PIPAAm

We have constructed thermoresponsive systems utillizing semitelechelic PIPAAm chains with freely mobile ends, synthesized by telomerization using telogens as a following reaction [6] nM -i- HS-X H-(M)n-S-X. 

Telomerization was effective to regulate quantitative incorporation of functional groups to one end of PIPAAm chains [7,8]. Molecular weight of the semitelechilic PIPAAm determined from GPC data was in good agreement with that determined by the end-group assay. This indicates that each macromolecule carries one amino or carboxyl end group. 

We have utilized thermoresponsive properties of PIPAAm and its gels as on-off switches for drug release [6,7], chromatography systems [9-11], and attachment/detachment of cells [12-14] (Scheme 1). Hydrophobic chains of collapsed PIPAAm above its LCST interact with cells and proteins. Although below the LCST, PIPAAms are highly hydrated flexible chains and... 

SYNTHESIS OF A-B BLOCK COPOLYMERS OF PIPAAm WITH VARIOUS HYDROPHOBIC SEGMENTS... 

Stearoyl-terminated PIPAAms (PIPAAm-CigH35) was obtained by the reaction of the primary amino end group of PIPAAm-NH2 with a large excess of acyl chlorides [23]. A block copolymer of PIPAAm and poly(styrene) (PIPAAm-PSt) was obtained by a condensation reaction... 

MICELLE FORMATION FROM PIPAAm BLOCK COPOLYMERS... 

Micelle solutions of PlPAAm-Ci8H35 was prepared by direct dissolution of the polymer in cold water (4°C) due to its good water solubility [23]. Each solution of PIPAAm-PSt, PlPAAm-PBMA, and PIPAAm-PLA was prepared by dissolving each copolymer in DMF, A-ethylacetamide, and DMAc, respectively. The solutions were put into a dialysis bag (MWCO = 13,000) and dialyzed against distilled water at 10°C, 20°C, and 4°C, respectively, for 24 hours. The micelles were purified with ultrafiltration membrane of 200,000 molecular weight cut off at 4°C. The aqueous solution was lyophilized to leave a white powder of micelles. 

In general, incorporation of hydrophobic groups into PIPAAm chains decreases the LCST [29-31]. Hydrophobic groups alter the hydrophilic/ hydrophobic balance in PIPAAm, promoting a PIPAAm phase transition at the LCST, water clusters around the hydrophobic segments are excluded from the hydrophobicaUy aggregated inner core. The resulting isolated hydrophobic micellar core does not directly interfere with outer shell PIPAAm chain dynamics in aqueous media. The PIPAAm chains of the micellar outer shell therefore remain as mobile linear chains in this core-shell micellar structure. As a result, the thermoresponsive properties of PIPAAm in the outer PIPAAm chains of this structure are unaltered [23-27,32]. 

Polymeric micelles with selected chemistries and molecular architecture of block copolymers, such as PIPAAm-CigHgs, PIPAAm-PSt, PIPAAm-PBMA, and PIPAAm-PLA micelles, showed the same LCST and the same thermoreponsive phase transition kinetics as those for PIPAAm irrespective of the hydrophobic segment incorporation. This confirms two points (a) that hydroxyl groups or amino goups of PIPAAm termini completely react with the hydrophobic segment end groups and (b) that the block copolymers form core-shell micellar structures with hydrophobic iimer cores completely isolated from the aqueous phase. 

Figure 1 Representative emission spectra for PC3P in alkyl-terminated PIPAAm aqueous solutions above the CMC (20,000 mg/L). = 333 nm, [PC3P] = 2.2 x 10 M, 20°C (Reference [23], p. 37).

Figure 2 Plot of the ratio of intensities (/1//3) of the vibrational bands in the pyrene fluoreseenee speetrum as a funetion of temperature for PIPAAm (a), PIPAAm-CigHjs and PIPAAm-PBMA (b), = 340 nm, [pyrene] = 1.6 x 10 M, l°C/min, [polymer] = 5,000 mg/L.

ADRIAMYCIN (ADR) INCORPORATION INTO PIPAAm-PBMA MICELLES... 

PIPAAm-PBMA block copolymers form a micellar structures by selfassociation of the hydrophobic PBMA segments in water, a good solvent for PlPAAm chains below the LCST but a nonsolvent for the PBMA chains. This amphiphilic system produces stable and monodispersed micelles from polymer/A-ethylacetamide (good solvent for the both polymer blocks) solutions dialyzed against water. Hydrophobic dmgs can be physically incorporated into the iimer micelle cores with PBMA chains by hydrophobic interactions between the hydrophobic segments and dmgs. 

Figure 4 Drug (ADR) release from thermoresponsive PIPAAm-PBMA micelles containing ADR [27]. 

Shown in Figure 6 is in vitro cytotoxic activity of PIPAAm-PBMA micelles loaded with ADR or micelles without ADR at 29°C (below the LCST) and at 37°C (above the LCST) compared with that of free ADR. In vitro cytotoxic activity was measured using bovine aorta endothelial cells. Bovine aortic endothelial cells were obtained as previously reported using dispase for cell dissociation from freshly harvested bovine aorta [13]. The cells plated at a density of 3 x cells/well, were exposed with free ADR or micelles loaded with ADR at below and above the LCST for 5 days. In order to assay cytotoxicity of the free ADR or micelles loaded with ADR, culture medium was replaced with 10% FBS-supplemented phenol red-free DMEM containing 10% alamar Blue, a dye that is subject to reduction by cytochrome c activity and changes the color from blue to red [38]. After 4-hour incubation, reduction of the dye was estimated by absorbance at 560 and 600 nm. PIPAAm-PBMA polymeric micelles loaded with ADR showed higher cytotoxic activity than that of free ADR at 37°C (above the LCST)... 

Figure 6 In vitro cytotoxicity of free ADR (0.1 jxg/mL) and thermoresponsive PIPAAm-PBMA micelles containing ADR (0.1 jxg/mL) against bovine aorta endodieUal cells at 29°C (below the LCST) and 37°C (above the LCST). Incubation time 4 days.

Thermoresponsive polymeric micelles with PIPAAm block copolymers can be expected to combine passive spatial targeting specificity with a stimuli-responsive targeting mechanism. We have developed LCSTs of PIPAAm chains with preservation of the thermoresponsive properties such as a phase transition rate by copolymerization with hydrophobic or hydrophilic comonomers into PIPAAm main chains. Micellar outer shell chains with the LCSTs adjusted between body temperature and hyperthermic temperature can play a dual role in micelle stabilization at a body temperature due to their hydrophilicity and initiation of drug release by hyperthermia resulting from outer shell structural deformation. Simultaneously, micelle interactions with cells could be enhanced at heated sites due... 

---