady release into the medium over an extended period (8 h).173 Stimuli-sensitive polymers that exhibit reversible conformational changes in response to external conditions such as temperature and pH are useful for drug delivery applications, giving the potential to unload cargo under specific conditions. Thermosensitive polymeric nanoassemblies can be applied in combination with regional hyperthermia as a strategy for tumor-specific drug delivery. For the creation of temperaturesensitive micelles/nanoassemblies, a common technique is to use a polymer that displays LCST behavior as the corona of a micelle. The most extensively used polymer for this purpose is PNIPAm, which has an LCST of ∼32 °C. Importantly, this transition temperature can be adjusted by copolymerization with hydrophilic/hydrophobic monomers in order to obtain a LCST value within a desired physiological range. Hydrophobically modified PNIPAm can produce micelles that are stable below the LCST, but when the temperature is raised above the LCST, this induces the entire system to become hydrophobic and precipitate out of solution, thus triggering release of the cargo.174 Thermally responsive cholesteryl end-capped p(Nisopropylacrylamide-co-N,N-dimethylacrylamide) and cholesteryl-grafted poly[N-isopropylacrylamide-co-N(hydroxymethyl)acrylamide] amphiphilic polymers were investigated for self-assembly behavior and their ability to encapsulate cyclosporin A and indomethacin. The LCST J


Review

Biomacromolecules

Figure 8. Poly(amidoamine) synthesis (a) from ref 181 and (b) from ref 183.

mice bearing mouse EL-4 T cell lymphoma showed slower blood clearance, enhanced tumor accumulation (via EPR) and significant antitumor activity, with up to 100% long-term survivors in the group of animals treated by the high-molecularweight cholesteryl substituted conjugate.179 A detailed structure analysis of the cholesteryl-based HPMA nanoparticles revealed that the size, anisotropy, and aggregation number of the nanoparticles increased along with cholesteryl content. A pearl necklace structure was proposed in which ellipsoidal pearls, mainly composed of cholesteryl groups, are covered by a HPMA shell that is uniformly distributed with Dox (Figure 7b).180 Poly(amidoamines) (PAAs) are synthetic degradable polymers obtained by Michael-type polyaddition of bis-primary or bis-secondary amines to bis-acrylamides, resulting in polymers with amide and tertiary amine groups along the main chain. These materials have developed as polymeric drug and gene carriers and biomaterials due to their low cytotoxicity and peptide-mimicking properties. Further, the amine groups along the backbone allow buffering capacity and pH-responsiveness. Amphiphilic PAA-cholesteryl conjugates, first reported by Ranucci et al., were synthesized in three steps (Figure 8a): PAA networks were first obtained by the use of cystamine as a cross-linking agent. These were then turned into linear PAAs with dithiopyridyl side groups by an exchange reaction with 2,2′-dithiodipyridine.181 A subsequent exchange reaction with thiocholesterol provides PAA in which cholesteryl moieties are linked to the hydrophilic PAA chain by S−S bonds. In aqueous media, the conjugates were found to self-assemble into nanoaggregates with inner cores comprised of cholesteryl domains. Because of the redox sensitivity of the S−S bond, the PAA-S−S-Chol particles are expected to be stable in blood but disrupted intracellularly to release their payload. An electrospray technique was used to produce tamoxifen-loaded PAA-S−

S-Chol nanoparticles with the ability to slowly release tamoxifen over time.182 Cheng et al. reported a pH and redox-responsive amphiphilic PAA-based drug delivery system incorporating cholesteryl side chains (Figure 8b). The polymer was synthesized in three steps.183 First the linear disulfide and secondary amine containing PAA precursor polymer was made via Michael addition polymerization, and then the secondary amines in the backbone were conjugated (first with PEG and then with cholesteryl groups) to form poly(BAC-AMPD)-g-PEG-g-CE. Micelles were formed via self-assembly in aqueous solution and used to load Dox. The loaded micelles showed fast redoxresponsive release of Dox, concurrent with formation of aggregates. However, the release profile was not greatly influenced by variations in pH. The Dox loaded micelles could deliver drugs into cancer cells and showed greater effectiveness in killing cancer cells than the free drug. Poly(β-amino esters) PAEs, obtained by Michael-type polyaddition of bis-primary or bis-secondary amines to bisacrylates have also been investigated as pH-responsive polymeric drug carriers for chemotherapy. The resulting polymers have both esters and tertiary amine groups along the main chain. Kim et al. reported on PAEs modified with PEG, cholesteryl, and biotin side groups.184 The biotinconjugated, pH-responsive micelles were found to release Dox under acidic conditions and are envisaged to increase drug accumulation at tumor sites or in tumor cells through receptormediated endocytosis. Senanayake et al. reported a nanoparticulate formulation for anticancer therapy containing a phosphorylated nucleoside.185 Drug conjugates were synthesized starting from cholesterylmodified poly(vinyl alcohol) (CPVA) or dextrin, followed by the covalent attachment of nucleoside analogues to the polymers through a degradable tetraphosphate linker. Association of cholesteryl moieties in water resulted in intramolecular K


Review

Biomacromolecules

Figure 9. Cholesteryl-modified PVA polymers conjugated to phosphorylated 5-fluoro-2′-deoxyuridine (CPVA-p4FdU) and their formation into compact nanogels. Adapted from ref 185. Copyright 2011 American Chemical Society.

