ELECTRICALLY RESPONSIVE, NANOPATTERNED SURFACE FOR TRIGGERED INTRACELLULAR DELIVERY OF BIOLOGICALLY ACTIVE MOLECULES

Abstract

Nano-patterned devices for triggered intracellular delivery of active materials are disclosed. The device may comprise a nano-sized polyelectrolyte multilayer (PEM) comprising at least one layer of an electroactive polyelectrolyte polymer, where the PEM is configured to hold or receive an active material to be disposed within the multilayer and to release the active material under an electric field.

Claims

1. A nano-sized polyelectrolyte multilayer (PEM) comprising at least one layer of an electroactive polyelectrolyte polymer, the PEM being configured to hold or receive an active material to be disposed within said multilayer and to release said active material under electric field.

2. The PEM according to claim 1, being a stacked nano-structure comprising a plurality of alternating layers of a positively charged polyelectrolyte polymer and a negatively charged polyelectrolyte polymer.

3. The PEM according to claim 1, being nano-patterned.

4. The PEM according to claim 1, comprising a plurality of stacked layers, at least one of said plurality of stacked layers is a layer of an electroactive polyelectrolyte polymer and at least one another of said plurality of stacked layers is a layer of a charged polyelectrolyte polymer, the PEM being configured to hold or receive at least one active material and to release said at least one active material under electric field.

5. (canceled)

6. The PEM according to claim 2, wherein the negatively charged polyelectrolyte polymer is selected from sulfonate-functionalized poly(3,4-ethylenedioxythiophene) (PEDOTS), sulfonate-functionalized poly(styrenesulfonic acid) (PSS), poly(2-acrylamido-2-methyl-1-propane sulfonic acid) (PAMPS), sulfonate-functionalized poly(ether ketone) (SPEEK), sulfonate-functionalized lignin, poly(ethylenesulfonic acid), poly(methacryloxyethylsulfonic acid), each of the aforementioned optionally provided in a salt form.

7. The PEM according to claim 2, wherein the positively charged polyelectrolyte polymer is selected from poly(ethylene imine) (PEI), poly(diallyldimethylammonium chloride) (PDAD) and copolymer thereof with polyacrylamide (PDAD-co-PAC), poly(vinylbenzyltrimethylammonium) (PVBTA), ionenes, poly(acryloxyethyltrimethyl ammonium chloride), poly(methacryloxy (2-hydroxy) propyltrimethyl ammonium chloride), and copolymers of any of the aforementioned; polyelectrolytes comprising pyridinium groups; poly(N-methylvinylpyridine) (PMVP), poly(N-alkylvinylpyridines) and copolymers thereof; protonated polyamines; and poly(allylaminehydrochloride) (PAH).

8. The PEM according to claim 1, comprising alternating layers of a sulfonate-functionalized poly(3,4-ethylenedioxythiophene) (PEDOTS) and poly(ethylene imine) (PEI).

9. The PEM according to claim 1, wherein the electroactive polyelectrolyte polymer is a negatively or positively charged polymer exhibiting a change in its charge upon stimulation with an electric field.

10. The PEM according to claim 9, wherein the electroactive polyelectrolyte polymer is a polymer selected from sulfonate-functionalized poly(3,4-ethylenedioxythiophene) (PEDOT), carboxylated or sulfonated derivatives of PEDOT, poly(3-hexylthiophene-2,5-diyl) (P3HT), poly(phenylene vinylene) (PPV) and polyacetylene.

11. The PEM according to claim 1, formed on a charged domain of a substrate having alternating charged and neutral domains.

12. A device comprising a surface region having alternating charged and neutral domains, or spaced-apart charged domains surrounded by neutral domains; and a polyelectrolyte multilayer (PEM) formed on at least one of the charged domains, the PEM comprising at least one layer of an electroactive polyelectrolyte polymer, the PEM being configured to hold or receive an active material disposed within said multilayer and to release said active material under electric field.

13. The device according to claim 12, wherein the charged domains are positively charged.

14. The device according to claim 12, wherein the charged domains are formed by nano-patterning the surface region or by causing the surface region material adopt a charge at nano-confined regions.

15. The device according to claim 14, wherein nano-patterning is achievable by using a block copolymer substrate.

16. The device according to claim 15, wherein the block copolymer is polystyrene-block-poly (2-vinyl pyridine) (PS-b-P2VP), polystyrene-block-poly(acrylic acid), polystyrene-block-poly(tert-butyl acrylate), polystyrene-block-poly(N-acrylamide), or polystyrene-block-poly(lactic acid).

