Yi-Fong Huang

Polymer Vesicles Containing Small Vesicles within Interior Aqueous Compartments and pH-Responsive Transmembrane Channels†

Intermolecular packing of amphiphilic block copolymers into vesicles is of particular interest, owing to the fundamental importance of such systems as a new class of polymer assemblies with well-controlled structures and potential biomedical applications. Similar to conventional liposomes, polymer vesicles usually form a continuous bilayer structure primarily consisting of the hydrophobic blocks of copolymers, but they exhibit markedly enhanced stability and feasibility of incorporating functional groups in response to external stimuli. However, the major limitation of polymer vesicles as biofunctional containers arises from the lack of permeation pathway for hydrophilic cargoes owing to the requirement to maintain the architectural integrity. The vesicles obtained from block co-polypeptides are imparted responsive channels upon the pH-induced conformational change of a polypeptide block. Redox control of the permeability of multilayer microcapsules containing poly(ferrocenylsiliane) was reported. Incorporating channelforming proteins into the vesicle membranes while fully retaining the protein functions represents an important paradigm of equipping polymer vesicles with transmembrane channels. Thus, the transport mechanism, being either size-selective or substrate-specific, can be tailored by the pore proteins selected. It is also desirable to have versatile vesicular assemblies that contain small vesicles within the interior aqueous compartments in a manner similar to discrete organelles within eukaryotic cells, which perform diverse functions and are one of the feature differences from prokaryotic counterparts. Unfortunately, such assembly structural control has not yet been achieved. Herein, we show the first example of polymeric multivesicle assemblies similar to the architectural arrangement of eukaryotic cells, in which both the vesicle membranes are equipped with pH-responsive channels permeable for hydrophilic solutes (Scheme 1). Copolymers comprising acrylic acid (AAc) and acrylate of 1,2-distearoyl-rac-glycerol (distearin acrylate, DSA) were obtained from partial transesterification of poly(N-acryloxysuccinimide) (poly(NAS)) with distearin and then thorough hydrolysis of the unreacted NAS to AAc units. Polymer vesicles were prepared by a double emulsion technique in a water/oil/water (w1/o/w2) system, in which the copolymer was dissolved in the organic phase prior to emulsification. The experimental methods are described in detail in the Supporting Information. THF/CH3Cl solutions of varying ratios, depending on the target vesicle size, were employed as the organic phase. Either water or buffers in the pH range of 4.0–5.5 were used as both the inner (w1) and outer (w2) aqueous phases. The vesicles formed upon the evaporation of organic solvents in w1/o/w2 emulsions. However, the copolymers assembled into micelles above pH 5.5 and large precipitates below pH 4.0. The vesicles were obtained mainly from copolymer with an average molecular weight of 2.97 ; 10 gmol 1 and a composition of 9.1 mol% DSA, unless stated otherwise. Figure 1a confirms that the resultant assemblies are unilamellar vesicles. The laser scanning confocal microscopy (LSCM) image of polymer vesicles in aqueous suspensions was revealed by the fluorescence of Nile red associated with the vesicle membranes. The lyophilized vesicles can be observed by scanning electron microscopy (see the Supporting Information). The fact that such polymer colloids maintain their structural integrity when subjected to transition from the aqueous to dried state reflects their robust stability. Transmission electron microscopy (TEM) examination of the sectioned specimens (ca. 60–90nm thickness) of polymer vesicles indicates that the wall thickness was approximately 25 nm (Figure 1b). The vesicle size can be controlled by adjusting either the THF/CH3Cl ratio used during emulsification or the DSA content of copolymers to give vesicles with diameters ranging from 1 to 15 mm. For example, changing the DSA content of copolymers from 9.1 to 13.1 mol% increases the vesicle diameter by 3–4 mm on the average. In contrast, increasing the THF content of the THF/CH3Cl solution from 2 to 20% (v/v) reduces the vesicle size significantly (Figure 2) because of the increased miscibility with water and the resulting decreased interfacial tension of the polymer-containing oil droplets in the aqueous phase. When the ionization of AAc residues increases to some extent with increasing pH value, the vesicles become equipped with transmembrane channels that are permeable for hydrophilic solutes. Figure 3 shows that, while transport of calcein (a water-soluble fluorescence probe) across the membrane was prohibited at pH 5.0, the probe molecules freely diffused into the vesicular aqueous compartment when the external pH value was increased to 8.0. Calcein was then confined within the compartment simply by adjusting the [*] Prof. H.-C. Chiu, Y.-W. Lin, Y.-F. Huang, C.-K. Chuang Department of Chemical Engineering National Chung Hsing University Taichung 402 (Taiwan) Fax: (+886)4-2285-2636 E-mail: hcchiu@dragon.nchu.edu.tw

