As noted throughout the web site one of the primary reasons for using a controlled radical copolymerization process for the preparation of a well defined macromolecule is that it is possible to incorporate site selected functional groups into the final copolymer. On this page we address how the group has worked with others to design materials that express specific bio-responsive properties, including antibacterial, protein conjugates, block copolymers designed for controlled drug delivery and materials designed for intra-cellular interactions.
Potentially harmful bacteria abound and pathogenic bacteria are frequently deposited onto surfaces subsequently touched by many people thereby spreading infection. It would be beneficial to have surfaces that kill bacteria on contact. Stable non-leaching antimicrobial surfaces are required for long term protection since leaching systems,see following schematic, are eventually exhausted, rendering the material ineffective and posing potential environmental risks.
Quaternary ammonium ion-containing polymers (PQA) are known to effectively kill cells and spores by disrupting cell membranes. Monomers, such as 2-dimethylaminoethyl methacrylate (DMAEMA), 4-vinyl pyridine (4-VP) and N-substituted acrylamides, that can be quaternized thereby providing biocidal activity, can be polymerized by ATRP. This means that stable antimicrobial surfaces can be prepared by grafting from(1-3) or grafting onto surfaces.(4)
Covalent attachment of PQA via "grafting onto" or "grafting from" provides long-lasting biocidal activity with no danger to the environment. Polymeric surfaces are the most common surfaces encountered in everyday life and recent work has focused on preparing biocidal polymer surfaces. A non-leachable biocidal polypropylene (PP) surface was created by chemically attaching poly(quaternary ammonium) (PQA) chains to the surface of PP.
A well-defined poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), a precursor of a PQA, was grown from the surface of PP via ATRP.(3) The tertiary ammine groups in PDMAEMA were subsequently converted to a quaternary amine groups in the presence of ethyl bromide
Polymeric surfaces are the most common surfaces encountered in everyday live and recent work has focused on preparing biocidal polymer surfaces. A non-leachable biocidal polypropylene (PP) surface was created by chemically attaching poly(quaternary ammonium) (PQA) chains to the surface of PP through a grafting from reaction. A well-defined poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), a precursor of a PQA, was grown from the surface of PP via ATRP.(3) The tertiary ammine groups in PDMAEMA were subsequently converted to quaternary amine groups in the presence of ethyl bromide providing an antibacterial surface that killed over 85% of contacting bacterial even at low graft density.
Successful surface modification was confirmed by ATR-IR, contact angle measurement, and an antibacterial activity test against Escherichia coli (E. coli), (see lower graph in schematic) showed that the biocidal activity of the resultant surfaces depends on the amount of the polymers grafted to the surface, i.e. the number of available quaternary ammonium units. Surfaces grafted with relatively high MW polymers (Mn > 10,000 g/mol) showed almost 100% killing efficiency, i.e. killing all of the added E. Coli (2.9 × 105) in the shaking test, whereas a lower biocidal activity (85%) was observed for the surface grafted with shorter PQA chains (Mn = 1,500 g/mol).
Many surfaces have inherent functional groups that can be employed to conduct a “grafting from” reaction, the only requirement is the ability to tether initiators to the target substrate. In the case of paper and glass this is readily accomplished by reacting surface hydroxyl groups with 2-bromoisobutyryl bromide.(2) ATRP of DMAEMA followed by quaternization with ethyl bromide provided effective tethered biocidal functionality. When paper was treated the modified surfaces were extremely effective at killing E.Coli, reducing the number of cells by four orders of magnitude, from 1.6 x 109 to 4.9 x 105, in one hour. The surface also showed activity against B. subtilis spores. The activity of a biocidal film on a glass surface survived repeated washing with aqueous detergent solution.
It is envisioned that such a permanent, nonleaching biocidal surface treatment would find utility in food packaging facilities, household items and military applications.
While grafting from is an efficient method of tethering quaternizable polymers to a substrate a more convenient approach for existing household equipment, and even hospital use, would be a "consumer friendly" "grafting onto" approach such as spraying a solution of a reactive copolymer onto a surface. In order to demonstrate the ‘grafting onto' approach PDMEAMA/PTMSPMA block copolymers of different molecular weights and different backbone topology were prepared via ATRP and immobilized on a glass surface by simple immersion of the glass slide in an aqueous solution of the copolymer.(4)
The efficiency of the tethering reaction was measured by addition of 1% of the sodium salt of fluorescein to the DMAEMA block prior to immersing the glass slide in the solution. Desorption of the dye after washing provided a measurement of the concentration of grafted chains on the surface. Grafting density was dependent on concentration and molecular weight of the copolymer as well as reaction time, with higher molecular weight copolymers providing lower graft density. Antibacterial activity depended only on the concentration of quaternary ammonium units tethered to the surface.