Figure 10. (a) chemical structure of CHP (cholesteryl-pullulan) and cCHP (cationic cholesteryl-pullunan). (b) Self-assembly of CHP polymers into nanogels. Reprinted by permission from Macmillan Publishers Ltd.: Nature Materials ref 200, Copyright 2010 Nature Publishing Group. http:// www.nature.com/nmat/journal/v9/n7/full/nmat2784.html.

trapping proteins inside their network structure, thus effectively preventing protein aggregation and allowing gradual release in native form.164,194 CHP nanogel has been exploited as a nanocarrier for protein delivery, especially in the area of cancer vaccine development.195,196 Successful clinical studies have shown that subcutaneous injection of CHP nanogel carrying the cancer antigen HER2 or NY-ESO-1 effectively induces antigen-specific CD8C cytotoxic T lymphocyte responses and antibody production.197,198 Nonetheless, a general observation by the authors was that intracellular uptake of the nanogels could be relatively low due to the nonionic character of the polysaccharide pullulan. To achieve higher cellular uptake of the nanocarrier, various cationic CHP derivatives were developed, which were found to complex with proteins through electrostatic as well as hydrophobic interactions.199 Recently, a cationic CHP (cCHP) nanogel was investigated as an adjuvantfree delivery vehicle for intranasal vaccination against infectious diseases (Figure 10b). In this application the cationic CHP was found to improve antigen delivery with enhanced uptake by nasal dendritic cells compared to the nonionic CHP carrier.200 Polymeric thin films can act as reservoirs for diverse cargo such as small molecule drugs, peptides, proteins, and gene cargo. Such films can be deposited as a multilayered coating on different substrates using the layer-by-layer (LbL) technique. This involves the sequential deposition of interacting polymers either through electrostatic attractions, hydrogen bonding, or covalent interactions.201 The LbL assembly of interacting polymers over a silica template, followed by the selective removal of the template core, produces hollow capsules that can be used as drug carrier vehicles or microreactors. An extension of LbL capsule technology involves the incorporation of structurally intact liposomes into the capsule providing subcompartments, these structures referred to as capsosomes. Cholesteryl-modified poly( L -lysine) (PLL c ) and poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc) were

polymer folding and subsequent formation of compact nanogel particles containing the embedded negatively charged, 5′phosphorylated nucleoside analogue (5-fluoro-2′-deoxyuridine (p4FdU)) within a hydrophilic polymeric shell (Figure 9). The particles could be further compacted by the addition of spermine. Upon hydrolytic or enzymatic degradation the polymeric drug conjugates release nucleoside analogues in active phosphorylated form. In cancer cells, this would provide a strong therapeutic advantage because the drug component does not have to pass through the phosphorylation step, which is known to be a rate-limiting step in biological activation of nucleoside analogues. In vitro evaluation against various cancer cell lines (including drug-resistant ones) demonstrated 50−100 times stronger cytotoxicity for the activated floxuridine polymer conjugate compared to the free nucleoside analogue. The therapeutic efficacy of polymeric conjugates was evaluated in subcutaneous tumor xenograft mouse models with encouraging results. Cholesteryl derivatives of antiviral nucleoside analogues have been used as amphiphilic antiviral prodrugs. The self-assembly characteristics, degradation, pharmacokinetics, tissue distribution, and antiviral behaviors of these prodrugs have been investigated both in vitro and in vivo.186,187 Cholesteryl-modified pullulan (CHP) is a well-studied system that self-assembles in water, forming monodisperse and stable nanogel particles in which the association of cholesteryl groups provide physical cross-links for the pullulan main chains (Figure 10a).188,189 The nature of the hydrophobic associations in water depends on concentration, since in the dilute regime distinct nanoparticles form. However, when the concentration exceeds 3.5% (w/w), macrogels form via interconnection of multiple CHP nanoparticles.190 A notable feature of CHP nanogels is their ability to trap proteins, primarily through hydrophobic interactions with cholesteryl groups.191−193 The nanogels exhibit chaperon-like activity, L