17. The device according to claim 12, wherein the active material is a drug, a therapeutic agent, an imaging agent, a neurotransmitter, a hormone, a growth factor, a peptide, a protein, lectin, an antibody, an enzyme, DNA, RNA antisense, iRNA, siRNA, microRNA, a ribozyme and combinations thereof.

18. A method for delivering an active material, or a biologically active material to a vicinity of living cells, or through a membrane of a living cell, the method comprising applying a voltage to a PEM positioned in contact with or in proximity to the living cells, the voltage being of a magnitude and applied for a duration sufficient to disassemble the PEM from the substrate on which it is present or disassemble the PEM multilayer, causing release of the active material into the vicinity of the living cells, thereby enabling penetration of the active material through the cell membrane.

19. (canceled)

20. A device comprising a surface region composed of polystyrene-block-poly (2-vinyl pyridine) (PS-b-P2VP) having alternating positively charged and neutral domains; and a polyelectrolyte multilayer (PEM) present on at least one of the positively charged domains, the PEM comprising alternating layers of a sulfonate-functionalized poly(3,4-ethylenedioxythiophene) (PEDOTS) and poly(ethylene imine) (PEI), at least one interface between said alternating layers comprising at least one biologically active material, the device being configured and operable to release said at least one biologically active material upon application of an electric field.

21. The method according to claim 17, for delivering an active material to a vicinity of a living cell or through a membrane of a living cell, the method comprising applying a voltage to the PEM device when positioned in contact with or in proximity to the living cell, the voltage being of a magnitude and applied for a duration sufficient to disassemble the PEM, release the active material from the device and cause a transient change to the membrane of the living cell, thereby enabling the material penetration through the cell membrane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0121] FIG. 1 depicts the components and production scheme of a PEM assembly according to an embodiment of the present invention.

[0122] FIGS. 2A-H present SEM images (A,D), SFM height images (B,E) and cross-sections (C,F) of the patterned substrate before (A-C) and after (D-F) deposition of the first PEDOTS layer. SEM images were taken at 26° tilt angle. SFM cross-sections represent averaging over 40 adjacent scan lines (800 nm×150 nm box). (G) XPS data of the block copolymer film (blue), the nano-patterned template (xBCP) formed after reaction with DIB (red), and the film after deposition of the first PEDOTS layer (black). (H) Cell proliferation and viability data on different substrate interfaces.

[0123] FIGS. 3A-D present fluorescence images (488 nm excitation) of the PEM assembled on the xP2VP film (A,B) and on the xBCP film (C,D), assembled with (B,D) and without Dox (A,C). The micro-scale pattern appearing in the xBCP images reflects thickness undulations in the film.

[0124] FIGS. 4A-F provide (A,B) Quantification of the amount (in nanomole per cm.sup.2 of film) of stored Dox in the polyelectrolyte films assembled on different substrates (P2VP homopolymer (xP2VP) vs. block copolymer (xBCP)) via its release in a 1 mL PBS using (A) sonication or (B) application of 20 cyclic voltammetry sweeps from −0.2 V to 0.8 V at 0.1 V/s rate. (C,D) Leakage test, performed by incubation of 3.5-bilayer PEMs: (C) amount of Dox released into a 1 mL PBS; (D) amount of Dox remaining in the multilayers, quantified after subsequent sonication into a fresh 1 mL of PBS. (E) Quartz crystal microbalance experiment, showing the buildup of the layers and their disassembly. Numerical labels denote the process steps: (1) introduction of 1 wt % PEI solution; (2) washing with water; (3) introduction of 1 wt % PEDOTS solution; (4) disassembly upon the application of 10 voltage cycles from −0.2 V to 0.8 V at 0.1 V/s rate. Washing cycles following deposition were associated by a slight increase in frequency, owing to the desorption of weakly associating polyelectrolytes. Insets show expanded regions of the disassembly step. (F) Quantification of Dox released from the multilayer assembled on the nano-patterned substrate into 1 mL of deionized water after different number of voltage cycles from −0.2 V to 0.8 V at 0.1 V/s rate.

