FUNCTIONAL NANO- AND MICROPARTICLES FOR DRUG, NUTRITION, AGRO AND ENVIRONMENTAL APPLICATIONS O. Kammona, K. Kotti, E. Dini, O. Kotrotsiou, S. Alexandridou and C. Kiparissides* Chemical Engineering Department and Chemical Process Engineering Research Institute, Aristotle University of Thessaloniki, P.O. Box 472, 54124, Thessaloniki, Greece Abstract The rapid evolution of nanotechnology is aiming to fulfill the goal of optimal drug delivery, for various types of diseases, through the development of advantageous targeted drug carriers (e.g., nanoparticles, liposomes, micelles) with properly modified surfaces in order to avoid interactions with other vehicles, cells and proteins and thus with increased lifetimes, as well as through the attachment of model ligands to drug vesicles for increased targeting efficiency and the synthesis of molecularly imprinted polymers (MIPs). In addition, nanotechnology is expected to meet the societal requirements for high-quality potable water and environmentally friendly industrial processes with minimal solid/liquid wastes through the development of specially designed functional nanoparticles, and their subsequent impregnation into inorganic substrates. Keywords Targeted drug delivery, functional nanoparticles, liposomes, molecular imprinting, channel proteins, water purification, electrodeposition. Introduction The on-going discovery of novel phenomena and processes at the nanometer scale is providing science with a wide range of tools, materials, devices and systems with unique characteristics. Nanostructured materials, nanodevices and novel nanofabrication processes have created great excitement in biomedical and environmental, research and technologies because of their numerous and diverse applications. The capability of synthesizing and processing nanoparticles with tailored structures and enhanced properties provide tremendous opportunities for designing novel materials of exceptional promise for biomedical and environmental applications. In the field of biomedicine, nanotechnology will help reach the elusive goal of active drug targeting to specific cells within the body by the development of miniature drug-carriers (e.g., nanoparticles, liposomes, micelles) with increased lifetimes, Alexandridou and Kiparissides (2002), as well as through the synthesis of molecularly imprinted polymers (MIPs). In the field of environmental technologies, nanotechnology is expected to contribute to a safer environment through the application of functional, tailor-made nanoparticles to water treatment processes and * To whom all correspondence should be addressed to various industrial processes (e.g., production of electrogalvanized steel, miniature connectors, etc). In the present study, some of the on-going and novel research activities of the Laboratory of Polymer Reaction Engineering in the field of biomedicine and environmental technologies are presented. Biomedical Applications Liposomes - Composite PLGA Microparticles Liposomes are microscopic and submicroscopic vesicles with sizes ranging from 10nm to 20µm. They are usually made up of phospholipids, although other amphiphiles such as nonionic surfactants can also be employed for their construction. When phospholipids are hydrated, they spontaneously form spherical lipid bilayers enclosing the aqueous medium and the solute. Liposomes offer several advantages over other delivery systems including biocompatibility, control of biological properties via modification of physical properties (e.g., lipid composition, vesicle size, lipid membrane fluidity etc.) and several modes for drug delivery to cells (e.g., absorption, fuse, endocytosis, phagocytosis) (Figure 1). 1 2004, Workshop of CPERI Liposomes can be classified according to the number of the lipid bilayers as unilamellar vesicles (ULVs) and multilamellar vesicles (MLVs). fuse endocytosis absorption of hydroquinone or of hydroquinone-loaded liposomes was added to a PLGA solution in dichloromethane resulting in the formation of a w/o emulsion, Hans and Lowman, (2002). The latter was then added to an aqueous PVA solution leading to the formation of a w/o/w emulsion. Simple and composite (Figure 3b), spherical PLGA microparticles were formed by solvent evaporation from the w/o/w emulsion at increased temperature. The release rate of hydroquinone from the composite PLGA microparticles was compared to that from the simple ones and was found to be significantly retarded. phagocytosis Figure 1. Drug delivery modes Functionalized liposomes can be synthesized using peptides and oligosaccharides (Figure 2) in order to achieve both targeting and circulation longevity. Peptides can be used in order to guide liposomes to desired receptors whereas, PEO-grafted phospholipids are known to dramatically increase liposome survival in the circulation. A surface modified liposomal drug delivery vehicle can be developed for selective targeting by coupling an RGD