The interaction between bacteria and poly(quaternary ammonium) was investigated and the ability of the modified surfaces to kill bacteria was tested. Treated glass samples were incubated with a suspension of E. coli. The test results showed that the treated glass killed 104 ~ 106 of the contacting bacteria. Biocidal efficiency increased with the amount of quaternary ammonium units on the surface. Rather surprisingly introduction of hydrophobic units into a poly(quaternary ammonium) segment led to 100 times enhancement of biocidal activity with a log/kill of 7.0.
Modification of surfaces with tethered/tetherable polymers is applicable to a variety of surfaces. Control of surface morphology can be achieved by patterning the initiator layer and subsequent graft polymerization. Further variation of surface morphology can be achieved by phase separation on the nanoscale thereby providing precision surface modification.
Another advantage of surface-initiated ATRP is that it allows one to precisely design the morphology and property of the tethered polymer brushes. Indeed a gradient surface with varied grafting density or molecular weight has been successfully prepared by surface-initiated ATRP.(7)
The dry layer thickness of the polymer brushes was controlled by polymerization time and/or initiator density on the surface.
This ability to control all aspects surface structure allows the properties of the antimicrobial polymer brushes to be rationally tailored. A combinatorial screening tool was developed to elucidate the role of chain length and chain density on cell kill in a single experiment.
The results indicate that surface charge density, is a critical element in designing a surface for maximum kill efficiency. The biocidal surfaces that were most efficient had charge densities of greater than 1 - 5 X 1015 accessible quaternary amine units/cm2.
An extension of the procedures used for the preparing biocidal surfaces was the preparation of antibacterial magnetic particles via "grafting from".(5) Considering that the EPA indicated that 90% of the world's water is contaminated the concept was to use these particles to purify water. They could be used to provide simple portable water purification systems for individuals or larger scale static systems for villages or towns. Highly efficient recyclable antibacterial magnetite nanoparticles consisting of a magnetic Fe3O4 core with an antibacterial poly(quaternary ammonium) (PQA) coating were prepared as shown in the following schematic. ATRP initiators were tethered to the surface of the magnetic Fe3O4 nanoparticles and used to graft PDMAEMA chains from the surface. AFM images show all particles with grafted chains. The grafted chains were converted to PGA coating by reaction with ethyl bromide.
The advantages that magnetic particle grafted with biocides possess over present procedures are that there would be no release of chemicals as the biocide, which was permanately attached to the particle, could kill bacteria with high efficiency due to the inherent large surface area and after or during use the particles could be recycled by application of a magnetic field.
This was confirmed when the PQA-modified magnetite nanoparticles were dispersed in water and exhibited a response to an external magnetic field, making the nanoparticles easy to remove from water after antibacterial tests.
The PQA-modified magnetite nanoparticles retained 100% biocidal efficiency against E. coli (105 to 106 E. coli/mg nanoparticles) during eight exposure/collect/recycle procedures without washing with any solvents or water.
Biocidal surfaces are not the only type of bio-responsive surfaces that can be created from tethered poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes.
Continuing the study of selectively modified surfaces, surface plasmon resonance was used to measure the binding of proteins from solution to poly(2-(dimethylamino)ethyl methacrylate (PDMEMA) brushes end-grafted from gold surfaces.(6) These brushes displayed a high capacity for electrostatically selective protein uptake. The net negatively charged protein bovine serum albumin (BSA) was taken up in amounts that approach its aqueous solubility limit in the case of surfaces modified with high grafting densities PDMAEMA brushes. These are among the highest reported protein binding capacities for ion exchange media. BSA binding scaled linearly with the mass of PDMAEMA grafted per unit area, with a constant ratio of approximately 120 DMAEMA monomer units per bound BSA molecule. The kinetics of BSA uptake on the tethered brush is considerably more rapid than the slow asymptotic approach to adsorption saturation that is often seen for BSA adsorption to a solid surface. The high affinity for BSA was evident in the complete lack of desorption from the brush when rinsing with dilute NaCl solutions. BSA desorption from the brush required changes in solution pH and/or ionic strength to eliminate its net electrostatic attraction to PDMAEMA. Desorption could be achieved by using pH and/or ionic strength changes to interfere with the electrostatic attraction for BSA.
In contrast, PDMAEMA had no affinity for the net positively charged protein lysozyme. These results indicate that PDMAEMA can be a charge selective, high capacity ion exchange medium for protein binding.
