Interfacial activity accounts for the high utility of amphiphilic block copolymers [8]
in the biomedical field as colloidal dispersants, micellar drug carriers [9], and surface modifiers, influencing block copolymer adsorption [10, 11] on solid surfaces.
A vast majority of diblock and triblock copolymers are used for the creation of micellar systems that are loaded with a biologically active substance and transport it, in a protected way, to the application site where the substance is then released at a controlled rate. Polymer micelles are prospective anticancer drug and radionuclide delivery systems, enabling efficient tumor accumulation due to their high apparent molecular weight. The passive tumor accumulation mechanism is called the enhanced permeability and retention (EPR) effect [12–14]; see Figure 8.3.
The high-molecular-weight species are accumulated in the solid tumor lesions as a consequence of the low-quality fenestrated neovasculature accompanied by poor or missing lymphatic drainage in such solid tumors.
Micelles exist in dynamic equilibrium with individual surfactant molecules (unimers) that are constantly being exchanged between the solvent and the micelles [5, 16]. The micelles themselves are also continuously disintegrating and reassem- bling. These two processes are characterized by relaxation times of the order of microseconds and milliseconds, respectively [17]. Unimers may have molecular weight below renal filtration threshold [18–20] (ca. 40–50 kDa depending on type of polymer) and thus may be eliminated rapidly from the organism by filtration in the kidneys after disassembly of the micellar system that fulfilled its task as a drug carrier.
k k healthy tissue
macromolecule
small molecules
active agent
malignant tissue
fenestration endothelial cells bloodstream lymphatic system
Figure 8.3 EPR effect [13]. Macromolecules and nanoparticles do not penetrate the compact endothelium but can enter the defective malignant tissue. (See color insert for color represen- tation of this figure).
A very important parameter in the block copolymer self-association process is the critical micelle concentration,cmc. It is the concentration that indicates the usually narrow range of concentrations separating the limits below which most of the copoly- mer is in the unimolecular state and above which virtually all additional copolymers enter the micellar state [21]. For block copolymers, thecmcis very low compared to low-molecular-weight surfactants, which is advantageous for biomedical applica- tions where the presence of a free nonassociated polymer must be avoided as much as possible [22]. For example, thecmcof a low-molecular-weight surfactant sodium dodecyl sulphate (SDS) iscmc =8 ×10−3mol/L [23], for the F127 copolymer it iscmc=1 ×10−4mol/L [24], and for block copolymers with longer hydrophobic blocks,cmcdecreases exponentially with chain length [25], and is, for example, 10−7 to 10−8mol/L for diblock copolymers with∼500–600 monomeric units [26]. Thecmc is also influenced by the ratio of block lengths, and the polydispersity and heterogene- ity of the block copolymer. A lowcmcfavors therapeutical applications of polymeric micellar systems, since the system would remain in a micellar state even after the critical dilution results from the injections into the bloodstream. Last, but not least, it is important whether or not the hydrophobic block(s) of the copolymer is/are com- patible/miscible with phospholipids forming cell membranes. On the one hand, if there is miscibility, one can expect cytotoxicity of such a macromolecular detergent due to membrane damage. On the other hand, block copolymers with a hydropho- bic block that is immiscible with the aliphatic part of the phospholipids—which is the case, for example, of Pluronic F127 with a poly(propylene oxide) hydrophobic central block—have only very little if any cytotoxicity.
k k The size of block copolymer micelles is dependent on the polymer characteris-
tics and ranges usually from 10 to 100 nm [27]. Therefore lipophilic agents, such as hydrophobic drugs, can by solubilized in the micellar core, significantly affecting their concentration in aqueous media. The encapsulation efficiency is dependent on several parameters, including drug and polymer solubility parameters, size, shape, and physical state of the hydrophobic guest molecule, block copolymer length and volume ratio.