Functionalized polymersomes with outlayered polyelectrolyte gels for potential tumor-targeted delivery of multimodal therapies and MR imaging

A novel tumor-targeting polymersome carrier system capable of delivering magnetic resonance imaging (MRI) and chemotherapy is presented in this study. The doxorubicin (DOX)-loaded magnetic polymersomes were first attained by the self-assembly of lipid-containing copolymer, poly(acrylic acid-co-distearin acrylate), in aqueous solution containing citric acid-coated superparamagnetic iron oxide nanoparticles (SPIONs), and followed by DOX loading via electrostatic attraction. To further functionalize these artificial vesicles with superior in vivo colloidal stability, pH-tunable drug release and active tumor-targeting, chitosan and poly(γ-glutamic acid-co-γ-glutamyl oxysuccinimide)-g-poly(ethyleneglycol)-folate (FA) were deposited in sequence onto the assembly outer surfaces. The interfacial nanogel layers via complementary electrostatic interactions and in-situ covalent cross-linking were thus produced. These nanogel-caged polymersomes (NCPs) show excellent anti-dilution and serum proteins-repellent behaviors. Triggerable release of the encapsulated DOX was governed by dual external stimuli, pH and temperature. When these theranostic NCPs were effectively internalized by HeLa cells via FA receptor-mediated endocytosis and then exposed to high frequency magnetic fields (HFMF), the combined effects of both pH and magnetic hyperthermia-triggered drug release and thermo-therapy resulted in greater cytotoxicity than the treatment by DOX alone. By virtue of the SPION clustering effect in the assembly inner aqueous compartments, the SPION/DOX-loaded NCPs displayed an r₂ relaxivity value (255.2 F emM⁻¹ S⁻¹) higher than Resovist (183.4 F emM⁻¹ S⁻¹), a commercial SPION-based T₂ contrast agent. The high magnetic relaxivity of the tumor-targeting NCPs coupled with their enhanced cellular uptake considerably promoted the MRI contrast of targeted cancer cells. These results demonstrate the great potential of the FA-decorated SPION/DOX-loaded NCPs as an advanced cancer theranostic nanodevice.

Dual stimuli-responsive polymeric hollow nanogels designed as carriers for intracellular triggered drug release.

Dual stimuli-responsive hollow nanogel spheres serving as an efficient intracellular drug delivery platform were obtained from the spontaneous coassociation of two graft copolymers into the vesicle architecture in aqueous phase. Both copolymers comprise acrylic acid (AAc) and 2-methacryloylethyl acrylate (MEA) units as the backbone and either poly(N-isopropylacrylamide) (PNIPAAm) alone or both PNIPAAm and monomethoxypoly(ethylene glycol) (mPEG) chain segments as the grafts. The assemblies were then subjected to covalent stabilization within vesicle walls with ester-containing cross-links by radical polymerization of MEA moieties, thereby leading to hollow nanogel particles. Taking the advantage of retaining a low quantity of payload within polymer layer-enclosed aqueous chambers through the entire loading process, doxorubicin (DOX) in the external bulk phase can be effectively transported into the gel membrane and bound therein via electrostatic interactions with ionized AAc residues and hydrogen-bond pairings with PNIPAAm grafts at pH 7.4. With the environmental pH being reduced (e.g., from 7.4 to 5.0) at 37 °C, the extensive disruption of AAc/DOX complexes due to the reduced ionization of AAc residues within the gel layer and the pronounced shrinkage of nanogels enable the rapid release of DOX species from drug-loaded hollow nanogels. By contrast, the drug liberation at 4 °C was severally restricted, particularly at pH 7.4 at which the DOX molecules remain strongly bound with ionized AAc residues and PNIPAAm grafts. The in vitro characterizations suggest that the DOX-loaded hollow nanogel particles after being internalized by HeLa cells via endocytosis can rapidly release the payload within acidic endosomes or lysosomes. This will then lead to significant drug accumulation in nuclei (within 1 h) and a cytotoxic effect comparable to free drug. This work demonstrates that the novel DOX-loaded hollow nanogel particles show great promise of therapeutic efficacy for potential anticancer treatment.

