ALEJANDRO SOSNIK
5.3 Temperature-sensitive Polymeric Micelles
5.3.3 Poly(N-isopropylacrylamide)
Together with PEO-PPOs, poly(isopropylacrylamide) (pNIPAAm) is one of the most popular thermo-responsive smart materials (Scheme 5.1C). From the reports by Tanaka in the late 1970s,185a broad spectrum of applications have been described for pristine pNIPAAm and its copolymers, such as diagnostics, separative chemistry, biosensors and drug delivery.186Another application of pNIPAAm gels is the engineering of cell sheets developed by Okano and collaborators,187which is covered in Chapter 23.
PNIPAAm displays a LCST around 32–331C in water, thus it is water soluble below this temperature and water insoluble above it. The mechanism involves the generation of hydrophobic interactions upon heating. This property was capitalized to develop polymeric micelles made of pNIPAAm as the hydrophilic component and different hydrophobic ones.188 The most common hydrophobic blocks were polystyrene,189dimethylacrylamide,190alkyl residues,191 PLA,192 butylmethacrylate,193 poly(N-vinylimidazole),194 acryl- amide195 and methylmethacrylate.196 In addition, pNIPAAm has been
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copolymerized with hydrophilic monomers to fine-tune the self-aggregation properties and the thermal responsiveness.197
The corona is highly hydrated below the LCST and it undergoes dehydration and shrinkage above it. Thus, these micelles can be accumulated in tumors by EPR and the heating and consequent shrinkage promote the fast release of the encapsulated drug in the target tissue/organ (Figure 5.3).193The LCST can be fine-tuned to be slightly higher than 371C and to release the drug only under hyperthermia.192,198 For example, Liu et al.195 developed a p(NIPAAm- co-acrylamide)-b-PLA copolymer with LCST of 411C for the encapsulation and thermal release of docetaxel.In vitro assays indicated that under hyper- thermia, micelles were more cytotoxic to different tumor cell lines and less toxic to human umbilical endothelial cells than a standard formulation, indicating the accelerated release of the encapsulated drug. Docetaxel-loaded micelles were more effectivein vivo.199,200When the temperature-dependent transition of NIPAAm is above 371C, triggering of drug releasein vivorequires the use of an external physical stimulus such as ultrasound.
One of the most appealing characteristics of pNIPAAm is the great chemical versatility to tailor novel copolymers with tunable self-assembly, drug encapsulation and release performance. The synthesis of random and block copolymers employing different modalities of free-radical polymerization with chain transfer agents, polycondensation and ring-opening polymerization Figure 5.2 (A) 3-in-1 PEG-PLA micelles containing paclitaxel/17-allylamino- 17-demethoxygeldanamycin/rapamycin and PEG-PCL micelles containing a carbocyanine dye. (B) Scheme of the tumor-primed delivery of the carbocyanine dye.
(Reproduced from Ref. 184 with permission of the American Chemical Society.)
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enabled the exquisite control of the molecular architecture, the copolymer composition and block arrangement and the physico-chemical properties. For example, the group of Leroux copolymerized NIPAAm, methacrylic acid (MAA) and octadecyl acrylate (ODA).201 NIPAAm and MAA formed the corona, while ODA formed the core. The incorporation of MAA pH-sensitive blocks resulted in dually responsive systems. Liu et al.202 synthesized pNIPAAm copolymers with poly(lactide-co-glycolide) (PLGA) blocks of different lengths for the encapsulation of doxorubicin. The drug-loading capacity increased with longer PLGA blocks. Interestingly, micelles were stable at 371C though they underwent deformation above body temperature leading to the accelerated release of doxorubicin. The synthesis and self- assembly of amphiphiles with more complex architectures such as poly(benzyl ether)-b-pNIPAAm dendritic linear diblocks displaying two collapse stages has also been reported.203
To overcome the physical instability of polymeric micelles, Wei et al.196 incorporated a small concentration of 3-(trimethoxysilyl)propyl methacrylate to pNIPAAm micelles in order to cross-link the corona by means of the sol-gel technology. Cross-linked micelles displayed greater encapsulation efficiency and were more physically stable than the non-cross-linked counterparts because they did not re-aggregate upon heating, a behavior that led to a sharp size growth of the non-cross-linked system. In addition, the release rate of encapsulated prednisone was 20 times slower above the LCST. In a more recent work, the same authors decorated the surface with biotin to confer the micelle pretargeting properties in cancer.204 Similarly, micelles were conjugated with folate for intra-cellular delivery of antitumorals in cells expressing the folate receptor.205
The modification of the precursors and the synthetic pathways results in copolymers with different organization of the blocks along the backbone. In this context, molecules where pNIPAAm is flanked by two terminal hydro- phobic arms of PCL or PMMA were also synthesized.206,207These copolymers formed ‘‘flower-like’’ micelles. When NIPAAm and e-caprolactone were Figure 5.3 Temperature-modulated drug release and interactions between micelles
with pNIPAAm as shell-forming segments and cells.
