4.425.2.2.3 Polymeric micelles

A micelle is generally defined as a collection of amphiphilic surfactant molecules that spontaneously aggregate in water in a concentration-dependent manner to produce a metastable aggregate. The core of the micelle is hydrophobic and therefore can sequester hydrophobic drugs. Conventional micelles are formed from small surface-active molecules that have a polar head group and a hydrophobic tail (amphiphiles). However, micelles can also be formed from large amphiphilic block copolymers. In aqueous media, amphiphilic block copolymers, which have a large solubility difference between hydrophilic and hydrophobic segments, spontaneously form polymeric micelles with a core–shell structure in which a hydrophobic inner core is surrounded by a hydrophilic shell or corona.¹¹⁴ In contrast to micelles from small surfactant molecules, polymeric micelles are generally more stable and can retain the loaded drug for a prolonged period.^115,116 Biocompatible PEG is often used as the hydrophilic segment to improve water solubility and to provide a ‘stealth’ corona to decrease nonspecific interactions, leading to increased blood circulation time.^117–119 Such polymeric micelles are usually large enough to escape renal excretion (15–30 nm). However, their size is controlled below 100 nm to allow extravasation and tumor accumulation, even though drug loading efficiencies at this size are generally low. These characteristics facilitate tumor accumulation by the EPR effect.¹²⁰

Ethylene Diamine Tetraacetate Acid Tetrasodium Salt (EDTA-4Na)

One example of polymeric micelles used in drug delivery is the anticancer drug doxorubicin (Dox). Dox-conjugated PEG-b-poly(aspartate) (PEG–PAsp) block copolymers spontaneously form polymeric micelles in aqueous media.^121–123 Dox was covalently conjugated to the side chains of the poly(aspartate) (PAsp) segment. This conjugation provided sufficient hydrophobicity in the PAsp segment due to Dox's hydrophobicity, thus forming a stable inner core of polymeric micelles. A Dox molecule can be physically entrapped in the micellar core by hydrophobic interactions. This physically entrapped Dox exerts the major cytotoxic function, while the conjugated Dox works mainly to increase micelle stability.¹²⁴ The PEG–PAsp(Dox) micelle showed prolonged blood circulation times and effective accumulation into a subcutaneous ectopic solid tumor (murine colon adenocarcinoma (C-26)).^125,126

Following the same concept, polymeric micelles were used for gene delivery. A polymeric micelle containing nucleic acids is formed by complexation between DNA or RNA and a block copolymer having a hydrophilic segment and a cationic segment. Complexation of plasmid DNA with PEG–poly(lysine) (PEG–PLys) occurs spontaneously.^127,128 The PEG shell promotes extended systemic circulation and thus tumor accumulation by the alleged EPR effect. Polyplex micelles containing pDNA indeed exhibited efficient gene introduction in cultured cells, achieving prolonged blood circulation.¹²⁹ These results demonstrate that polymeric micelles might also serve as promising gene carriers in gene therapy.

Polymeric micelles can be regarded as a first-generation system, as they are mostly monofunctional.¹³⁰ Supramolecular multifunctional nanodevices prepared with smart multifunctional polymeric micelles designed with stimuli-responsive properties may provide promising systems with increased targeting ability and reduced toxicity.¹³⁰ For example, active targeting using specific ligands can increase gene delivery efficiency and reduce side effects. Antibodies and their fragments, transferrin, folate, sugars, and peptides are commonly used to achieve active tumor site targeting.¹³¹ One example of such targeted smart nanocarriers is the polymeric micelle encapsulating pDNA targeted to hepatocytes bearing abundant ASGP receptors bind lactose moieties.¹³² The transfection of these micelles in HepG2 cells showed that lactose-bearing micelles were significantly more efficient at transfection than micelles lacking lactose.¹³³ Another example involves targeting vascular cells near tumors to inhibit formation of new blood vessels, thus cutting off the nutritional supply to cancer cells. The cyclic RGD peptide (c(RGDfK)), which specifically target integrin receptors on vascular cells, was conjugated to PEG–PLys block copolymers,¹³⁴ showing increased transfection efficiency in vitro compared to ligand-free polyplex micelles.

A second approach to prepare smart multifunctional polymeric micelles involves the use of stimuli-responsive biodegradable micelles. Nanocarriers must be sufficiently stable to resist extracellular degradation and dissociation. However, they must also release their nucleic acid payload near their intended site of action. One class relies on acid-dependent degradation of polymeric micelles. Nanocarriers internalized through endocytosis experience a pH drop in endosomes. Thus, drug carriers with pH-sensitive linkages based on hydrazone, cis-aconityl, or acetal groups that degrade at low pH can be prepared. Such carriers are stable at physiologic pH but degrade under acidic conditions inside cells. As an example of acid-sensitive micelles, a triblock copolymer was designed for micelle formation by two cationic segments in a single polymer strand.¹³⁵ In this triblock copolymer, each segment has its own distinctive role: a PEG segment for biocompatibility, a second segment composed of low pK_a amines (poly[(3-morpholinopropyl) aspartamide] (PMPA)) for buffering capacity to enhance endosomal escape, and a third segment composed of high pK_a amines (PLys segment) for DNA payload binding. The transfection activity of the triblock system exhibited one order of magnitude higher transfection compared with the PEG–PLys block copolymer with similar PLys unit length. This transfection efficiency is comparable with that of the well-known PEI/pDNA polyplex, but with a remarkably lower cytotoxicity.

To find a suitable cationic segment, PEG block copolymers with a series of cationic side chains were synthesized. A polymer library was prepared, from which it was found that PEG–polyaspartamide with an ethylenediamine unit as a side chain (PEG–PAsp(DET)) had high transfection efficiency as well as low cytotoxicity.¹³⁶ This polymeric micelle provided gene expression in primary cells as well as in in vivo disease models with low toxicity. The polyplex micelle prepared with PEG–PAsp(DET) and pDNA was also applied to vascular lesions with good results.¹³⁷ The micelle was instilled intravascularly into rabbit carotid arteries with neointima, exhibiting appreciable reporter gene transfer into vascular lesions without any vessel occlusion by thrombus. The PEG–PAsp(DET) polyplex system was also applied for in vivo gene transfer in a bone-defect model of mouse skull bone.¹³⁸ The polyplex micelle was mixed with a calcium phosphate cement scaffold and installed into the bone defect. Sustained release of PEG–PAsp(DET) from the scaffold and transfection of surrounding cells were observed. Transfection of constitutively active activin receptor-like kinase 6 (caALK6) and runt-related transcription factor 2 (Runx2) induced osteogenic differentiation in mouse calvarial cells with no signs of inflammation.

In a similar way, stimuli-responsive smart nanocarriers can be prepared with a disulfide bond, which is known to be stable in the extracellular environment yet readily cleaved inside the cell due to the increased concentrations of glutathione. In this regard, bioresponsive smart polymeric micelles that dissociate inside cells in response to chemical stimuli were designed. The inner core of the micelle was cross-linked through disulfide bonds for cleavage inside the cell, increasing the stability of the micelle. At the same time, the efficient release of packaged pDNA was demonstrated in response to reducing reagent (dithiothreitol), mimicking the intracellular environment.¹³⁹ Gene transfection was higher by an order of magnitude than that of the non-cross-linked system, with the cross-linked micelle exhibited appreciable gene expression in parenchymal cells of the mouse liver through intravenous injection.¹⁴⁰ The disulfide cross-linking micelle maintained the original transfection capacity even after freeze–thaw treatment without the use of any protective reagents, while the non-cross-linked micelles showed significantly lower transfection efficiency after the same treatment.