Many micelles with a thermoresponsive core and hydrophilic corona have the additional advantage in that they can be simply prepared by heating the thermore- sponsive polymer in aqueous solution to a temperature above their cloud point temperature, CPT [28–30]. Among the polymeric micellar drug delivery systems, poly(ethylene oxide), PEO, is most often employed because of its high hydrophilicity and biocompatibility, long experimental record, and FDA approval [31]. However, careful studies of PEO-derived systems over recent decades have revealed some drawbacks, namely interactions with various immunological entities and superflu- ous accumulation in the body [32]. Another important reason for the search for alternatives is the wide range of patent coverage for nearly all PEO drug carriers and therapeutics. The PEO alternatives poly[N-(2-hydroxypropyl)methacrylamide], HPMA, polyvinylpyrrolidone, PVP, and poly(2-alkyl-2-oxazoline)s, POX, have been examined [33].
Because of their unique self-assembly properties, such micellar systems have been found suitable for a variety of applications such as in gene and drug delivery, chemosensors/biosensors, cell adhesion, protein-ligand recognition, and molecular separation, and their functions have been evaluated in vitro and in vivo [34–36].
Special attention has been devoted to micelles formed from hydrophilic/hydrophilic copolymers (also called double-hydrophilic copolymers, or DHCs) consisting of two or more water-soluble monomers with different chemical properties [37, 38].
The DHCs typically exhibit properties of reversible micellization in water upon temperature variation when one of the blocks presents a lower critical solution temperature (LCST) above room temperature. In addition, there are cases where one of the blocks promotes their dissolution but the other block interacts with a substrate or is sensitive to the influence of an external stimulus such as changes in temperature, pH, or ionic strength [36, 39].
Because the micelle is supramolecular aggregate of tens to hundreds of macro- molecules, one can easily construct hybrid micelles containing several types of moi- eties and polymer blocks in the desired ratio by simply mixing several appropriate block copolymers and forming the mixed micelle afterward from the mixture. The only prerequisite for the formation of such macromolecular modules is compatibility of the copolymers with each other, which may be ensured, for example, by using an identical hydrophobic core-forming block.
A novel polymeric micellar pH-sensitive system for delivery of doxorubicin (Dox) was prepared by the self-assembly of amphiphilic diblock copolymers in aqueous solutions [40, 41]. The copolymers consist of a biocompatible hydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic block containing covalently bound anthracycline antibiotic Dox. The starting block copolymers
k k poly(ethylene oxide)-block-poly(allylglycidyl ether) (PEO-PAGE) with a very
narrow molecular-weight distribution (Mw/Mn ca. 1.05) were prepared by anionic ring-opening polymerization using a sodium salt of poly(ethylene oxide) monomethyl ether as the macro initiator and allyl glycidyl ether as the functional monomer; the double bond of the allyl glycidyl ether moiety was further functionalized to form the polymer hydrazide. The hydrazide was coupled with Dox, yielding pH-sensitive hydrazone bonds between the drug and carrier. The resulting conjugate containing ca. 3 wt% Dox formed micelles with Rh∼100 nm in phosphate-buffered saline.
Thermoresponsive polymer micelles for applications as drug and radionuclide carriers [41] were prepared [1] from ABA triblock copolymers poly[2-methyl-2 -oxazoline-block-(2-isopropyl-2-oxazoline-co-2-butyl-2-oxazoline)-block-2-methyl- 2-oxazoline]. These polymers are molecularly dissolved in an aqueous milieu below the cloud point temperature (CPT) of the thermoresponsive central (2-isopropyl- 2-oxazoline-co-2-butyl-2-oxazoline) block, and above the CPT they form polymeric micelles with a diameter of ∼200 nm. The phenolic moiety introduced into the copolymer allowed radionuclide labeling with iodine-125 with sufficient in vitro stability under model conditions. Various iodine radioisotopes are suitable for both the diagnosis and the radionuclide therapy of the solid tumors. The composition of these triblock copolymers is more complex than that of the vast majority of poly(2-alkyl-2-oxazoline)s: a statistical thermoresponsive (iPrOx) and hydrophobic (BuOx) central block with terminal hydrophilic blocks (MeOx). As the temperature increases, nanoparticles form in a process starting with single molecules that become loose aggregates and end with the formation of compact nanoparticles. It has been proved [42] that the iPrOx/MeOx ratio determines the value of the cloud point temperature, whereas the different BuOx−iPrOx blocks determine the character of the process.
