Structurally Inspired Biomaterial Virosomes

Liposomes virosomes polymersomes

FIGURE 3.1 Structurally inspired biomaterials. Liposomes, virosomes, and polymersomes are examples of simple cell mimics without internal contents. They have been used for vaccines and targeted drug delivery systems. Peptide nucleic acids mimic both proteins and DNA, and peptoids are mimics of proteins.

FIGURE 3.2 Functionally inspired biomaterials. Mussels and barnacles secrete proteinaceous bioglues for holdfasts on both organic and inorganic surfaces in aqueous environments. Posttranslationally modified proteins mediate this strong adhesion and serve as models for biomimetic materials.

TABLE 3.1 Clinically Available Liposomal Pharmaceutics

Therapeutic Name

Company

Applications

Daunorubicin Nexstar Pharmaceuticals, 1995

Doxorubicin Sequus Pharmaceuticals, 1997

Amphotericin Fujisawa USA Inc./Nexstar pharmaceuticals, 1997

Doxorubicin Liposome, 2000

Kaposi's sarcoma Kaposi's sarcoma

Fungal infections in immunocompromised patients Metastatic breast cancer interactions between polar lipid head groups and the aqueous phase. The physical and chemical principles that explain the whole self-assembly processes include intermolecular forces (hydrophobic, electrostatic, hydrogen bonding), thermodynamics, and surfactant number theory. Here, we begin with a brief introduction to the intermolecular forces.

3.2.1.1.1 Intermolecular Forces and Thermodynamics

Hydrophobic interaction between hydrocarbon chains is the major driving force for self-assembly and consists of several factors, which under appropriate conditions makes the assembly processes energetically favorable. First is the hydrophobic energy, which is defined as the energy required to transfer a hydrocarbon chain from an aqueous environment into the aggregation region (AG < 0). Second is the contact energy, which is derived from the physical interaction between hydrophobic chains and the surrounding solvent (AG > 0). The final energetic term is the hydrophobic packing energy, which describes the decrease in entropy associated with the loss of conformational freedom within the aggregate (AG > 0).

Upon the introduction of amphiphilic molecules into an aqueous environment, intermolecular aggregation is observed above a critical concentration due to the minimization of free energy of the system. This critical concentration, called critical aggregation concentration (CAC) or critical micelle concentration (CMC), is a critical concentration above which the self-assembly process proceeds. The self-aggregation process becomes favorable above CMC-CAC when the balance of the following interactions gives rise to a net decrease in free energy: an increase in hydrophobic interactions (aggregation), a decrease in entropy due to the elongation of the core segments (hydrocarbon chains), and an increase in the steric repulsion at the interface between individual molecules participating in the self-assembly process.

3.2.1.1.2 Surfactant Number Theory

To successfully formulate a vesicle, it is particularly important to consider the steric repulsion between molecules. This consideration leads us to the surfactant number, a dimensionless quantity that predicts the shapes of self-assembled supramolecules. The surfactant number is defined as follows:

N v s ao l where v is the volume of the tail-groups, a0 is the head group area, and l is the length of the fully extended hydrocarbon tail. In other words, this number represents a ratio of surface areas between a hydrophobic tail (v/l) and a hydrophilic head group (a0). By estimating Ns for a range of molecules, the morphology of assemblies can be predicted from spherical micelles (Ns < 1 /3) to planar bilayer (Ns ~ 1) or even inverted micelle structures (Ns > 1) [2] (Figure 3.3). This theory has been confirmed by several systematic studies in which phases were correctly predicted by the surfactant number [3,4].

3.2.1.2 Poly(Ethylene Glycol)-Liposomes

Despite the initial scientific excitement during the early years of liposome research, several drawbacks of liposomes became apparent, such as short half-life during in vivo circulation and physical instability during storage. Studies later showed that liposomes rapidly activated the complement immune system, a major player for clearing injected circulating liposomes in the blood. This drawback of immune system activation by liposomes resulted from nonspecific interactions between biomacromolecules (proteins) and lipo-somal surfaces, ultimately triggering the rapid clearance of liposomes from the bloodstream by the reticuloendothelial system (RES) [5].