Review

Biomacromolecules

Figure 11. Schematic illustration of capsosome assembly. Reprinted from ref 202. Copyright 2009, with permission from Elsevier.

the lipophilic matrix of the cell membrane by themselves, and as such particle based delivery provides a significant opportunity.208−210 During endocytosis material becomes progressively enclosed by a small portion of the plasma membrane, which first invaginates the substance and then pinches off to form an endocytic vesicle containing the ingested material.211 The process can be subdivided into two main types, distinguished broadly by the size of the endocytic vesicles formed. Phagocytosis is the uptake pathway specific to neutrophils and macrophages, and is responsible for the uptake of solid large particles, such as bacteria and dead cells (generally >250 nm in diameter). Pinocytosis involves the ingestion of fluid and solutes via small pinocytic vesicles (∼100 nm in diameter). The study of different pinocytotic pathways is still an evolving field, and no current classification system is completely satisfactory. However, several subdivisions are generally well accepted based on the vesicle coat proteins that are involved. Clathrin-dependent endocytosis (CDE) normally occurs for particles up to 200 nm in size, and begins with clathrin-coated pits which are specialized domains of the plasma membrane that invaginate the particle into the cell and pinch off to form a clathrin-coated vesicle. In most animal cells, clathrin-coated pits and vesicles provide an efficient pathway for taking up specific macromolecules from the extracellular fluid in a process called receptor-mediated endocytosis. A particularly well-understood and physiologically relevant example is the process whereby mammalian cells take up cholesterol via the low density lipoprotein (LDL) receptors. Understanding of this process was developed through the classic studies of Brown and Goldstein, for which they were awarded the 1985 Nobel Prize in Medicine (Section 8).212 The caveola-mediated endocytosis pathway involves deeply invaginated, flask-shaped structures on the cell membrane called caveolae (50−80 nm). These structures consist of the cholesterol-binding wedge-shaped protein, caveolin, which is embedded into a bilayer enriched in cholesterol and glycolipids. Calveolin proteins bind cholesterol in the membrane and form hairpin structures embedded in the bilayer. Polymerization of

synthesized as precursor and capping layers on the silica templates and used for sandwiching liposomes in between the polyelectrolyte layers (Figure 11).202,203 In this system, cholesteryl moieties were used to anchor the polyelectrolytes on the surface of the liposomes which in turn allowed the liposomes to become anchored into the polymer shell of the capsules. Cholesteryl moieties were found to be superior over oleyl groups at promoting anchoring of liposomes to polymer layers.204 It was also demonstrated that the number of subcompartments within a capsosome could be controlled by the alternate layering of liposomes and polymer separation layers.205 Poly(N-vinylpyrrolidone)-block-(cholesteryl acrylate) (PVPc), a block copolymer consisting of a short block of cholesteryl acrylate and a longer PVP segment capable of adsorbing via hydrogen bonding onto poly(methacrylic acid) PMA, was synthesized via RAFT and used to anchor liposomes to a PMA/PVP capsule film. Non-cross-linked layers were then sacrificed using poly(methacrylic acid) deprotonation, leaving behind capsosomes with “free-floating” liposome compartments entrapped within PMA hydrogel capsules, (as opposed to being located within the capsule film).206 It was also further demonstrated that antitumor drugs could be efficiently loaded into the membrane of the liposomal subcompartments. The number of liposome layers provides a way to tune the drug loading of the capsosomes.207 In these examples, cholesterylmodified polymers are the key building blocks for creating capsosomes efficiently loaded with liposomal subcompartments.

7. CHOLESTERYL-BASED NANOMEDICINES FOR DRUG DELIVERY TO CYTOPLASM 7.1. Endocytosis: Involvement of Membrane Cholesterol. While small molecules can be transferred via pumps or channels into cells, macromolecules generally require an endocytosis process to breach the cell membrane. The endocytic pathway is responsible for the entry of nanoparticles that deliver drugs or highly polar agents such as plasmids or oligonucleotides. Such materials could not ordinarily penetrate M


Review

Biomacromolecules

Figure 12. Membrane destabilization capability of poly(methacrylic acid)-co-poly(cholesteryl methacrylate) (P(MAA-co-CMA)) copolymers, analyzed using liposomal leakage assays and surface plasmon resonance experiments. Reproduced from ref 241. Copyright 2012 American Chemical Society.