One important advantage of grafting from method via CRP, specifically ATRP is the ease with which specific surfaces can be functionalized with initiating moieties for the preparation of well-defined high-density of polymer brushes. The highest grafting density can reach up to 1 chain/nm2. In that case, the polymer chain has a stretched conformation which is quite different from those of the semi-dilute polymer brushes previously studied, leading to very unique properties.
The field of polymer bioconjugation (i.e. covalent attachment of synthetic polymers to biological entities such as nucleic acids, oligopeptides, proteins, enzymes, carbohydrates, viruses or cells) has evolved rapidly during the last decade.(8-9) Polymer bioconjugates were initially developed by biochemists and had been exclusively studied for bio-medical applications. However, within the last few years the utility of this novel class of macromolecules has expanded and they are being examined in many emerging areas of materials science.(10-13) Both the bio-component and the attached polymer based segment can span a range of complexity and function(14) and the synthesis of polymer bioconjugates has emerged as a central topic in polymer chemistry.
A review paper which describes recent changes and progress in the field of polymer bio-conjugation; i.e. covalent attachment of synthetic polymers to biological entities such as nucleic acids, oligopeptides, proteins, enzymes, carbohydrates, viruses or cells, has been provided by two former group members.(12) The covalent bioconjugation approach can utilize either “grafting from” (I in following schematic) or “grafting to” (“click” reaction in following schematic) in which a preformed polymer with a reactive chain end is reacted with the targeted protein.(15)
The advantages of prior grafting to techniques are the ability to form protein-polymer conjugates with predefined polymers while the disadvantages are that the procedures provide non-uniform products and the products are hard to purify. Nevertheless modern copper catalyzed synthetic methods such as ATRP and ‘‘click’’ chemistry, a simplified schematic is shown below, have been recently proven to be extremely versatile tools for the preparation of tailor-made polymer bioconjugates.(17-18) Procedures for preparing such bioconjugates include preparation of functional telechelic polymers for direct attachment to the target bioresponsive molecule(19) or transformation of terminal groups on polymers prepared by ATRP for subsequent click reactions.(20-22)
The reverse approach is functionalization of a targeted bio-responsive molecule with an initiator for an ATRP followed by tethering a copolymer of desired composition and molecular weight is also receiving attention,(16,23-26) shown in the lower section of the following schematic which illustrates Maynard’s initial work grafting from a tetra-functionalized streptavadin molecule.
In “grafting from” procedures the initiator is immobilized onto the protein and polymerized in situ providing advantages of simple purification and high yields but poor reproducibility due to limited understanding of kinetics of the reaction. The grafting from method was employed in the group using a genetically incorporated initiator site at the 134 aspartic acid residue in a green fluorescent protein (GFP).(16) Polymerization initiated from the green fluorescent protein genetically modified at position 134 by incorporation of an amino acid modified to contain an ATRP initiator resulted in formation of a conjugate containing a single well-defined polymer chain per protein molecule with high MW.(16) that exhibited 95% initiator efficiency with retention of tertiary structure.
The advantages of genetically engineering proteins are that they maintain the highest possible protein functionality, 95% with retention of tertiary structure, by protecting active sites and avoid structural weak regions and protection of immune active sequences while allowing precise control of initiator loading via amber codon. The resulting GFP1-poly(OEOMA) showed thermoresponsive behavior and retained activity.
The protein conjugate could be incorporated into nanogels to assist in cellular delivery. This was accomplished by conducting a copolymerization of PEGMA and a PEG-dimethacrylate coinitiated by GFP1-initiator and a PEG-Br. The resulting nanogels provided a unique platform for protein delivery and enzymatic solid support systems. The successful incorporation of GFP1 demonstrates that proteins can be encapsulated whilst maintaining tertiary structure. N another example retained terminal functionality was used to attach catalase which converts hydrogen peroxide into water and oxygen thereby providing a simple assay for activity.
Composite structures prepared by this route include synthesis of near-uniform protein-polymer conjugates by initiating atom transfer radical polymerization of monomethoxy poly(ethylene glycol)-methacrylate from 2-bromoisobutyramide derivatives of chymotrypsin (a protein-initiator).(8) Protein-polymer conjugates synthesized by this novel technique retained 50-86% of the original enzyme activity.
Advantages of genetically engineering proteins are that one maintains highest possible protein functionality by protecting active sites thereby avoiding any structurally weak regions and protection of immune active sequences through precise control of initiator loading via amber codon.