[0125] FIGS. 5A-F provide fluorescence images of a live/dead assay of fibroblast cells cultured for 2 days on PEDOTS/PEI multilayers that were assembled on the block copolymer template: (A) without Dox; (B) without Dox, after 20 cycles of electrochemical stimulation.sub.; (C) with Dox, without electrochemical treatment; (D-F) with Dox, after 1, 5, and 10 cycles of electrochemical stimulation.

[0126] FIGS. 6A-C provide fluorescence images of a live/dead assay of fibroblast cells cultured for 2 days on PEDOTS/PEI multilayers that were assembled on the xP2VP template: (A) without Dox; (B) without Dox, after 20 cycles of electrochemical stimulation; (C) with Dox, without electrochemical treatment.

[0127] FIGS. 7A-D provide (A,B) Overlaid optical microscopy and fluorescence images (λ.sub.ex=481 nm) showing extent of Dox internalization in NIH3T3 fibroblast cells after 24 and 36 h incubation in 17 μM Dox solution (for comparison, the Dox concentration that was released from 1-cm.sup.2 nano-patterned PEM to 1-mL solution was ˜14 μM as shown in FIG. 4F). Cells were cultured for 2 days prior to incubation. (C,D) Live/dead assays corresponding to the images shown in (A,B).

DETAILED DESCRIPTION OF EMBODIMENTS

Nano-Patterned Substrate Characterization

[0128] FIGS. 2A-F present the scanning electron microscopy (SEM) and scanning force microscopy (SFM) images of the cross-linked, nano-patterned block copolymer template before and after the deposition of the first PEDOTS layer. A strong increase in height contrast (from ca. 4 nm to ca. 15 nm) indicates that the PEDOTS adsorbed specifically to the positively-charged xP2VP domains. X-ray photoelectron spectroscopy (XPS) measurements performed on the block copolymer film, the crosslinked template (xBCP), and the template after the deposition of the first PEDOTS layer (FIG. 2G) shows a decrease in the intensity of the nitrogen peak after reaction with DIB, which is attributed to the conversion of the surface pyridine groups into alkylated pyridinium ions. Additionally, a strong decrease in the intensity of the iodine peak as well as an evolution of a sulfur peak is noted after PEDOTS deposition, which corroborates the displacement of iodide anions with the PEDOTS during the electrostatic self-assembly process. Lastly, water contact angle measurements show that the PEDOTS layer renders the substrate more hydrophilic (static contact angle decreased from 66.0°±0.3° on the xBCP template to 49°±2° on the xBCP-PEDOTS surface).

[0129] Biological testing of cell proliferation and viability were performed on the different substrates (FIG. 2H). Cells did not proliferate on the PS substrate, but adhered nicely on the xP2VP substrate. The amount of cells on the xBCP substrate was about half that on the xP2VP substrate, an intermediate value between PS and xP2VP substrates, reflecting the areal fraction occupied by the xP2VP domains in the xBCP template. However, deposition of PEDOTS increased the number of live cells by about 65% compared to the number of cells cultured on the xP2VP substrate, demonstrating the biocompatibility of PEDOTS. Interestingly, the PEDOTS coating on the xP2VP domains of the xBCP template diminishes the effect of the presence of the PS domains, despite the fact that microscopy data indicates highly selective deposition on the xP2VP domains.

[0130] Assembly and quantification of doxorubicin inside the polyelectrolyte multilayers. Polyelectrolyte multilayers consisting of 3.5-bilayers incorporating Dox were assembled on the homogeneous xP2VP and on the nano-patterned xBCP substrates. After the initial PEDOTS layer was deposited, we first tried to absorb the Dox by dipping the substrate into Dox solutions after each polyelectrolyte deposition step. However, only limited amount of Dox was absorbed onto the PEDOTS or PEI top layer. In order to increase the Dox loading within the PEMs, layers of Dox were spin-coated after each polyelectrolyte deposition. Probing Dox fluorescence in the films (λ.sub.ex=481 nm) shows increased emission from the films after deposition of 3.5 bilayers containing Dox (FIG. 3). Quantification of Dox inside the layers at different stages of the multilayer buildup was performed by releasing Dox into solution using two independent techniques, namely sonication and the application of voltage scans (FIGS. 4A-B; see Experimental Section). Both techniques yielded rather similar values, showing a consistent increase in the amounts of stored Dox with the number of bilayers on both types of substrates. However, the amounts of Dox stored in the nano-patterned multilayers are considerably higher than that in the multilayers assembled on the laterally homogeneous xP2VP substrates.