caveolins is thought to bend the membrane to form caveolae.213 Caveola are therefore considered to be a specialized form of lipid raft. Although they have been implicated in cholesterol intracellular transport and signal transduction, the functions of caveolae remain poorly understood. A recent review by Rewatkar et al. highlights a number of issues in the literature regarding engineering nanomaterials for caveolae-mediated uptake.214 Most of the data reported on the cellular uptake of nanoparticles, particularly for gene-delivery vectors, has come from studies using chemical inhibitors of endocytosis. A depletion of cholesterol from the membrane may be implicated in the inhibition of pinocytosis,215 particularly caveola and clathrin-coated pit internalization pathways.216,217 However, an increasing volume of evidence suggests that several chemical agents used for probing specific pathways lack inhibitory specificity and are highly cell-line dependent, and as such, results should be treated with caution.218,214 Further, the proposition that caveola entry is advantageous for drug or gene delivery because it avoids lysosomal degradation has been discredited.219 Macropinocytosis is a nonselective endocytic mechanism that is not induced by cargo but rather derives from membrane ruffles generated upon stimulation by growth factors or other signals. The process can form a vesicle up to 5 μm in diameter which pinches off into the cell. Due to the relatively large size of these vesicles, macropinocytosis is often overlooked as a route for nonselective endocytosis of macromolecules.220 A recent quantitative examination of the endocytic pathways involved in lipid nanoparticle uptake showed stimulated macropinocytosis to be one of the entry pathways.221 The contributions of certain endocytosis pathways to the uptake of lipid- and polymer-based delivery vehicles are not well understood, let alone for those incorporating cholesteryl moieties. Overall there are many complex interplays that influence nanoparticle endocytosis, which require consideration when engineering nanomaterials for effective cell entry. The dominating interactions, often described as occurring at the “nano−bio interface”, encompass the effect of particle size, particle surface charge, particle shape, and cell type, in addition to the cell culture conditions.222 These are too complex to discuss at length for the purposes of this review, and interested readers are directed to comprehensive reviews published specifically on this topic.223,224 Once formed, endocytic vesicles either continue down the endocytic pathway or recycle their contents back to the cell surface. As such, the fate of the endosome, rather than the cell uptake pathway, is likely to have a greater impact on the outcome for endocytosed nanoparticles. Drug cargo associated with the nanoparticles that is susceptible to degradation by

enzymes and the acidic conditions found in lysosomes, and for which function relies upon interaction with subcellular targets, require an endosomal escape route. To this end, cholesterylbased agents have been developed, which can enhance the transport from the endosomal compartments into the cytoplasm. 7.2. Membrane-Disruptive, pH-Responsive Anionic Carriers. Certain viruses, such as the influenza virus, have evolved specific hydrophobic fusion peptide domains in their protein coat that become protonated upon acidification in the endosome, leading to membrane interactions that eventually cause destabilization. This mechanism has inspired novel approaches that aim to facilitate endosomal release, and so enable intracellular delivery of drugs. To this end, liposomes have been developed, which are able to adopt a stable uni/multi lamellar state at conditions of neutral pH outside the cell, but which fuse with adjacent membranes when exposed to the inherently acidic environment of the endosome.225 Cholesteryl hemisuccinate (CHEMS), which consists of succinic acid esterified to the hydroxyl group of cholesterol, is an acidic lipid that undergoes pH-dependent morphological transitions as a function of its protonation state, and which therefore changes around its pKa.226 Cation binding to deprotonated CHEMS (which increases the effective headgroup volume and changes lipid shape) may also be a factor that leads to morphological phase transitions.227 CHEMS, together with DOPE has been used to prepare pH-sensitive liposomes in which CHEMS acts as the liposomal stabilizer for DOPE.228,229 While lamellar CHEMS/DOPE systems can be prepared at neutral or slightly alkaline pH, these systems become unstable at acidic pH. This can be rationalized by the ability of the anionic form of CHEMS to stabilize DOPE in a bilayer (lamellar) vesicle organization, whereas when CHEMS is protonated, the stabilizing ability is compromised and DOPE reverts to the nonlamellar, inverse hexagonal phase, promoting fusion and destabilization.230,231 Liposomes containing CHEMS and increasing amounts of the permanently charged cationic lipid, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) have also been shown to exhibit pH-sensitive fusion properties, with the pH at which fusion occurs being dependent on the content of cationic lipid.232 Synthetic polymers containing hydrophobic side chains and ionizable carboxyl groups have been shown to mimic the pHdependent, membrane-lytic behavior of endosomal fusogenic lipids and peptides.233,234 Biomimetic, hydrophobically associating polymers undergo a change of conformation from extended charged chains to globular hydrophobic structures upon a reduction of pH below their pKa ranges.235 Membrane destabilization at acidic pH is thought to arise from a number of N


