Micellar System - an overview (2024)

D Which formulation principles are used?

At early stages stable solutions are preferred because of their defined physical state assumed as to be monomolecularly dispersed systems.

Simple micellar dispersed solutions are also directly usable by the experimenter.

Complex micellar systems like microemulsions or special solid systems (suspensions or solid dispersions) usually require a basic pharmaceutical development strategy and sufficient understanding of the technical concept to ensure appropriate quality and reproducibility of the formulation. These systems can usually be properly elaborated only at later development stages because of a lack of sufficient drug substance at early stages!

In early preclinical drug testing, it is preferable to use excipients that can also be used in later development phases, including clinical development. Whenever possible, the same formulation principle should be used throughout preclinical testing. This applies to all studies in experimental animals including pharmacological, toxicological and pharmaco*kinetic studies.

3.4 The multifunctional design of the micelle nanomedicine platform for cancer

Multifunctional micelles made up of PEO-b-PCL block copolymers were investigated with useful modifications on all the blocks [4]. They could codeliver siRNA and doxorubicin via passive/receptor-mediated targeting to the cellular component and give a pH-sensitive drug release in the endosomes. The PCL core contains short polyamines (spermine) to conjugate MDR1 siRNA, and conjugates doxorubicin through a pH-sensitive hydrazone bond. Fluorescent imaging probes were conjugated to trace the micelles in vitro and in vivo. An active targeting moiety, RGD4C unique for integrin (αvβ3) receptors, and a cell-penetrating TAT-peptide encouraged passive targeting [17].

Bay and coworkers investigated the application of multifunctional polymer micelles composed of PEG-p(Asp-Hyd-ADR). These micelles showed folate receptor-mediated targeted delivery and pH-responsive doxorubicin release. This multifunctional platform was found to be efficient (approximately 10-fold post 24hours exposure) for the treatment of KB cells [46].

Nasongkla et al. reported multifunctional cRGD-DOXSPIO micelles. They showed effective tumor targeting ability and theranostic efficacy [47].

A recent study reported multifunctional pluronic P123/F127 mixed micelles in a photodynamic therapy against PC3 and MCF-7 cancer cells [48].

8.6.1 pH-Responsive Micelles

The concept of using pH as a means of cellular delivery possibly originates from the observation that certain enveloped viruses developed strategies to make use of the acidification of the endosomal lumen to infect the host cells (Miyauchi et al., 2011). Although the human body maintains a steady pH of 7.4 in case of most fluid compartments, nevertheless, both at the macro (e.g., various segments of the gastrointestinal tract) and at the micro levels (e.g. lysosome), variations do exist. Several pathological conditions also contribute to this natural variation in pH. For example, the intra-tumoral environment has a more acidic pH because of the lactic acid production, owing to the under-perfused and hypoxic environment within the tumor. Similarly, pH within inflamed tissue is also found to be lowered, as in the case of joints affected by rheumatoid arthritis (Farr et al., 1985). Nanocarriers also encounter a cascade of declining pH values along their journey from tissue interstitium to cytoplasm (early endosome > late endosome > lysosome). In the case of the above situations, a pH or proton concentration-triggered drug release can serve as an effective way of enabling higher drug accumulation at the disease site.

Depending on ionizable groups employed to impart pH-responsiveness, amphiphilic polymers of this category may be either anionic or cationic. Polymers bearing pendant acidic functional groups protonate at low pH, yielding neutral molecules that are relatively hydrophobic. At neutral or high pH, the molecules can release protons and become hydrophilic. Such polymers include polyacrylic acid, polymethacrylic acid, polyglutamic acid, and those containing polysulfonamide groups.