[0131] The observation that the multilayers assembled on the homogeneous xP2VP substrate stored considerably less Dox compared to the amount stored in the nano-patterned xBCP substrate is rather surprising; considering the areas available for polyelectrolyte assembly on both substrates (i.e., the xP2VP domains in the nano-patterned substrates compared to the entire substrate in the xP2VP homopolymer), one would expect the opposite. Moreover, incubation of both 3.5-bilayer Dox-containing films in 1 mL of phosphate buffer saline (PBS; pH 7.5) for extended periods of time revealed that multilayers assembled on the un-patterned homopolymer substrate was considerably less stable in terms of retaining the Dox, which leaked out from the multilayer within ˜12 hours (FIG. 4C,D). In comparison, the multilayer assembled on the xBCP retained its stored Dox for prolonged time, with less than 5% Dox lost after 3 days of continuous incubation (an average leakage rate of 1.6% per day; FIGS. 4C,D). We propose two possible explanations for such a behavior. The first relates to our previous observation that polyelectrolytes deposited over the interface between the PS and xP2VP domains fold back into the xP2VP domains during the drying stage. This possibly helps encapsulating the adsorbed Dox molecules within the PEM and retaining them for prolonged times. The second explanation relates to possible leakage of the Dox from defect points in the PEM caused by dust particles. Whereas a single defect point may, in principle, drain an entire continuous multilayer, only a few domains would be affected by it in a nano-patterned PEM, which consist of isolated domains. Although we cannot provide direct evidence to support these explanations, the ability to store and retain high amounts of bioactive molecules is a clear advantage of nano-patterned devices.

[0132] Stimulated release of encapsulated doxorubicin. Direct evidence into the mechanism of release was obtained by quartz crystal microbalance (QCM) experiments performed on a PEDOTS/PEI multilayer assembled on both xP2VP- and xBCP-coated QCM resonator (FIG. 4E). Initially, multilayer buildup is demonstrated by the decrease in resonator frequency every time a new polyelectrolyte solution is injected. Washing cycles resulted in a small increase in the frequency owing to desorption of non-specifically adsorbed polyelectrolytes. Application of 10 voltage cycles (from −0.2 V to 0.8 V at 0.1 V/s rate) caused an abrupt increase in the frequency, reaching the original level, indicating complete disassembly of the multilayer. We observed that the multilayer built on xP2VP disassembled more rapidly compared to that on xBCP, which also supports our previous findings on the increased stability of PEMs on nanopatterns.

[0133] FIG. 4F shows the extent of Dox released to solution after different number of cycles of cyclic voltammetry sweeps from −0.2 V to +0.8 V at 0.1 V/s rate. Saturation is reached already after 10 scans, suggesting the complete disassembly of the multilayer and release of all the stored Dox.

[0134] The viability of NIH3T3 fibroblast cells cultured on nano-patterned multilayers were probed (FIG. 5). The cells were cultured for 2 days on the PEM, the multilayer was then subjected to electrochemical treatment, and live/dead assay was performed after 6 additional hours of culturing. FIGS. 5A,B show that cells assembled on nano-patterned PEI/PEDOTS multilayer that did not contain Dox thrive even when voltage scans were applied to the PEM. This indicates that the electrochemical treatment itself does not harm the cells adsorbed on the PEM. Cells adsorbed on a nano-patterned multilayer that contained Dox also thrived as long as no voltage was applied (FIG. 5C), in accord with our previous findings on the ability of the nano-patterned multilayer to retain the stored Dox (FIG. 4C,D). For comparison, cells adsorbed on the Dox-containing PEM assembled on xP2VP did not survive (FIG. 6), owing to Dox leakage from such multilayers (FIGS. 4C,D). Applying a single voltage scan resulted in ˜5% cell death (FIG. 4d); additional scans annihilated the entire population (FIGS. 5E,F). These experiments demonstrated that cell death occurred only because of the triggered release of Dox.

[0135] It is noted that the effect of the released drug on the cells is rather quick; much longer time was needed for the drug to penetrate the cell membrane when the cells were incubated with a similar concentration of drug in solution (FIG. 7). This could be attributed to the presence of high local concentrations of the released Dox in the vicinity of the cells, but may also relate to a certain change in membrane permeability induced by the applied voltage.