Subsequently, coating the outer surface of liposomes with chemically inert molecules was suggested as a solution to reduce clearance by the RES. The most popular molecule for this purpose is poly(ethylene glycol) (PEG), which significantly inhibited liposome-induced complement activation [6,7]. A comparison study showed that liposomes coated with PEG exhibited significantly prolonged in vivo circulation time compared to liposomes without PEGs. The ability to inhibit nonspecific adhesion of proteins at the solid-liquid interface arises from the entropic repulsion of PEG: the loss of con-formational entropy creates a repulsion force counteracting molecular attraction. In a similar manner, proteins covalently conjugated with PEG molecules showed improved drug efficacy due to the increased in vivo circulation time [8-11].

3.2.1.3 Stimuli-Responsive Liposomes

Smart liposomes have been developed in which the liposomal contents were released in response to external stimuli (temperature, light, pH, etc.) [12-15]. The mechanisms of reagent release include phase transitions (lamellar to micellar or lamellar to hexagonal), phase segregation, and bilayer fusion. Here we describe several examples of triggerable liposomal systems.

3.2.1.3.1 pH-Sensitive Liposomes

Perhaps the most extensively studied release mechanism involves a pH-induced liposomal destabil-ization. Liposomes with pH-sensitivity can be utilized for the delivery of therapeutics to acidic tissues, such as inflammatory tissues and solid tumors [16,17]. Upon entering the target tissue, pH-sensitive liposomes undergo a phase transition to release the internal contents. Four designs have been proposed to achieve this: neutralization of negatively charged lipids, protonation of anionic polymers or peptides, acid-catalyzed hydrolysis of liposomal molecules, and ionization of neutral surfactants into positive species.

The first two approaches result in liposomal destabilization by using negatively charged groups at neutral pH, which are subsequently protonated in acidic conditions depending on their pKa values. An example, is given by dioleoylphosphatidylethanolamine (DOPE), which is a pH-titratable lipid that undergoes a pH-dependent phase transition from lamellar to hexagonal, which can be used as a triggering mechanism [18]. Other liposomes modified with negatively charged peptides or polymers lose their structural integrity by lysis, pore formation, or fusion depending on the nature of the integrated nonlipid molecules [19]. A drawback of these approaches was severe adsorption of proteins

Sphere

Tube

Sphere

Cone

Tube

Truncated cone

Cone

Inverse micelle

Truncated cone

Inverse micelle

Bilayer

Bilayer

Truncated cone

FIGURE 3.3 Morphology of self-assembled structures. The dimensionless surfactant number, a ratio of surface areas between hydrophobic and hydrophilic regions, predicts a variety of self-assembled structures: sphere, tube, bilayer, and inverse micelle. The liposomes, virosomes, and polymersomes discussed in the text are mimics of bilayer structures.

on liposomal surfaces that led to rapid elimination from the blood circulation. As a result, these approaches are often integrated with stealth methods i.e., PEG-liposomes (see Section 3.2.1.2).

Acid-induced cleavage is an attractive alternative to circumvent the use of negatively charged lipids. In this approach, lipids are designed with uncharged functional groups in which their hydrolysis is accelerated by acidic conditions. The key chemistry of this mechanism has been well established in organic chemistry [20]. Mono- and diplasmenyl lipids with acid-sensitive vinyl ether linkages located between head group and hydrocarbon chains were synthesized. Cleavage of one or more hydrocarbon chains catalyzed by low pH resulted in structural defects that allowed the release of liposomal contents [21,22]. Attempts have been made to incorporate other functional groups into lipid chains to achieve more rapid destabilization kinetics. For example, lipids containing ortho ester groups have been developed for this purpose [23-25]. To enhance in vivo performance, ortho ester containing lipids were combined with a PEG moiety (MW = 2000) [26]. The imidazole group found in the side chain of histidine amino acid has been used as a pH-titratable head group as it becomes cationic at acidic pH [27,28].