For drug targeting, with due regard to the pathological conditions existing within the tumor microenvironment, the more useful pH-sensitive polymers are those bearing cationic groups, since these would tend to become protonated to the charged species at lower pH. Examples of such polymers include poly(N,N′-dimethyl aminoethyl methacrylate) (PDMAEMA), poly(4-vinylpyridine), poly(histidine), and poly(β-amino ester) (PAE), of which the latter two are of major interest owing to their biocompatibility. pH-sensitive PEG–PAE-based amphiphiles can be synthesized via a Michael addition reaction between monoacrylated PEG, 1,6-hexane diol diacrylate, and 4,4′-trimethylene dipiperidine (Gao et al., 2010a). Micelles based on these amphiphiles have been investigated for diversified applications, including delivery of doxorubicin to cancerous tissue (Wu et al., 2010), imaging agents like ferric oxide for magnetic resonance imaging (MRI) of acidic ischemic regions in the brain (Gao et al., 2011), tetramethyl rhodamine for imparting discernible fluorescence to the tumor site (Min et al., 2010), and photosensitive agents such as the hydrophobic protoporphyrin (PpIX) for tumor-targeted theranostic applications (Koo et al., 2010).

Similarly, micelles based on amphiphilic polymer composed of pH-sensitive poly(histidine) as hydrophobic block and PEG as hydrophilic block have been probed for targeted delivery. Copolymerization of histidine (His) with another amino acid l-phenylalanine (Phe) is reported to reduce the pKa of resulting PEG-b-PHis/Phe based micelles (Kim et al., 2005). Triblock copolymer composed of poly(l-lactic acid) (PLLA)-b-PEG-b-pHis is reported to form micelles at physiological pH; however in acidic pH (6.6), the micelles swell to release the loaded doxorubicin (Liu et al., 2011). The addition of a hydrophobic PLLA segment stabilizes the micellar state (Lee et al., 2007).

pH sensitivity can also be imparted by tethering appropriate pendant groups to the amphiphile backbone. Micelles were designed using di-block copolymer of poly(ethylene glycol) and poly(γ-benzyl l-glutamate), with N,N-diisopropyl ethylenediamine (DIP) conjugated to poly(l-glutamic acid) (PGA) blocks as side groups. N,N-diisopropyl ethylenediamine enhances the pH-sensitivity of poly(ethylene glycol)-block-poly[N-(N′,N′-diisopropylaminoethyl) glutamide]. This PEG-PGA(DIP) polymer can self-assemble into micelles with an acid-responsive PGA-DIP core and hydrophilic PEG corona. The authors have reported faster acid response with increased grafting percentage of DIP. In acidic condition, the hydrophobic block PGA (DIP) transforms to a hydrophilic block, leading to a gradual expansion and disassembly of micellar structure, resulting in the release of entrapped doxorubicin (Li et al., 2016).

The complexity of the disease condition prompts the need for more effective control on drug release to enhance therapeutic outcome. Therefore, besides the pH-based systems discussed, literature also abounds in studies where dual, or sometimes multiple, approaches are simultaneously explored to achieve greater drug targeting potential of the developed system(s); pH change is also sometimes an arm of this multi-pronged approach. Tian K et al. developed a multi-responsive (pH-sensitive, reduction-sensitive and folic acid-mediated targeting) micelles for targeted delivery of anticancer drug doxorubicin. Here, a double end functionalized PEG with folic acid as one end group served as the hydrophilic part to which a hydrophobic block of t-butyl acrylate was added via atom transfer radical polymerization. A definite proportion of N, N’-bis (acryloyl) cystamine was incorporated in the hydrophobic block to enable the core to undergo disulfide-mediated crosslinking and consequent stabilization. The core cross-linked micelles were finally subjected to hydrolysis for removal of t-butyl group leaving the acrylic acid groups, which further conferred pH sensitivity in the core. The micelles loaded with doxorubicin exhibited only 13.4% premature drug release in 36 hours, while release in media stimulating a tumor microenvironment: acidic and high Glutathione (GSH) level; was observed to be 78.7% (Tian et al., 2016a). In vitro studies in ovarian carcinoma cell line SKOV3 demonstrated the ability of the micelles to accumulate doxorubicin into the cell nuclei.

Mixed micelles, composed of (PEG-poly(d,l-lactic acid) block copolymer) bearing a tumor-targeting peptide as one component and the other comprising of pH-sensitive methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE) have been prepared. Both tetramethylrhodamine (TRITC) as fluorescent marker and doxorubicin as chemotherapeutic agent were separately entrapped in the micelles. In vivo studies in tumor-bearing mice demonstrated excellent anticancer efficacy, compared to free doxorubicin and doxorubicin-encapsulated MPEG-PAE micelles (without targeting ligand) which provides further justification for the use of multiple strategies in achieving selective tumor targeting (Wu et al., 2010).