CONCLUSIONS

[0136] A new platform is disclosed herein that is based on a nano-patterned polyelectrolyte multilayer that enables triggered drug delivery to adsorbed cells. The nano-pattern multilayer is furnished by a hierarchical construction approach, where a microphase-separated block copolymer film serves as a template for the selective deposition of the functional components. The multilayer consists of an electroactive polyelectrolyte, which inverts its charge upon the application of voltage and thus leads to multilayer disassembly and release of an embedded drug.

[0137] One of the most interesting and non-trivial attributes of the nano-patterned multilayer is its ability to retain the drug better than the corresponding homogeneous (i.e., non-patterned) multilayer. This ability is explained by the different average conformation of polyelectrolytes when adsorbed on nano-patterned substrates, which may assist in encapsulating the drug in the multilayer, and by the isolation of nano-patterned PEM domains, which reduces leakage from defect points caused by dust particles.

[0138] The other important attribute of our system is the relatively high efficacy of drug delivery to cells adsorbed on the surface compared to the delivery efficacy of similar concentration of drug to cells suspended in solution. Two reasons may account for this behavior: a high local concentration of the drug, which is released in close vicinity to the cells, and a possible enhancement in cell permeability caused by the application of voltage.

[0139] Overall, we have developed a delivery platform which efficiently encapsulates high loading of biologically active ingredients and controllably releases them upon the application of an external electrical stimulation. Utilizations of this platform technology for cell reprogramming, therapeutic implants and tissue engineering are currently underway.

EXPERIMENTAL

[0140] Preparation of block copolymer templates. Silicon wafers coated with 5 nm titanium adhesion layer and 45 nm gold were pre-cleaned in sulfuric acid-NoChromix (purchased from Sigma-Aldrich) bath overnight and then rinsed with triply distilled water. Thin films of PS, P2VP and PS-b-P2VP were prepared by spin casting from the respective 0.39 wt % chloroform solutions on each substrate at 3000 rpm for 30 s. The films thicknesses (29.5±0.5 nm) were determined by ellipsometry before annealing. The block copolymer films were solvent annealed in a closed petri dish under saturated chloroform vapour for 25 min under ambient conditions. Microphase separation in the BCP films led to the formation of lying P2VP cylinders in PS matrix, which gave rise to surface patterns of alternating stripes of ca. 36 nm width. Films were crosslinked with DIB for 42 hr at 75° C. These conditions led to quaternization of ca. 22% of all pyridine rings (in the volume sampled by an X-ray photoelectron spectroscopy beam) and degree of cross-linking of ˜16%. Fresh films were dipped for 10 min in PEDOTS (10 mM repeat unit concentration, prepared in ultrapure water, 0.055 mSiemens/cm conductivity), rinsed in ultrapure water, and dried by spinning at 2000 rpm for 30 s followed by nitrogen blowing.

[0141] Preparation of PEMs on gold electrodes. xP2VP and xBCP films were alternatively immersed in 1 wt % aqueous solutions of PEDOTS and PEI. Each immersion cycle was performed for 15 min and was followed by rinsing with deionized water and then introduction of Dox (by spin coating a 1 mg/mL solution at 2000 rpm for 30 s). The deposition of the last layer (PEDOTS) was followed by drying under nitrogen flow. The fabricated electrodes were stored under 4° C.

[0142] Electrochemical setup and disassembly conditions. The electrochemical cell consisted of an Ag/AgCl reference electrode and a Pt counter electrode, and was connected to a potentiostat station (Autolab, Metrohm). The gold electrode coated with Dox-containing 3.5-PEDOTS/PEI bilayer was immersed into PBS (10 mM, pH 7.5) and connected as the working electrode. Cyclic potential sweeps from −0.2 V to +0.8 V were applied at a scan rate 0.1 V/s.

[0143] QCM study of multilayer buildup and disassembly. Gold coated QCM sensors were cleaned by RCA-1 procedure (NH.sub.4OH:H.sub.2O.sub.2:H.sub.2O=1:1:4) at 80° C. for 15 min, then rinsed with deionized water and dried under nitrogen flow before use. The cleaned QCM sensors were coated with either P2VP or BCP films, annealed, reacted with DIB, and then coated with the first PEDOTS layer. A coated sensor was mounted into an electrochemical cell and connected as the working electrode; a leakless miniature Ag/AgCl electrode was used as the reference electrode. Polyelectrolyte solutions and deionized water were introduced at a constant flow rate of 50 μL/min. The experiments were started by running deionized water on the chip, and each solution was introduced after the resonance frequency stabilized.