3.2.1.3.2 Temperature-Sensitive Liposomes

The second mechanism of releasing liposomal contents is by temperature. Clinical applications of temperature-sensitive liposomes include mainly local drug delivery to tumors and remineralization of dental hard tissues [29,30]. Two approaches have been used to introduce the thermosensitive property to liposomes: (1) control of lipid chain melting temperature (Tm) with the use of lipid mixtures and (2) the modification of liposomal surfaces with temperature-sensitive polymers. Release of entrapped compounds can be induced either by local hyperthermal tissues, or by simply taking advantage of the rapid warming of liposomes to body temperature after injection into a tissue.

Early studies by Yatvin et al. [31] and Weinstein et al. [32] demonstrated the use of increased permeability near the lipid bilayer Tm for the purpose of drug release. The enhanced permeability at Tm allowed entrapped small molecules to be released by diffusion down to a concentration gradient. In these studies, dipalmitoyl phosphatidylcholine (DPPC) was the primary lipid, which underwent a phase transition (Tm) at 41°C. This Tm was modulated by coformulation with lysolipids such as distearoyl phosphatidylcholine (DSPC). DSPC is a hydrophilic lysolipid compound, which decreases the phase transition temperature to ~39°C at 5% coformulation. Later studies used the lysolipids monopalmi-toylphosphatidylcholine (MPPC) and monostearoylphosphatidylcholine (MSPC) to obtain better temperature tuning as well as a rapid release of liposomal contents [33]. Liposomes formulated with DPPC and MPPC or DPPC and MSPC (10 mol%) showed an approximately 80% release of drug during a very short period of time (<60 s) at 40°C.

In addition to drug delivery through local hyperthermia of tissues, Messersmith and coworkers have used thermosensitive liposomes for site-directed formation of polymer, ceramic, and polymer-ceramic composite biomaterials. This technique was first developed for the remineralization of dentin and enamel surfaces [34,35]. Liposomes loaded by calcium and phosphate ions were prepared separately and then mixed. When heated to Tm, the liposome contents were rapidly released, upon which they reacted to form calcium phosphate minerals. In their studies, saturated phosphatidylcholines were used to tailor Tm by changing the fatty acid chain length. For example, a 9:1 molar ratio of DPPC and DSPC resulted in Tm around body temperature (~37°C). Further analysis showed that the mineral phases after the reaction composed of apatite and brushite phases. This approach can be potentially used for the in situ mineralization in dental or bone repair applications. Messersmith and coworkers subsequently utilized a similar strategy for inducing rapid formation of polymer hydrogels [36], self-assembled peptide hydrogels [37], and mineral-collagen composite hydrogels [38].

Another strategy for thermosensitive liposomal formulation is the incorporation of temperature-sensitive polymers to surfaces of liposomes. Poly(N-isopropylacrylamide) [poly(NIPAAm)] is a water-soluble polymer that exhibits a hydrophilic to hydrophobic transition at a characteristic temperature called the lower critical solution temperature (LCST). The LCST of poly(NIPAAm) homopolymer is

31°C but is increased by copolymerization with other hydrophilic monomers [39,40]. Ringsdorf and coworkers synthesized a random copolymer of NIPAAm and N-[4-(1-prenyl)-butyl]-N-n-octadecyl-acrylamide at a molar ratio of 200:1. The octadecyl-pyrene groups were inserted into liposome layers by hydrophobic interactions, which could be monitored by a remarkable decrease of pyrene excimer emission. This anchorage fixed the thermosensitive polymer chains on the liposomal surfaces and led to temperature-induced liposomal release at LCST [41]. Subsequent studies with end-functionalized poly(NIPAAm) resulted in liposomal content release within a narrow temperature range [42]. The end-located pyrene molecule was less sterically hindered so that the poly(NIPAAm)-co-pyrene showed better incorporation of pyrene into the bilayer [43].

3.2.2 Virosomes

3.2.2.1 Introduction

A key issue in the development of second-generation vaccines involves targeting and delivery of antigens to antigen-presenting cells (APCs). Remarkable advances have been achieved in vaccine preparation techniques and among them, virus-mimicking nanoparticles (virosomes) have demonstrated potency in eliciting immune responses while minimizing side effects.