peptide to the liposome through a PEO spacer, Lestini et al., 2002. Hydrated oligosaccharide interface Drug Covalently coupled peptides 8080-100 nm Figure 2. Functionalized liposome In the present study, MLVs were synthesized using hydration, followed by sonication and extrusion. Various types of phospholipids (e.g., Phospholipon 80, 80H, 90 and 90H) and cholesterol were employed for their synthesis. Hydroquinone, a hydrophilic drug used for skin whitening was employed as the active ingredient. The morphology of the MLVs after the hydration step was examined by means of optical microscopy (Figure 3a) and their size distribution was measured using dynamic light scattering. The size of the MLVs was found to depend on the preparation method, the type of the phospholipid and the pore size of the membrane used during the extrusion process. The hydroquinone-loaded MLVs were subsequently encapsulated in PLGA microparticles employing a complex solvent evaporation process. PLGA microparticles containing hydroquinone were also prepared using the same technique. An aqueous solution 2 (a) (b) Figure 3. Optical micrograph of (a) MLVs prepared by hydration and (b) composite PLGA particles Antibody-directed Enzyme Prodrug Therapy(ADEPT) Antibody-directed enzyme prodrug therapy (ADEPT) is a two-step, therapeutic strategy, which aims to improve the selectivity of anticancer drugs. In ADEPT, an enzyme is linked to an antibody (Ab) (Ab-enzyme conjugate) that binds to an antigen preferentially expressed on the surface of tumor cells. Subsequently, the enzyme activates and converts a nontoxic prodrug injected at the tumor, into a cytotoxic drug (Figure 4). The enzymes used for ADEPT must be stable under physiological conditions and able to catalyze a scission reaction of the prodrug. In addition, their catalytic properties should be different from those of any circulating endogenous enzyme and ideally, they should be able to activate a panel of anticancer prodrugs. The main requirement of Ab-conjugates is that they must localize on the tumor with high affinity and have minimum binding to normal sites. In addition, the covalent binding of the enzyme should not destroy the ability of the Ab to bind to its associated antigen, nor should it alter the enzyme activity and there should be a rapid clearance of the Ab-enzyme conjugate from the body fluids. Finally, the prodrugs designed for ADEPT must be chemically stable with good pharmacological and pharmacokinetic properties, less cytotoxic than their corresponding active drugs and suitable substrates for the activating enzyme, Niculescu-Duvaz and Springer (1997). Advanced Materials and Nanotechnology immobilized enzyme inactive prodrug immobilized antibody antigen binding site (a) (b) Figure 5. Reconstitution of channel proteins in (a) ABA triblock copolymer membranes and (b) nanoreactors active drug target tumor cell active drug inside the cell Figure 4. Schematic representation of ADEPT Channel Proteins Spherically closed triblock copolymer membranes can be prepared in dilute aqueous solutions using an amphiphilic ABA triblock copolymer, consisting of a flexible, hydrophobic poly (dimethylsiloxane) (PDMS) middle block and two water-soluble poly(2-methyloxazoline) (PMOXA) side blocks. The PMOXA-PDMS-PMOXA triblock copolymer vesicles thus formed carry methacrylate end groups, which can be polymerized within these self-assembled structures under preservation of the characteristic membrane structure. As a result of the crosslinking polymerization, the individual triblock copolymer molecules are covalently linked together, which leads to a considerable mechanical stabilization of the membranes. The PMOXA-PDMS-PMOXA triblock copolymer membranes can be employed for the reconstitution of transmembrane proteins (e.g., outer membrane protein F (OmpF) and maltoporin) The latter form trimeric channels which allow the diffusion of small solutes like ions, nutrients or antibiotics across the polymeric membrane (Figure 5a) (Meier et al., 2000). Channel proteins can also be reconstituted in enzyme containing polymer-stabilized liposomes or triblock copolymer nanocapsules (e.g., nanoreactors) in order to control the rate of the reaction in the interior (e.g., hydrolysis of β-lactam antibiotics like ampicillin by the enzyme β-lactamase) by controlling the permeability of the solute (e.g., ampicillin) (Figure 5b) (Winterhalter et al., 2001). Molecularly Imprinted Polymeric Nanoparticles Molecular imprinting of synthetic polymers is a process where functional and cross-linking monomers are co-polymerized in the presence of the target analyte i.e., the imprint molecule, which acts as a molecular template. The functional monomers initially form a complex with the imprint molecule, and following polymerization, their functional groups are held in position by the highly cross-linked polymeric structure. Subsequent removal of the imprint molecule reveals binding sites that