Besides the approaches outlined above, change in pH as a trigger to bring about other changes in the micelle properties have also been explored. e.g., for removal of the PEG segment from the micelles. PEGylation offers advantages of longer systemic circulation (avoid RES uptake) and selective accumulation at tumor site by EPR effect. However, its utility is rendered limited by recent reports that PEGylation inhibits cellular internalization and subsequent endosomal escape (Hatakeyama et al., 2013). Therefore, attempts are in progress to design “sheddable” micelles by conjugating PEG in the polymer through a pH-sensitive linker. Zhao and coworkers conjugated phosphatidylethanolamine with PEG using benzoic imine. The PEG chains offer the steric hindrance and protect micelles at physiological pH, whereas disassembly of the PEG chain occurs in an acidic microenvironment of lysosomes after intracellular uptake. Although complete removal of PEG was not observed in extracellular pH, in vitro cell uptake was significantly higher when incubated at pH 6.8, compared to pH 7.4 (Zhao et al., 2016).

6.8.8 Stability of polymeric micelle

Micelle stability is indicated by thermodynamic and kinetic stability. The micellar system can be considered thermodynamically stable if the amount of polymer in the aqueous solution is above the CMC. As the micelle is administered i.v., the system undergoes extreme dilution; hence, it is necessary to know the CMC of the system. It is necessary that the system remains in the micellar form in the systemic circulation until it reaches the targeted site. Kinetic stability is determined by the exchange rate of the polymer chain between the micelle and the bulk. Kinetic stability is important to predict the release of the physical entrapped drug from the polymeric micelle. Block copolymers are found to produce a micellar system which is kinetically stable because of the presence of more number of the hydrophobic site available for interaction. Lack of kinetic stability may lead to leakage of the drug from the micellar system in the systemic circulation before reaching the target site. Hence to enhance both the thermodynamic and kinetic stability, several strategies have been employed, such as strategies to reduce the CMC, cross-linking in the core, and formation of heterocomplex micelles. Aromatic groups have been found to decrease the CMC and also help to strengthen the interactions leading to increased stability of the micelle (Kulthe et al., 2012).

Renneting Properties

The renneting properties of sheeps' milk are affected by a number of factors such as pH, physicochemical composition, micellar system, salts equilibrium, calcium concentration, and the temperature and time of heating. Sheeps' milk coagulates well and is indeed very suitable for making high-quality cheeses. It coagulates faster than cows' milk, and less rennet is needed to coagulate it in the same time as cows' milk. Curd formation is also faster than in cows' milk, but syneresis takes longer. The reason for this difference may be that sheeps' milk contains more casein, ionic calcium, and colloidal calcium than cows' milk. The renneting time and rate of firming are practically unaffected by the addition of calcium.

Renneting Properties

The renneting properties of sheep’s milk are affected by a number of factors such as pH, physicochemical composition, micellar system, salts equilibria, calcium concentration, temperature and time of heating. Sheep’s milk coagulates well and is indeed very suitable for making high-quality cheeses. Coagulation is faster than in cow’s milk, and less rennet is needed to coagulate in the same time as for cow’s milk. Curd formation is also faster than in cow’s milk, but syneresis takes longer. The reason for this difference may be that sheep’s milk contains more casein, ionic calcium, and colloidal calcium than those contained in cow’s milk. Renneting time and rate of firming are practically unaffected by the addition of calcium.

The coagulation properties of sheep’s milk are less affected than those of cow’s milk by storage at 4°C or by heat treatment. In fact, although severe treatments (90°C, 10min) delay coagulation of sheep’s milk, gelation does take place. In cow’s milk, on the other hand, heating for 1min at 90°C prevents gelation. The reason for this difference in the behavior of cow’s and sheep’s milks could be that in the latter, when heated to 85 or 90°C, the micelles grow considerably in size, attaining 1.75 times the size of the micelles in fresh milk.