(Reproduced from Ref. 193 with permission of Elsevier.)
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reacted via an atom transfer radical polymerization instead of ring opening polymerization, copolymers having a pNIPAAm-PCL-pNIPAAm structure that self-assembled into regular polymeric micelles.208 Other groups designed copolymers having more complex molecular arrangements such as graft209,210 and Y-211,212and star-shaped micelles, each system displaying advantages and drawbacks.213,214For example, Liuet al.215synthesized two pNIPAAM graft copolymers with negatively and positively charged backbones made of methacrylic acid and fully quaternized 2-(dimethylamino)ethyl methacrylate, respectively. In water, electrostatic interaction between both copolymers led to the generation of micelles with a polyion complex core and a thermo-responsive corona. The incorporation of azide moieties along the main chain allowed the cross-linking of the core to stabilize the micelles physically. PNIPAAm copolymers were also co-micellized with other types of amphiphiles to produce mixed micelles combining improved physical stability, targeting properties and imaging features.216–218 Findings suggest that when engineered well, these micelles could be used in diagnosis, targeting and therapy.
As mentioned above, different hydrophobic blocks have been combined with PEG to produce self-assembly amphiphiles. Since pNIPAAm turns from hydrophilic at low temperature to hydrophobic above the LCST, the potential application of PEG-pNIPAAm copolymers has also been explored.219–222 In general, most of these novel systems capitalized on the same pNIPAAm thermal transition to adjust the release rate of the encapsulated drug, and they have been evaluated in vitroandin vivo. Even if these works provide further evidence of the versatility of these biomaterials, the use of pNIPAAm and other non-biodegradable polymers by the parenteral route (e.g. intravenous) should be appropriately pondered, becausein vitro cytotoxicity studies are certainly insufficient to ensure their biocompatibility and to demonstrate elimination from the body. In general, these polymers are eliminated by renal filtration as previously described for PEG.72–74
Recently, Bertrand et al.223 investigated for the first time the fate of pNIPAAm polymers in vivo after intravenous administration. The effect of three key molecular properties, (i) molecular weight, (ii) amphiphilicity and (iii) LCST, on the elimination, the biodistribution and the accumulation was assessed (Table 5.1). In general, the greater the molecular weight, the longer the circulation time is; e.g. 30% of the administered dose of a copolymer of molecular weight 40 kDa was in the systemic circulation after 48 h (Figure 5.4).223 Conversely, the fastest clearance from the bloodstream was observed for copolymers of (i) very low molecular weight (P-5k-NA, 5 kDa) that did not aggregate or (ii) high molecular weight that were insoluble in water (P-40k-L) at 371C, the former showing the greatest accumulation in the extravascular space. Copolymers of low molecular weight that formed micelles (P-5k) or of intermediate molecular weight showed intermediate elimination rates. In addition, findings indicated that the glomerular filtration cut-off of pNIPAAm molecules would be approximately 32 kDa; this value being slightly greater than that established for PEG.72–74LCST and HLB also affected the process. Nevertheless, data were not always consistent and Temperature- and pH-sensitive Polymeric Micelles for Drug Encapsulation 131
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clearance from the bloodstream did not necessarily mean elimination from the body. These aspects are very relevant, especially for systems envisioned for multiple administrations and where toxicity due to accumulation could take place. For an extensive overview of the most relevant developments in pNIPAAm micelles, readers are referred to the review of Weiet al.188