are complementary in size and shape to the analyte (Figure 6). Three particular features have made molecularly imprinted polymers (MIPs) the target of intense investigation: i) their high affinity and selectivity, which are similar to those of natural receptors, ii) their unique stability which is superior to that demonstrated by natural biomolecules and iii) the simplicity of their preparation. Molecularly imprinted polymers can be prepared in a variety of physical forms to suit the final application desired (Byrne et al., 2002; Allender et al., 2000). Complex Formation Functional monomers Template molecule Polymerization Extraction Figure 6. Schematic representation of molecular imprinting In the present study, precipitation polymerization was employed for the synthesis of MIP nanoparticles to be used as synthetic receptors selective for theophylline (Ye et al., 2000; Ciardelli et al., 2004) and simazine (Matsui et al., 1995). Non-imprinted polymeric nanoparticles were also prepared using the same technique. The surface examination of the polymeric particles revealed that both MIP and non-imprinted particles exhibit a rough, porous surface (Figure 7) indicating that the presence of the template does not influence significantly the polymer morphology. UV spectroscopy was employed to measure the affinity and selectivity of the MIP particles. A small amount of nanoparticles (e.g., 0.1gr) were incubated 3 2004, Workshop of CPERI overnight at room temperature in print molecule/ acetonitrile solutions of known concentrations (e.g., 1.4 µmole of theophylline/ml of solvent and 0.8 µmole of simazine/ml of solvent). The binding capacity of the MIP particles was compared to that of the non-imprinted polymeric nanoparticles. It was shown that the nanoparticles, which were imprinted with theophylline adsorb 14.57 µmole of theophylline per 1gr of polymer whereas, those imprinted with simazine were found to adsorb 2.2 µmole of simazine per 1gr of polymer. When non-imprinted polymeric nanoparticles were used, 3.12 µmole of theophylline per 1gr of polymer were adsorbed, whereas, in the case of simazine, minimal binding of the target analyte was observed. Competitive analysis was also performed employing caffeine, an analyte which is chemically-related to theophylline, in order to examine the selectivity of the theophylline imprinted polymeric receptors. It was shown that the MIP nanoparticles adsorb 1.79 µmole of caffeine per 1gr of polymer thus, proving the selectivity of the artificial receptors towards the template molecule. (a) In the present study, poly (styrene/β-cyclodextrin) P(St/β-CD) and highly crosslinked poly(styrene/metadiisopropylbenzene) P(St/mDIB) porous nanoparticles were prepared employing emulsifier-free emulsion polymerization and a single-step swelling and polymerization process (Cheng et al., 1992; Ogino et al., 1995) to be used as hosts for the recovery of organic components from potable water. The effect of the crosslinker concentration on the particle morphology was examined experimentally. The surface morphology of the polymeric particles was assessed by scanning electron microscopy (SEM) (Figure 8) and their pore size distribution was determined by nitrogen adsorption. A GC-FID method was employed to measure the adsorption efficiency of the particles in various pollutants. The adsorption experiments were carried out in a batch mode and the Freundlich constant (KF) was calculated in terms of the initial and equilibrium pollutant concentrations, volume of the aqueous solution and mass of the adsorbent (Jung et al., 2001). Adsorption measurements with activated carbon (AC) were also performed for comparison purposes. It was shown that the particles examined have a moderate affinity for styrene, a low affinity for chloroform and dibromochloromethane and a high affinity for trichloro-ethylene and tetrachloroethylene higher than that of activated carbon (Table 1). (b) (a) (b) (c) Figure 7. SEM photomicrographs of (a) non-imprinted polymeric nanoparticles and nanoparticles imprinted with (b) theophylline and (c) simazine (c) Environmental Applications Water Purification Activated carbon has been traditionally used for water purification. However, despite its relatively broad range of effectiveness in adsorbing organic substances from aqueous solutions, activated carbon cannot be considered as a water treatment panacea, Weber (1973). Thus, there is an emerging need for the development of improved, alternative purification systems (e.g., porous polymeric nanoparticles, composite membranes etc.). 