Review

Biomacromolecules

trafficking. Altogether, these remain major challenges to surmount for effective gene therapies.245 7.3.1. Cholesteryl Cationic Lipids. Cationic lipid/nucleic acid complexes (lipoplexes) have been extensively developed as gene carriers, primarily in the form of liposomes.246,247 Such liposomes typically contain at least two components: a cationic lipid and a neutral lipid (which is sometimes called “helper” lipid). Cationic lipids (lipoamines) are positively charged amphiphilic molecules that contain three sections: (i) a polar headgroup, which is positively charged via protonation of one or more amino groups; (ii) a hydrophobic segment composed of a steroid or saturated or unsaturated alkyl chain(s); and (iii) a linker that connects the cationic headgroup with the hydrophobic segment and which influences the stability and biodegradability of the formed carrier. DOPE and cholesterol are often used as neutral (helper) lipids in liposomal compositions. When combined with a cationic lipid, DOPE can participate in bilayer formation and is thought to enhance endosomal escape of the lipoplexes into the cytoplasm due to fusogenic properties. Significantly, cholesterol-containing cationic liposomes exhibit enhanced stability in physiologic media, thereby enabling the lipoplexes to reach their target tissue intact, protecting the nucleic acid from degradation, and eventually facilitating transfection. Widely used benchmarks for cationic lipid transfection include the lipoamines, dioctadecylamido-glycylspermine (DOGS)248 and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), as well as commercial cationic liposome formulations, Lipofectamine 2000 (L2k) and Oligofectamine. DC-Chol (3β(N-(N′,Ndimethylaminoethyl)carbamoyl)cholesterol)249 is an early example that had success as a lipid-based gene delivery system, which motivated interest in the development of other cholesteryl-based cationic lipid systems. Such systems include those based on spermine cholesteryl carbamate and spermidine cholesteryl carbamate, each of which has more than one protonatable nitrogen.250 Cationic liposomes prepared from TMAEC-Chol [3β(N-(N′,N′,N′-trimethylaminoethane)carbamoyl)cholesteroliodide] and TEAPC-Chol [3β(N′, (N′,N′,N′-triethylaminopropane)-carbamoyl)cholesterol iodide] (i.e., cholesterol-based cationic lipids containing a quaternary ammonium group), have also been reported.251 More recent developments of cholesteryl-based polyamine lipids include N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9diamine (CDAN),252 and 3β[L-ornithinamide-carbamoyl]cholesterol (O-Chol)253 among other carbamate-linked polyamine cholesteryl examples.254 Vigneron et al. synthesized guanidinium cholesteryl lipids and reported their use in the transfection of various mammalian cell lines.255,256 The cationic lipid bis-guanidinium-trencholesterol (BGTC), in combination with the neutral colipid DOPE, was found to confer increased liposomal stability upon nebulization. This enabled effective gene delivery of pDNA by the aerosol route to respiratory sites, as examined in mice.257 Choi et al. reported cholesteryl-based cationic lipids with cationic head groups based on L-lysine and L-ornithine, these being afforded by a solid phase technique. The resulting cationic liposomes self-assembled with pDNA and displayed a relatively high capacity to transfect various mammalian cells in vitro.258 Plasmid DNA and siRNA are known to formulate differently with cationic lipids in liposome formulations. Despite progress in cationic liposome-mediated delivery, the reduced cellular

interdependent factors: (i) increased polymer binding, as a result of hydrophobic interactions between the hydrophobic domains and lipid bilayer membranes; (ii) hydrogen bonding between protonated carboxyl groups and lipid phosphodiester groups; and/or (iii) pH-dependent changes in polymer conformation.236−238 Further, pore formation and binding to the membrane to form phospholipid-polymer micelles239 are thought to be possible endosomal escape mechanisms. One polymer that has been investigated for possible application in endosomal escape situations is poly(propylacrylic acid) (PPA). PPA is thought to undergo a conformational shift at endosomal pH and so cause membrane disruption, due to the gradual protonation of the carboxylic acid residues along the polymer backbone.240 Copolymerization with hydrophobic monomers can be used to tune the pH range of this transition. Bulmus et al. applied similar concepts to study the ability of poly(methacrylic acid)-co-poly(cholesteryl methacrylate), P(MAA-co-CMA), copolymers to destabilize the endosomal membrane (Figure 12).241 RAFT polymerization was used to generate statistical copolymers with varying cholesteryl content (2, 4, and 8 mol %). Surface plasmon resonance (SPR) experiments were performed to monitor (in real-time) the binding of the cholesteryl polymers to lipid bilayers, which mimic both the cell plasma membrane and the endosomal membrane. For liposome leakage assays, liposomes were prepared to mimic the cell membrane lipid composition. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol % CMA displayed the greatest polymer−lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer surfaces and demonstrating the highest membrane destabilization potential. Overall, results suggest that membrane destabilization ability of the copolymers depends on the hydrophobic content and pH of the environment, which is in line with what others have found for synthetic membrane-destabilizing polyanions.233 A series of anionic P(MAA-co-CMA) copolymers produced by the same authors were evaluated as gene delivery vehicles with cationic oligolysine introduced into the mixture to form ternary complexes with pDNA. However, DNA transfection studies revealed the P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhered to the cell surface, as opposed to being effectively internalized. 7.3. Approaches Used for Gene Delivery. Delivering nucleic acids such as plasmid DNA (pDNA) and small interfering RNA (siRNA) to control gene expression in cells and so confer a therapeutic effect, has attracted growing attention in the medical arena. The concept requires that pDNA produce its encoded protein, and that siRNA silence mRNA translation into protein in a sequence specific manner.242 It is generally accepted that delivery of nucleic acids for gene therapy is only possible with an efficient carrier for protection and transportation. Specifically, an appropriate delivery system must be suitable for overcoming both extracellular and intracellular obstacles to gene delivery, transcription, and translation. Typical extracellular barriers include loss of nucleic acid by phagocytosis, or degradation by enzymes, while intracellular obstacles include lysosomal degradation.243,244 As such, the objectives of using an effective delivery system would be (i) to improve the stability in the body, (ii) to improve the pharmacokinetics and biodistribution, (iii) to deliver specifically to the desired site, (iv) to facilitate the uptake within target cells, and (v) to promote intracellular O