Superparamagnetic Hollow Hybrid Nanogels as a Potential Guidable Vehicle System of Stimuli-Mediated MR Imaging and Multiple Cancer Therapeutics

Hollow hybrid nanogels were prepared first by the coassembly of the citric acid-coated superparamagnetic iron oxide nanoparticles (SPIONs, 44 wt %) with the graft copolymer (56 wt %) comprising acrylic acid and 2-methacryloylethyl acrylate units as the backbone and poly(ethylene glycol) and poly(N-isopropylacrylamide) as the grafts in the aqueous phase of pH 3.0 in the hybrid vesicle structure, followed by in situ covalent stabilization via the photoinitiated polymerization of MEA residues within vesicles. The resultant hollow nanogels, though slightly swollen, satisfactorily retain their structural integrity while the medium pH is adjusted to 7.4. Confining SPION clusters to such a high level (44 wt %) within the pH-responsive thin gel layer remarkably enhances the transverse relaxivity (r2) and renders the MR imaging highly pH-tunable. For example, with the pH being adjusted from 4.0 to 7.4, the r2 value can be dramatically increased from 138.5 to 265.5 mM(-1) s(-1). The DOX-loaded hybrid nanogels also exhibit accelerated drug release in response to both pH reduction and temperature increase as a result of the substantial disruption of the interactions between drug molecules and copolymer components. With magnetic transport guidance toward the target and subsequent exposure to an alternating magnetic field, this DOX-loaded nanogel system possessing combined capabilities of hyperthermia and stimuli-triggered drug release showed superior in vitro cytotoxicity against HeLa cells as compared to the case with only free drug or hyperthermia alone. This work demonstrates that the hollow inorganic/organic hybrid nanogels hold great potential to serve as a multimodal theranostic vehicle functionalized with such desirable features as the guidable delivery of stimuli-mediated diagnostic imaging and hyperthermia/chemotherapies.

Nano-scaled pH-responsive polymeric vesicles for intracellular release of doxorubicin

Polymeric vesicles produced by spontaneous self-association of poly(acrylic acid-co-distearin acrylate) (poly(AAc-co-DSA)) with varying ratios of AAc and DSA units in aqueous solution of pH 5.0 exhibit the pH-regulated drug release behavior. Through the electrostatic interaction with ionized AAc residues, doxorubicin (DOX) molecules can be highly accommodated onto either the inner or outer surfaces of vesicles when the pH is adjusted from 5.0 to 7.4. The extent of DOX encapsulation is dependent largely on the structural transition of vesicles in response to the pH change. While the pH-evolved drug release profile varies to some extent with the distribution of DOX molecules within vesicles, the drug release from vesicles is accelerated significantly via the disruption of the electrostatic interaction of DOX species with ionized AAc moieties at pH 5.0. The DOX-loaded polymeric vesicles show promoted cellular uptake and cytotoxicity comparable to free DOX for HeLa cells. This indicates that they are probably taken up by the cells via the lipid raft-mediated endocytosis.

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX) were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid) (poly(γ-GA)), followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide)-g-monomethoxy poly(ethylene glycol) in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs) showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37°C) at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

pH-responsive hierarchical transformation of charged lipid assemblies within polyelectrolyte gel layers with applications for controlled drug release and MR imaging contrast

A cationic lipid-embedded poly(acrylic acid) (PAAc) gel layer coated on chitosan/superparamagnetic iron oxide nanoparticle (SPION) nanohybrid surfaces effectively modulates drug release and MR imaging contrast by pH-responsive morphological transformation and hierarchical alignment of the lipid assemblies.