Polymers that are pH-sensitive have attracted considerable research interest because the differences in pH between normal tissue and cancer tissues create an opportunity to design pH-sensitive drug delivery systems that can target tumors and release loaded drugs at the tumor site [43, 44]. Much effort is being devoted to biodegradable polymeric micelles because their degradability prevents micelle accumulation that can result in toxicity in the long term. The employment of pH-responsive micelles is another approach because the polymers can be triggered and destabilized in response to the slightly acidic tumor microenvironment, enabling rapid release of active agents upon arrival at the desired site and simplifying renal clearance. In such way, we recently devised block copolymers to produce nanoparticles comprising a PDPA (poly[2-diisopropylaminoethyl methacrylate]) core (Figure 8.4). PDPA is a promising smart material for the construction of tumor-targeting drug delivery polymeric nanocarriers because it is able to encapsu- late hydrophobic anticancer drugs and it undergoes a sharp hydrophobic-hydrophilic pH-induced transition within a pH range that is desirable for tumor-targeting drug delivery [45]. PDPA is one of the few polymers that can quickly release hydrophobic guest molecules at specific tumor sites via a pH-triggered pathway at slightly acidic conditions [46, 47].
k k PEG-b-PDPA
hydrophobic drug
O m
b
O O
N nBr
PDPA
Figure 8.4 Schematic representation of a pH-responsive PEG-b-PDPA drug-loaded block copolymer micelle and the molecular structure of the block copolymer PEG-b-PDPA.
The block copolymer poly(ethylene oxide)-b-poly(glycerol monomethacrylate)-b- poly[2-diisopropylaminoethyl methacrylate] self-assembles in a phosphate buffered saline (PBS) into highly regular spherical micelles. The micellar size (diameter∼42 nm) and micellar molecular weight (Mw >100 kDa) are in the range intended to avoid renal clearance in order to provide for a long blood circulation time. Their size is below the cutoff size of the leaky pathological vasculature (d < 200 nm), making them candidates for the use in cancer therapy based on the EPR effect.
The pH-responsive PDPA core could be loaded with the poorly water-soluble anticancer drug paclitaxel (PTX) whose encapsulation efficiency is∼70% and drug loading content∼7%. Release experiments have evidenced that approximately 90%
of the encapsulated PTX is sustained at the PDPA micellar core within the first 9 hours at pH 7.4, while only 18 hours were required for complete drug release at pH 5.0. Thus the micellar dissociation could be triggered at the slightly acidic tumoral extracellular environments. In contact with human plasma or human serum albumin (HSA) diluted in PBS, the micelles are stable for up to 48 hours; long blood circulation time of the nanoparticles at serum environments is a prerequisite for drug delivery applications. Cell viability experiments also demonstrated that such (drug-free) block copolymer micelles are nontoxic.
The stability and interaction of block copolymer micelles with model proteins (e.g., lysozyme or bovine serum albumine, BSA) was studied [48] for micel- lar systems featuring a hydrophobic (poly[2-diisopropylaminoethyl methacrylate]) (PDPA) core and hydrophilic coronas comprising poly(ethylene oxide)/poly(glycerol monomethacrylate) (PEO-b-PG2MA) or poly[2-(methacryloyloxy)ethyl phosphoryl choline] (PMPC). The results revealed that protein size and hydrophilic chain density play important roles in the observed interactions. The nanoparticles proved to be stable, and protein adsorption was prevented at all the investigated protein environments. The successful protein-repellent characteristic of these nanoparticles is attributed to a highly hydrophilic surface chain density (>0.1 chains per nm2) and to the length of the hydrophilic chains.