Review

Biomacromolecules

alternative route to compacted nanoparticles suitable for gene transfection. These so-called “polyplexes” form by electrostatic interactions between cationic amine groups of the polymer and anionic phosphates of the nucleotides.271 Cationic polymers for gene therapy272,273 include poly(ethylenimine) (PEI),274 poly(amidoamine) (PAMAM),275 poly(2-N-(dimethylaminoethyl) methacrylate) (P(DMAEMA)),276 synthetic amino acid polymers (poly(histidine)277,278 and poly(L-lysine)279 or natural polymers such as chitosan, 280 to name only a small selection.272,281 While it is still debated, one proposed mechanism by which cationic polymers escape from endosomal compartments is via the “proton sponge effect”. This involves unprotonated amines in the polymers buffering the endosomal vesicle to such a degree that it leads to endosomal swelling and lysis, releasing nucleic acids into the cytoplasm in the process. In addition to the proton sponge effect, release of complexes into the cytoplasm is also thought to occur as a result of local membrane damage.282 Moreover, cationic nanoparticles may destabilize the membrane via the formation of pores in the membrane.283 Binding to the membrane is initially driven by nonspecific electrostatic interactions between charged polymer units and oppositely charged lipid head groups. Following the initial interaction, the nanoparticles may be able to exert a force on the membrane via a steric crowding mechanism, thus increasing membrane surface tension and leading to the formation of transient pores.284 A significant disadvantage of many cationic polymers is that they can be quite cytotoxic. A well-known example in the field is PEI, which is often considered the “gold standard” cationic polymer for gene transfection because of its early endosomal release capabilities (which prevents nucleic acid degradation in the late endosomes). PEI has been investigated by many research groups, and its transfection efficiency has been studied over a wide range of molecular weights and with a range of structures (such as linear and branched).285,286 Godbey et al. showed that transfection efficiency of PEI polyplexes increases with increasing molecular weight (from 600 to 70 000 Da). However, longer PEI chains are known to aggregate more easily into huge clusters on the outer cell membrane, and this can induce membrane damage and necrosis.287,288 In the quest to find a highly efficient and low-cytotoxicity polymeric vector various modifications have been explored. Among these, conjugation of hydrophobic segments to the polycations has been increasingly considered, largely inspired by the membrane disruptive behavior of cationic lipids. Lipid and hydrophobically modified gene carriers have been the subject of a review by Incani et al., which focuses on mechanisms of internalization, binding and dissociation of nucleic acids with different carriers and possible interactions of carriers with the membrane.271 Liu et al. also published a comprehensive review in the area of hydrophobic modifications of cationic polymers for gene delivery.289 Here crucial aspects of the gene delivery process are discussed, with reference to hydrophobic modifications to several classes of polymers including PEI, chitosan, etc. 7.3.2.1. Serum Protection. Anionic proteins abundant in serum can adsorb onto positively charged polycations in the polyplex, and this can lead to diminished endocytosis and gene expression. Serum has therefore been found to reduce the transfection efficiency of cationic carriers. Hydrophobic moieties on the polycation are thought to shield the polyplexes from the serum and prevent dissociation of the complex. This could explain the enhanced serum compatibility observed for