2 Enzymatic Considerations for Kinetic Analysis of Proteolysis in Detergent Micelle Systems

In addition to the inherent properties that surfactants impose, intramembrane proteases behave differently in detergent vs membrane systems: GlpG, for example, generates incorrect cleavage sites in substrates, has a 10-fold higher turnover constant (k_cat), and even cleaves some nonsubstrates in detergent systems (Dickey et al., 2013; Moin & Urban, 2012). This is because the membrane environment plays an active role in “tempering” enzyme and substrate dynamics, while both are radically changed in detergent systems (Moin & Urban, 2012). Inhibitor accessibility to the active site is also altered in detergent vs membrane systems. For example, the small-molecule inhibitor JLK6 (7-amino-4-chloro-3-methoxy-isocoumarin) has a >10-fold more potent EC₅₀ in detergent vs membrane with exactly the same substrate/enzyme (Fig. 6A). Finally, most medically relevant rhomboid proteases are inactive in detergent systems; the only eukaryotic rhomboid protease that has been characterized biochemically, Drosophila melanogaster Rhomboid-4 (Baker & Urban, 2015), absolutely requires reconstitution for activity. Ascribing enzymatic properties to rhomboid proteases as a family based on analysis of a minority that remains active in detergents introduces a serious sampling bias.

10.6.4.2.7.2 Thermo-sensitive micelles

Thermo-sensitive polymeric micelles are prepared in order to achieve temporal drug delivery control through variations in the temperature of the environment slightly below or above the lower critical solution temperature. This leads to the micellar system destabilization and triggers a burst-like release of the loaded drugs. Hyperthermia is used for this purpose in the case of targeted drug delivery to tumors. Poly(N-isopropylacrylamide) is the most widely used thermo-sensitive polymer. It exhibits lower critical solution temperature at 33°C in aqueous solution, below which it is water soluble.

Changes in temperature occur either due to internal triggers like hyperthermia in inflammation, or can be applied externally. When ultrasound or high frequency is applied locally, the generation of heat takes place and results in the oscillation of target-accumulated magneto-sensitive micelles. Poly(N-isopropylacrylamide-co-acrylamide)-b-poly(d,l-lactide) copolymer micelles have been used for tumor targeting of the drug docetaxel (Mourya et al., 2011). It was found that hyperthermia increased the targeting efficiency of drug-loaded micelles with their reduced toxicity.

8.7.1 Phospholipid Stability

Membranes in liposomes are primarily constituted of phospholipids, and these phospholipids consist of ester bonds, which are sensitive to hydrolysis [122]. The organization of the lipid assembly can change from lamellar to a micellar system because of the chemical hydrolysis of the liposomal phospholipids [123]. Lysophosphatidylcholine and fatty acids are formed [124] and membrane permeability is increased [125] when these transformations occur. The peroxidation of unsaturated acyl chains is generally accompanied by phospholipid degradation [125]. The permeability of the bilayer was increased by the lipid peroxidation. The degradation process resulted in a number of products with highly different chemical natures [122,125]. As a result of these degradation processes, phospholipid use in such formulations is very limited and is currently substituted with nonionic surfactants to circumvent degradation problems [125].

18.2.3.2 Bile salt-phospholipid mixed micelles

When combined with polar lipids, traditional surfactants, or amphiphilic medicines, bile acids can form mixed micelles with lower critical micelle concentrations and greater solubility than the individual entities. There is also the option of making mixed micellar systems without the use of nonpolar solvents that are often used in pharmaceutical formulations to improve or increase the bioavailability of poorly soluble drugs.^34,35 A phospholipid like lecithin can also buffer bile salt detergent characteristics and reduce their cytotoxic properties by forming mixed micelles. a phospholipid like lecithin can also neutralize bile salt detergent qualities and minimize potentially harmful effects by generating mixed micelles. Combination micelles of glycocholic acid and soy lecithin are utilized as a solubilization agent for diazepam, a hydrophobic benzodiazepine.³⁶ Amphotericin B is available as a micellar suspension with sodium deoxycholate for parenteral administration.³⁷