The term virosome was coined by Almeida et al. [44] in 1975 due to the unexpected finding of a viruslike structure during the visualization of liposomal particles composed of lipids, hemagglutinin (HA), and neuraminidase (NA). These were called influenza virosomes because the key protein components, HA and NA, were derived from the influenza virus. Since this first report, virus-mimicking envelopes have been reconstituted by molecules derived from other viruses, such as the Sendai virus [45], Rubella virus [46], hepatitis virus [47], and vesicular stomatitis virus [48]. None of these has been as well-studied as the influenza virosomes, and will be the main focus of this section.

The influenza virus, which causes the common flu, is notorious for its variability due to mainly two membrane proteins, HA and NA. These proteins create morphological changes on the viral coat membranes by trimerization of HA and tetramerization of NA. The biological function of HA is the initiation of cell internalization by specific interactions between HA and sialic acids from cell surface glycoproteins. After binding, receptor-mediated endocytosis occurs so that the receptor-virus complexes are located within an endosome. Due to the slightly acidic endosomal pH (5.5-5.8), HA undergoes a conformational change that induces the fusion of the viral and endosomal membranes [49]. HA has been shown to mediate membrane fusion in virosomes. This fusion process can be monitored by fluorescence change from the fluorophore (pyrene-phosphatidylcholine). The fluorescence study revealed that endosomal fusion was completely a ligand-receptor-medicated process and also was not affected by virosome contents (peptides, proteins, or DNA) [50,51].

3.2.2.2 Applications

3.2.2.2.1 Antigen Delivery

Virosomes are ideally suited for inducing cellular responses, which require delivery of protein antigens to the cytosol of APCs. For example, virosomes can activate both major histocompatibility complex (MHC) class I and II as well as dendritic cells (DC), the key orchestrator of T-cell response. The mechanism for the antigen presentation through MHC class I is composed of four steps. First, virosomes bind to the APC membrane through HA and receptor complexation. Second, the virosomal complex is internalized by receptor-medicated endocytosis and subsequently undergoes a fusion into endosomal membranes, releasing virosomal contents to the cytosol. Third, the released proteins are enzymatically processed in proteosomes. Finally, the processed peptides are loaded onto MHC class I molecules and the antigen-presented MHC complexes presented outside the cell membrane. The mechanism of MHC class II presentation is similar with the exception that enzymatic processing occurs within lysosomes because not all virosomes fuse into endosomal membranes. Proteins not released by the fusion process are eventually degraded in lysosomes and subsequently MHC class II receptor-antigen binding occurs. This complex is relocated in the membrane of APCs [52,53]. In addition, due to the fact that reconstituted viral envelopes mimic the outer surface of a virus, virosomes can be used for mild induction of antibody responses against a native virus (for example, influenza virosomes can be used as a flu vaccine). Antigen delivery using virosomes is efficient enough to elicit a measurable immune response, and this approach has been developed into two commercially available vaccines: Epaxal for hepatitis A and Inflexal V for influenza from Berna Biotech in 1994 and 1999, respectively.

3.2.2.2.2 DNA Delivery

Historically, gene therapy has been recognized as a promising way to treat difficult human diseases through the introduction of new DNA as a source material. However, it is a difficult challenge to develop DNA vectors with complete fidelity so that every single cell suffering from a nonfunctional gene can be cured. The most efficient vector systems developed so far are of viral origin, but their use often raises safety concerns. Accordingly, nonviral delivery techniques, such as liposome and polymer vectors, have been developed. However, these nonviral systems typically exhibit relatively low transfection efficiency. Virosomes have been proposed as vectors to solve the problems associated with both viral and nonviral delivery systems. Virosomes are attractive in this respect because of the potency of their internalization combined with their ability to encapsulate DNA.

The incorporation of cationic lipids has been the main approach to entrap DNA. Many cationic lipids have been used successfully, and the two most commonly used one are dioleyloxypropyltrimethylam-monium methyl sulfate (DOTAP) and dioleoyl-dimethylammonium (DODAC). Cationic virosomes showed a gene expression level comparable to the efficiency of viral vectors with a HA-dependent fusogenic activity [51]. Several studies demonstrated that immune responses upon the administration of DNA-virosomes were moderate [54-56].