[0144] For the disassembly process, the buffer system was first changed to PBS (10 mM, pH 7.5) until a stable frequency measurement was obtained, and then 10 cyclic potential sweeps were applied. The QCM-D signal of the disassembly process was acquired during continuous buffer flow.

[0145] Quantification of the amount of stored doxorubicin. Fluorescence images of Dox inside the multilayers were taken using an Olympus BX53 microscope at 488 nm excitation. The amount of Dox stored in the 3.5 bilayer films was quantified by releasing the Dox into 1 mL deionized water using either 30 min sonication or electrochemical scans (see above). The amount of Dox released was quantified using absorption spectroscopy (λ.sub.max=481 nm; ε=10410 M.sup.−1cm.sup.−1). Data represent averages of 3 repetitions.

[0146] Leakage test. Electrodes coated with 3.5-bilayer films were immersed into 1 mL PBS for 0, 6, 12, 24, 48 and 72 h. Samples were then removed from solution, sonicated in PBS as described above, and the concentrations of Dox in both types of solutions were analyzed by absorption spectroscopy. Data represent averages of 3 repetitions.

[0147] Cell culturing. NIH3T3 mice fibroblast cells were cultured for two days on the coated electrodes in the Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) at 37° C. under 5% CO.sub.2 atmosphere. Samples were subjected to experiments when cell coverage reached 85-90% confluence.

[0148] Cell Viability on coated electrodes. Cell viability before and after multilayer disassembly was performed using live/dead cell double staining kit (purchased from Merck/Sigma-Aldrich), which simultaneously stains viable and dead cells with green and red fluorescence tags, respectively. The stain solutions were added at 37° C. to the cell-covered electrode for 15 min, and images were then taken using a fluorescence microscope (Olympus BX53) with excitation wavelength at 488 nm and 545 nm to differentiate the viable/dead cells. Data represent averages of 3 repetitions.

[0149] Triggered drug release and viability assay. Doxorubicin was released from the 3.5-bilayer-coated gold electrode covered with cultured cells using the same conditions described above. After the electrochemical treatment, 2 mL of DMEM were added into the chamber and culturing was continued for additional 6 h at 37° C. under CO.sub.2 atmosphere. The electrodes were then rinsed with PBS three times and stained with live/dead assay kit to determine the amount of viable and dead cells. Data represent averages of 3 repetitions.

Additional Experimental Details

[0150] Instruments. Scanning force microscopy (SFM) images were acquired using a Dimension 3100 scanning probe microscope with a Nanoscope V controller (Veeco, USA). Images were corrected by first-order flattening and processed by the Nanoscope Analysis Program (V1.40, Bruker). BCP height images and film thicknesses were analysed by the built-in depth analysis according to the procedures described elsewhere. High resolution scanning electron microscopy (HR-SEM) images of the films were acquired with a Sirion microscope (FEI Company) at 5 kV acceleration voltage. XPS spectra were recorded on a Kratos ultra axis spectrometer (Kratos Analytical) using mono-energetic Al Kα.sub.1,2 irradiation (1486.6 eV) with a total power of 144 W (12 kV). .sup.1H and .sup.13C NMR spectra were recorded with Bruker AVA-300 spectrometer and chemical shifts were measured in δ (ppm) with residual solvent peaks as internal standards. Multilayer buildup was monitored on a Qsense quartz crystal microbalance (Biolin Scientific). Absorption spectra were recorded on a Cary 8454 UV-Vis spectrometer equipped with a diode array (Agilent). Film thicknesses measured using a Rudolph monochromatic ellipsometer operating at 633 nm.