4 Figure 8. SEM photomicrographs of the (a,b) P(St/mDIB) and (c) P(St/βCD) nanoparticles The porous nanoparticles were subsequently deposited onto ceramic carriers (e.g., SiC/TiO2, alumina), resulting in the formation of hybrid membranes. It was shown that the P(St/mDIB) and P(St/β-CD) particles were distributed rather homogeneously in the ceramic filters and a high percentage of coverage was achieved (Figure 9). Advanced Materials and Nanotechnology Table 1. Adsorption efficiency of the polymeric nanoparticles in various pollutants Sam ple Kf 1/n Styrene EP-PSmD IB -009 38.6456 SW P-PSm D IB -001 58.3579 EP-PSβCD -001 27.7140 0.88694 0.57390 0.92570 Trichloroethylene EP-PSmD IB -009 203.376 SW P-PSm D IB -001 156.460 EP-PSβCD -001 132.620 Activated Carbon 89.39 0.43484 0.78340 0.44104 0.61253 unfriendly pre-treatment process, presently used to ensure good paint adhesion onto electrogalvanized steel. The polymerization experiments were carried out in laboratory-scale glass reactors and the most promising recipes were successfully scaled-up in a fully automated pilot-scale reactor. The polymer containing zinc coatings were produced at K.U. Leuven by electrolytic codeposition of the particles from an acid zinc plating bath using a rotating disk electrode (RDE). The coatings thus prepared (Figure 11), were subsequently painted and subjected to a number of corrosion resistance and paint adhesion tests, which gave rather promising results. Tetrachloroethylene EP-PSmD IB -009 201.604 EP-PSβCD -001 155.310 SW P-PSm D IB -001 62.216 Activated Carbon 111.276 0.4408 0.9283 0.8284 0.7913 Chloroform EP-PSmD IB -009 EP-PSβCD -001 Activated Carbon 3.3083 0.1332 5.9786 1.2812 1.5630 0.9854 D ibrom ochlorom ethane EP-PSmD IB -009 EP-PSβCD -001 Activated Carbon 10.0299 9.9977 4.6419 0.86406 0.76424 1.2024 (a) (b) (c) (a) Figure 10. SEM photomicrographs of the (a) PS, (b) P(MMA/DMAEMA) and (c) PGMA nanoparticles (b) Figure 9. Alumina filter containing P(St/β-CD) particles: (a) peripheral surface, (b) horizontal section. Figure 11. Codeposition of P (St/2-HEMA) nanoparticles from an acid zinc-plating bath using an RDE Electrogalvanized Steel with Improved Properties Monodisperse polymeric nanoparticles with diameters in the range of 60 - 1400 nm, with various surface charges and functional groups were prepared by (emulsifier-free) emulsion polymerization (Figure 10) and incorporated into electrolytic zinc coatings aiming to improve the corrosion resistance of painted and unpainted electrogalvanized steel and to eliminate phosphating, an environmentally 5 2004, Workshop of CPERI References Alexandridou, S., Kiparissides, C., Proceedings of the EC-NSF Workshop on “Nanotechnology - Revolutionary Opportunities and Societal Implications”, January 31February 1, 2002, Lecce, Italy. Allender C.J., Richardson C., Woodhouse B., Heard C.M., Brain K.R. (2000). Pharmaceutical Applications for Molecularly Imprinted Polymers. Int. J. Pharm., 195, 39. Byrne M.E., Park K., Peppas N.A. (2002). Molecular Imprinting within Hydrogels. Adv. Drug Delivery Rev., 54, 149. Cheng, C.M., Micale, F.J., Vanderhoff, J.W., El-Aasser, M.S. (1992). Synthesis and Characterization of Monodisperse Porous Microparticles. J. Pol. Sci.: Part A: Pol. Chem., 30, 235. Ciardelli G., Cioni B., Cristallini C., Barbani N., Silvestri D., Giusti P. (2004). Acrylic Polymeric Nanospheres for the Release and Recognition of Molecules of Clinical Interest. Biosens. Bioelectron (in press). Hans M.C., Lowman A.M. (2002). Biodegradable Nanoparticles for drug Delivery and Targeting. Curr. Opin. Solid State Mater. Sci., 6, 319. Jung, M.-W., Ahn, K.-H., Lee, Y., Kim, K.-P., Rhee, J.-S., Park, J.T., Paeng, K.-J. (2001). Adsorption Characteristics of Phenol and Chlorophenols on Granular Activated Carbons (GAC). Microchem. J., 70, 123. Lestini B.J., Sagnella S.M., Xu Z., Shive M.S., Richter N.J., Jayaseharan J., Case A.J., Kottke-Marchant K., Anderson J.M., Marchant R.E. (2002). Surface Modification of Liposomes for Selective Cell Targeting in Cardiovascular Drug Delivery. J. Controlled Release, 78, 235. Matsui J., Miyoshi Y., Doblhoff-Dier O., Takeuchi T. (1995). A Molecularly Imprinted Synthetic Polymer Receptor Selective for Atrazine. Anal. Chem., 67, 4404. Meier, W., Nardin, C., Winterhalter, M. (2000). Reconstitution of Channel Proteins in (Polymerized) ABA Triblock Copolymer Membranes. Angew. Chem. Int. Ed., 39, 4599. Niculescu-Duvaz, I., Springer, C.J. (1996). Antibody-directed Enzyme Prodrug Therapy (ADEPT): A Review. Adv. Drug Delivery Rev., 26, 151. Ogino, K., Sato, H., Tsuchiya, K., Suzuki, H., Moriguchi, S. (1995). Preparation and Characterization of Monodisperse Oligo(ethylene glycol) dimethacrylate Polymer Beads for Aqueous High-performance Liquid Chromatography. J. Chrom. A, 699, 59. Weber, W.J. (1973). “The Prediction of the Performance of Activated Carbon for Water Treatment”, Activated Carbon in Water Treatment, A Water Research Association Conference, University of Reading. Winterhalter, M., Hilty, C., Bezrukov, S.M., Nardin, C., Meier, W., Fournier, D. (2001). Controlling Membrane Permeability with Bacterial Porins: Application to encapsulated Enzymes. Talanta, 55, 965. Ye, L., Weiss, R., Mosbach, K. (2000). Synthesis and Characterization of Molecularly Imprinted Microspheres. Macromolecules, 33, 8239. 6