uptake efficiency of siRNA in the presence of serum proteins is still considered a major drawback. In fact siRNA is thought to form less condensed complexes with cationic liposomes than plasmid DNA, which may make it more vulnerable to attack by nucleases in the serum.252 With a view to increasing serum stability and therefore transfection efficiency, cationic lipidbased delivery systems for siRNA have been developed using cholesteryl-based cationic lipids such as cholesteryloxypropan1-amine (COPA).259 One of the reasons put forward for the enhanced delivery of siRNA via COPA liposomes in the presence of serum is the strong electrostatic interaction between COPA and siRNA which hinders serum-mediated dissociation.260 Further, several examples of lipoamines consisting of both a cholesteryl ether tail and another aliphatic tail attached to an ionizable amine headgroup have been investigated for siRNA delivery.261,262 Disulfide-containing cationic lipids have also been proposed as improved reagents for gene transfection.263 A disulfide bridge between a lipid and the cationic headgroup can be selectively cleaved by reductive media, such as the enhanced glutathione concentration within the cytosol. The reduction affects the binding between the cationic liposomes and the attached pDNA, thus facilitating migration into the cytoplasm. Bioreducable cholesterol-disulfide cationic linear conjugates have therefore been studied as vectors for pDNA.264−266 In a more recent example, cholesterol was coupled to a disulfide bridge via a carbonate bond and the disulfide was subsequently attached to a variety of different cationic headgroups including L-lysine. The prepared lipids demonstrated low cytotoxicity, strong pDNA binding affinity, high transfection efficacy, and specific cellular localization of pDNA at the periphery of cell nuclei.267 The mechanism by which cationic lipids destabilize cell membranes to facilitate intracellular delivery of nucleic acids was proposed Cullis et al.232 It is thought that a key process is the formation of neutralized ion pairs between the cationic lipids and anionic lipids located in the endosomal membrane. The formation of nonbilayer lipid structures is induced, which can destabilize the organization of the endosomal membrane, leading to membrane disruption and unbinding of nucleic acids from the lipoplex. It is also thought that the anionic lipids are initially located in the cytoplasmic face of the endosome membrane268 and that the helper lipids (chol and DOPE) assist in inducing the formation of nonbilayer lipid structures during the disruption process.232 Crocker et al. investigated the structure−activity relationship of lipid-biomembrane interactions using a combination of small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Specifically, these authors evaluated the interaction between the cholesteryl conjugated ionizable amino lipids and biomembranes.269 The cholesteryl conjugated amino lipids were found to be effective in increasing the order of biomembranes and were also highly effective in inducing phase changes in biological membranes in vitro (i.e., the lamellar bilayer to inverted hexagonal phase transition). The induction of morphological changes to the biomembrane is now a wellaccepted mechanism for destabilization of endosomal membranes for cystolic delivery. Reviews are extensive in the area of lipid-based systems for nucleic acid delivery carriers, and these can be sourced for further reference.270 7.3.2. Cholesteryl Cationic Polymers. Complexation between cationic polymers and nucleic acids provides an P


Review

Biomacromolecules

7.3.2.2. Complex Unloading versus Complex Formation. The high affinity of polycations for nucleic acids presents a significant hurdle in successful transfection because of difficulty in separating the nucleic acid from the carrier. While complex formation should be reasonably strong outside a cell, dissociation of the complex inside the cell should also be efficient: this dichotomy of requirements is one of the challenges for nonviral vectors. Hydrophobically modifying the primary amines in the polyamine used for polyplex formation (usually via acylation) can enhance transfection by affecting electrostatic interactions between the polycation and the nucleic acid, and so favor dissociation of the polyplex inside the cell. Partially acetylating the primary amines of PEI was found to increase gene delivery effectiveness by up to 21-fold, compared to unmodified PEI, both in the presence and absence of serum. The enhancement may be caused by more effective polyplex unpackaging.294 Kurisawa et al. also found that transfection efficiency increased by incorporating hydrophobic butyl methacrylate monomer units into polymeric gene carriers based on pDMAEMA, due in part to the decreased stability and size of DNA−polymer complexes.295 The relatively low cationic density of the oligosaccharide chitosan makes the corresponding polymer/DNA complexes less compact compared to complexes with other cationic polymers, which is unfavorable for transfecting animal cells. To address this, cholesteryl groups have been introduced into chitosan to efficiently condense plasmid DNA into nanosized ion-complexes.296 Cholesteryl modification provided amphiphilic character for self-assembly, and this resolved the lack of complexing ability of the chitosan. Consequently, selfassembled nanoparticles based on cholesteryl-modified chitosan have been applied for drug delivery.297 It has also been reported that adding cholesteryl groups into peptide vectors can induce the formation of micelle-like nanoparticles with increased local cationic charge density, thus facilitating better complexation with DNA. Self-assembling amphiphilic cholesteryl-peptides containing positively charged histidine residues have been designed and evaluated in vitro as gene delivery carriers.278,298 Qin et al. also studied a series of amphiphilic cholesteryl peptides that were found to self-assemble into compacted micelle-like structures in aqueous solution. These materials significantly promoted siRNA condensation and effectively protected siRNA from degradation in rat serum up to 3 days. Furthermore, the cholesteryl peptides were found to efficiently transfect siRNA into different cancer cells inducing a potent gene silencing effect, whereas peptides without cholesteryl modification were ineffective for delivering siRNA into the cells.299 Cationic micelles were formed using a biodegradable amphiphilic copolymer based on poly(N-methyldietheneamine sebacate) (PMDS). A quaternization reaction between a bromoethyl derivative of cholesterol, N-(2-bromoethyl)carbarmoyl cholesterol, and amine groups in the PDMS main chain produced the cationic amphiphilic copolymer, poly(Nmethyldietheneaminesebacate)-co-[(cholesteryl oxocarbonylamidoethyl)methylbis(ethylene) ammonium bromide]sebacate] (P(MDS-co-CES) (Figure 14).300 In this system the main chain is a polyester that renders the copolymer potentially degradable in vivo. The nanoparticles were developed for codelivery of paclitaxel and DNA to enhance gene expression and achieve the synergistic/combined effect of both drug and gene therapies.301 This application demonstrates the potential of well-designed core−shell nanoparticles to carry