k k The fouling properties of nanoparticles based on block copolymer of poly(lactic
acid), PLA and poly(𝜀-caprolactone), PCL, have been evaluated [49] by placing them in contact with the model proteins. Block copolymer NPs were produced through the self-assembly of PEO-b-PLA and PEO-b-PCL. This procedure yielded nanosized objects with distinct structural features, depending on the length of the hydrophobic and hydrophilic blocks and the volume ratio. The protein adsorption events were examined in relation to size, chain length, surface curvature, and hydrophilic chain density. Fouling by BSA and lysozyme was considerably reduced as the length of the hydrophilic PEO-stabilizing shell increased. In contrast to the case of hydrophilic polymer-grafted planar surfaces, the hydrophilic chain density did not seem to markedly influence protein fouling. The protein adsorption took place at the outer surface of the NPs, since neither BSA nor lysozyme was able to diffuse within the hydrophilic layer due to geometric restrictions. Protein binding is an exothermic process, and it is modulated mainly by polymer features.
A new biocompatible and biodegradable diblock copolymer [50] that contains a specific acid-labile degradable linkage (acyclic ketal group) between the hydropho- bic poly(𝜀-caprolactone) (PCL) and the hydrophilic poly(ethylene oxide monomethyl ether) (MPEO) block was prepared. Upon dissolution in a mild organic solvent, the MPEO-b-PCL block copolymer self-assembled in water–PBS into regular, spherical, stable nanoparticles. Furthermore, the presence of the acid-labile ketal linker enabled the disassembly of these nanoparticles in a buffer that simulated acidic cytosolic or endosomal conditions in tumor cells. This disassembly led to hydrolysis profiles that resulted in neutral degradation products.
The diblock copolymer monomethoxy-PEG-b-poly(D,L-lactic acid) (MPEG- PDLLA) is used in the approved micellar formulation of the anticancer drug pacli- taxel for the treatment of cancer in clinical studies [1]. In a similar way, paclitaxel- loaded micellar systems prepared from PEG-poly(aspartic acid) (PEG-b-Pas) are used in clinical trial of gastric cancer [1]. Diblock copolymers made of 6-O-methacryloyl-1,2:3,4-di-O-isopropylidene-D-galactopyranose (MAIGal) and 2-dimethylaminoethyl methacrylate (DMAEMA) were used to construct spherical brushes with a crosslinked polystyrene core [53]. The variation of pH makes it possible to reversibly expose to the external medium the inner block attached to the nanoparticle surface. This property makes these nanoparticles a very useful model of a delivery system specifically targeted to the liver for treatment of hepatocellular carcinoma. In the bloodstream at pH∼7.4, the PDMAEMA block is collapsed; the glycopolymer block is then exposed to the outside and is selectively accumulated in liver. After contact with the tumor at pH ∼5, the PDMAEMA block will expand (Figure 8.5), and its newly exposed cationic surface will damage the anionic surface of the cancer cells.
Melatonin-loaded vesicular nanocarriers were prepared [54] by interfacial de- position using a blend of an amphiphilic diblock copolymer, poly(methyl methacry- late)-block-poly(2-dimethylaminoethyl methacrylate), PMMA-b-PDMAEMA, with poly(ε-caprolactone), PCL. Dynamic light scattering showed the nanocarriers to have hydrodynamic radii between 100 and 180 nm, with unimodal particle size distribution for each formulation. The standard TEM for nanocapsules
k k
pH = 7.4 pH = 5.0
Figure 8.5 Response of the MAIGal-b-DMAEMA diblock copolymer attached to the surface of a polystyrene nanoparticle to change of pH.
showed an oily core surrounded by a thin layer composed by PCL/PMMA- b-PDMAEMA. Encapsulation efficiencies of ca. 25% were determined by assaying the nanoparticles using HPLC. Platinum nanoparticles were incorporated into the nanocarrier as evidenced by the TEM, which opens up the possibility for promising applications like monitoring the encapsulated drug in the body.
Polymer vesicles called also polymersomes [55–58] are an important type of self-assembled block copolymer system and hold promise for the development of robust nanocontainers for both hydrophobic and hydrophilic bioactive molecules.
Figure 8.6 schematically shows a polymersome structure where the hydrophilic drug is loaded into the aqueous core of the polymersome and the hydrophobic drug is inserted in the lipophilic interlayer [62].
Block copolymer polymersomes can be also prepared with compartimentalized inner structure that can be considered as a model of more complex biological environment [60–62].