3.2.3 Polymersomes: Toward a Synthetic Cell 3.2.3.1 Motivation, Development, and Properties

So far, we have discussed lipid-based vesicles in the form of liposomes and virosomes. These vesicles closely mimic membrane dynamics as well as physicochemical properties of biological membranes. However, these properties result in poor physical stability of lipid-based vesicles, which is a major drawback as a drug encapsulator and carrier. These drawbacks motivated researchers to find alternative ways to construct bilayer vesicles with better stability. The use of amphiphilic polymers is one approach of increasing stability [57]. By tailoring the ratio and composition of hydrophilic and hydrophobic groups in these block copolymers, the construction of self-assembled polymer-based vesicles is possible.

The first polymer-based vesicles (polymersome) were reported by Discher and coworkers [58] in which they synthesized a diblock copolymer of poly(ethyleneoxide)-co-poly(ethylethylene) (PEO40-PEE37) and found that this block copolymer was self-assembled in H2O into vesicle-like structures with polymer membranes. This study suggested that a large hydrophilic fraction in a diblock copolymer was a critical requirement for polymersome assembly. They also found several unexpected characteristics of the poly-mersomes: (1) they were 10 times tougher than liposomes, (2) a fluid-like phase of the polymersome membrane, and (3) a significant retention time (~10 x longer) of encapsulated materials (globin and dextran). Subsequently, other diblock copolymers, such as polyethyleneoxide-polybutadiene (PEO-PBD) and polyethyleneoxide-polypropylenesulfide (PEO-PSS), have been demonstrated to form polymer vesicles in aqueous solutions [59,60]. These studies strongly indicate that, generally, linear diblock copolymers can form bilayer structures in a self-directed way by an appropriate amphiphilic polymer design. Recently, biodegradable polymersomes were formulated in which poly(lactic acid, PLA) and poly(carprolactone, PCL) are located in hydrophobic cores, gradually destabilizing the vesicle structures by hydrolytic degradation [61-63]. Regarding drug encapsulation efficiency of polymersomes, it appears to be independent of molecular weight of model drugs and otherwise comparable to liposomes. When loaded with therapeutics, polymersomes have longer retention of internal content than liposomes, which is a significant advantage from a pharmaceutical point of view. Another advantage is that the stealth is integrated into the polymersome design by virtue of the presence of PEO as a main building block.

Research has shown that polymersomes are stable for longer than 1 month in a physiological saline solution, and have shown to be stable in the presence of plasma [59]. Permeation of water through the polymersome membrane is significantly decreased compared to liposomes [58], suggesting that polymersomes are excellent alternatives for controlled drug delivery [64]. Future research targets may include the development of triggerable polymersomes upon external stimuli, such as pH, temperature, or light.

3.2.3.2 Polymersomes from Triblock Copolymers

Polymersomes may also be formed by triblock copolymers [65,66]. Triblock copolymer (PEO5-PPO68-PEO5) yielded vesicles with a relatively thin membrane (3-5 nm). In another study, a triblock copolymer with two identical water-soluble end blocks of poly(2-methyloxazoline, PMOXA) and a midblock of poly(dimethylsiloxane, PDMS) formed polymersomes, and even a pentablock copolymer was reported to form polymersome [67]. However, insufficient information about phase transitions and membrane structures of the multiblock copolymers in polymersomes requires further studies.

In an effort that perhaps will lead to a synthetic cell, a membrane protein was successfully integrated into PDMS-PMOXA polymersomes [68]. This result was surprising in the sense that the thickness of polymersome membranes (~10 nm) was significantly greater than the length of a transmembrane domain (3-5 nm). One possible explanation for this is that the local membrane experiences membrane compression and the polydispersity of constituted polymers made the protein integration feasible. Spontaneous DNA transfer inside polymersomes was achieved by mimicking viral translocation mechanisms. A channel protein derived from bacteria (LamB) was integrated into polymersome membranes, which mediated viral DNA transfer into polymersomes by protein interactions between viral coat proteins and LamB [69]. This study demonstrated that a simple biological process can be reconstituted in a totally synthetic way in vitro, further suggesting a possibility to mimic more complex biological processes.

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