[0151] Materials. PS-b-P2VP (M.sub.n 185 kDa, PDI 1.24, 67 wt % PS, 73 nm lamellar period, as determined by small-angle X-ray scattering) was synthesized by standard anionic polymerization using sec-butyllithium in tetrahydrofuran (THF) under nitrogen atmosphere. The molecular weight, size distribution and polystyrene weight fraction were all determined by gel permeation chromatography (GPC) in tetrahydrofuran (THF) against PS standards for the PS block and comparison of the .sup.1H NMR signals for phenyl and pyridine groups, respectively, for the P2VP block. P2VP (M.sub.w 6.2 kDa, polydispersity 1.04) and PS (M.sub.w 9.5 kDa, polydispersity 1.05) were purchased from Polymer Source, Inc. Poly(ethylene imine) (PEI, M.sub.w ˜25 kDa by LS, M.sub.n ˜10 kDa by GPC) was purchased from Sigma-Aldrich. 1,4-diiodobutane (DIB) and FeCl.sub.3 were purchased from Alfa-Aesar and used as received. 2,3-Dihydrothieno[3,4-b][1,4]dioxin-2-yl methanol (EDOT-OH) and sodium hydride 60% mineral oil suspension were purchased from Angene and Merck/Sigma-Aldrich, respectively. Sodium persulfate and 1,4-butane sulfone were purchased from SHOWA and Acros, respectively. These chemicals were used as received without further purification.

Synthesis of butanesulfonate-3,4-ethylenedioxythiophene (PEDOTS) Polymer

[0152] ##STR00001##

[0153] Monomer synthesis. The monomer (4-(2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxybutane-1-sulfonic acid sodium salt; EDOTS) was synthesized as previously described..sup.2EDOT-OH (2.5 g, 14.5 mmol) and NaH (0.9 g, 23 mmol) were mixed in a dry 250 mL two-neck round-bottom flask, and dry toluene (50 mL) was added after removing the air by nitrogen blowing. The resulting light brown solution was refluxed at 80° C. for 1.5 h. A solution of butane sulfone (1.98 g, 14.5 mmol) in 15 mL dry toluene was added slowly via syringe after the reaction mixture was cooled to ambient temperature, and then refluxed for another 2.5 h. After cooling to ambient temperature, acetone (150 mL) was poured into the reaction mixture under vigorous stirring. The resulting suspension was filtered, washed with acetone repeatedly and dried to yield light brown powder (4.45 g, 93%). .sup.1H NMR (300 MHz, D.sub.2O), δ: 6.53 (d, 1H, J=3.6 Hz), 6.51 (d, 1H, J=3.3 Hz), 4.48-4.42 (m, 1H), 4.28 (dd, 1H, J=12.0, 2.1 Hz), 4.11 (dd, 1H, J=11.7, 6.9 Hz), 3.76 (t, 2H, J=4.2 Hz), 3.65-3.60 (m, 2H), 2.94 (t, 2H, J=6.9 Hz), 1.82-1.70 (m, 4H). .sup.13C NMR (75 MHz, D.sub.2O) δ: 140.72, 140.57, 100.43, 100.27, 72.78, 71.00, 68.54, 65.64, 50.70, 27.49, 20.87.

[0154] Polymer Synthesis

[0155] Poly(4-(2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxybutane-1-sulfonic acid sodium salt) (PEDOTS, M.sub.n 12,000, PDI ˜3) was synthesized according to a literature procedure using FeCl.sub.3/Na.sub.2S.sub.2O.sub.8. EDOTS (0.4 g, 1.21 mmol) was dissolved in 6 mL of DI water. A solution of FeCl.sub.3 (0.01 g, 0.06 mmol) and Na.sub.2S.sub.2O.sub.8 (0.58 g, 2.44 mmol) in water (4 mL) was added dropwise to the above solution and stirred at room temperature (4 h). The reaction mixture was quenched by acetone (50 mL). The precipitated product was centrifuged (5 min, 4000 rpm), separated from the supernatant liquid, re-dissolved in water (10 mL), and precipitated from acetone. This procedure repeated until a clear solution was obtained. Finally, the polymer was dialyzed against deionized water for three days (changing the water every 24 h) using a 1000 g/mol cutoff membrane to yield PEDOTS polymer (51% conversion) after drying.

[0156] Molecular weight averages of PEDOTS polymer were determined using a Shimadzu liquid chromatography system (LC-2030C Plus) equipped with a MCX column (Polymer Standards Service, 8×300 mm, 10 μm bead diameter, 10.sup.5 Å pore size) and an RI detector. The analysis was performed at 23° C. using 0.08 M Na.sub.2HPO.sub.4 aqueous solution as eluent at 0.8 mL/min. The molecular weight values were determined with respect to poly(styrene sulfonate) sodium salt standard kit (Polymer Standards Service) ranging from 1,100-976,000 g/mol.