lipopolymers based on low molecular weight PEI (Mw = 800, 1200, and 2000 Da), conjugated with cholesteryl groups via an ether linkage. These were studied for gene transfection activities in HeLa cells and as coliposomes with DOPE. Application of lipopolymers led to improved transfection and serum compatibility compared to commercially available PEI25KDa.290 One micelle system that has been applied in complexation and delivery of pDNA is (PEG)-block-poly(N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide, [PEG-PAsp(DET)].291 This polyplex system exhibits pH-selective membrane destabilization suitable for late endosomal or lysosomal escape with limited cytotoxicity. The transfection efficiency of the PEG-PAsp(DET) polyplex system was improved further for use as an in vivo systemic vector by introduction of a cholesteryl group into the ω-terminus of PEG-PAsp(DET), to give PEG-PAsp(DET)Chol (Figure 13).292 Introduction of the cholesteryl group led

Figure 13. Synthesis of cholesteryl end functionalized (PEG)-blockpoly(N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide (PEG-PAsp(DET)-Chol) as reported in ref 292.

to increased stability in protein media and also in the bloodstream after systemic injection, compared to PEGPAsp(DET) micelles formed without a cholesteryl terminus. Increased micelle stability of PEG-PAsp(DET)-Chol polyplex and enhanced association of block copolymers with pDNA were thought to be the primary reasons for the high in vitro gene transfection, even at relatively low concentrations. Moreover, suppression of tumor growth was demonstrated following intravenous injection into mice. In a further study, the molecular weight of PEG in the PEGylated polyplex micelle was increased up to 20 kDa (from 12 kDa) to allow increased retention in blood circulation by virtue of enhanced PEG shielding.293 Cyclic RGD peptide (cRGD) (a ligand to integrin receptors), was also installed at the distal end of PEG in order to facilitate accumulation at the tumor site. The cholesteryl conjugation was thought to compact the polyplex and synergistically increase PEG density on the surface. The cRGD conjugated polyplex micelle, incorporating a cholesteryl group and longer PEG chain, achieved potent tumor growth suppression by expression of antiangiogenic protein (sFlt-1) at the tumor site. Q


Review

Biomacromolecules

was also used for siRNA delivery. The polymer was able to form stable nanocomplexes and exhibited in vitro VEGF gene silencing and in vivo tumor growth inhibition when formulated with VEGF-siRNA. Imaging demonstrated escape from endosomes; however, it is uncertain to what extent this process was assisted by disulfide bond cleavage.304 7.3.2.4. Reduced Toxicity. Although the mechanisms that govern the cytotoxic effects of vectors remain uncertain, a strong hypothesis suggests that it is mediated by ionic interactions between anionic groups on the cell surface and cationic moieties present on the vector. This is thought to induce polyplex aggregation and accumulation at the cell surface, which can severely impair membrane function and ultimately lead to cell death.305 It is generally known that short PEI chains, such as bPEI-0.8K and bPEI-2K, are not as cytotoxic in the normal concentration range used for the gene transfection in comparison to longer chains such as long branched PEI (bPEI-25K). However, longer free chains are much more effective than their short counterparts in promoting effective gene transfection.306 Low molecular weight PEIs (

Cholesterol Modified Self-Assemblies and Their Application to

Recommend Documents