Increasing the number of conjugated 2-bromoisobutyramide initiators per molecule of protein increased the MW and Mw/Mn of the final protein-polymer conjugates. The generic nature of this technique was demonstrated by initiating polymerization of nonionic, cationic, and anionic monomers from the protein-initiator. The technique described herein should be useful in synthesizing well-defined protein-polymer conjugates exhibiting a wide range of physical and chemical properties.
Functional nanoparticles have also been conjugated with bioresponsive molecules.(9) The synthetic procedure involved combining surface-initiated polymerization from magnetic nanoparticles with subsequent conjugation of the biologically active molecules generated materials. The products exhibited good separation capability and binding-specificity for bio-molecules. Polymeric shells of non-biofouling poly(poly(ethylene glycol) methacrylate) were initially introduced onto the surface of magnetic nanoparticles by surface-initiated ATRP. (25) This step was followed by successful post-functionalization via activation of the polymeric shells and bioconjugation of biotin. The resulting hybrids showed a bio-specific binding property for streptavidin and could be separated by magnet capture.(26)
One non-covalent bioconjugation approach is based on formation of an ionic complex through electrostatic interactions.(27) The requirements for delivery are that the bioconjugate forms a stable delivery system that allows prolonged circulation in the blood stream while incorporating functionality for targeted delivery through incorporation of specific ligands for receptors on target cells. The size of the nanogels control cellular uptake and incorporation of biodegradable links allows for controlled release of the delivered biomolecules.(28)
Another approach is the non-covalent approach which involves preparation of delivery systems that do not require direct linkage of the biologically active agent to the synthetic polymer includes synthesis of degradable star polymers for siRNA delivery systems.
Short Interfering RNA, (siRNA) was discovered in 1999.(29)siRNA is a double stranded RNA with 20-25 nucleotides with a negative charge due to the presence of phosphate groups. There has been progress in developing cationic vectors for non-viral systemic gene therapy for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized.(28) Dendrimers have been developed for siRNA delivery across the cell membrane(30) which would indicate that star macromolecules(31) which possess a similar core/shell structure with multiple arms, multiple surface functionality and a globular topology should also work.
The arm first route to star synthesis(32-33) was employed to prepare PEG-star macromolecules with a degradable core in addition to incorporated peripheral functionality to tailor the stars for siRNA delivery to MC3T3-E1.4 cells.(34) Successful degradation of PEG stars was demonstrated using glutathione and under a physiological concentration of GSH demonstrated. siRNA formed a complex with the cationically charged DMAEMA/QDMAEMA core and confocal microscopy analysis showed complete cellular uptake in 24 hours.
Another approach demonstrated was the preparation of N3-PEG Stars for siRNA delivery which is being conducted in collaboration with Leroux’s group in Switzerland. The targeting agent is “clicked” to the star prior to siRNA complexation.
The final “star” topic to be discussed in this section is preparation of surfaces for cell tissue engineering. A series of photo-cross-linkable thermoresponsive poly(oligo ethylene oxide)methacrylate star polymers were designed for control of cell-surface interactions. The star macromolecules were prepared and deposited on a polystyrene surface. The star polymers covalently attached onto surfaces allowed a control of cell shrinkage and attachment in response to temperature changes.(35)
Future work will focus on preparation of protein-polymer hybrids utilizing biologically compatible ATRP systems to overcome disadvantages of grafting-from methods. This requires development of ATRP systems with very low amounts of catalyst, solvent, and monomer polymerizable at room temperature, eATRP is one approach being evaluated. siRNA delivery systems are being developed that include biodegradable polymer scaffolds or biodegradable cationic nanogels.
Acrylate-based block copolymers were prepared by ATRP as matrices for paclitaxel delivery from coronary stents.(36-37) A series of multiblock acrylate-based copolymers, synthesized by ATRP were evaluated as drug delivery matrixes for the controlled release of paclitaxel from coronary stents. The copolymers consisted of poly(butyl acrylate) or poly(lauryl acrylate) soft blocks and hard blocks composed of poly(methyl methacrylate), poly(isobornyl acrylate), or poly(styrene) homo- or copolymers. Depending on the ratio of hard to soft blocks in the copolymers, coating formulations were produced that possessed variable elastomeric properties, resulting in stent coatings that maintained their integrity when assessed by SEM imaging of over-expanded stents. In vitro paclitaxel release kinetics from coronary stents coated with these copolymers typically showed an early burst followed by sustained release behavior, which permitted the elution of the majority of the paclitaxel over a 10-day time period. It was determined that neither the nature of the polyacrylate (butyl or lauryl) nor that of the hard block appeared to affect the release kinetics of paclitaxel at a loading of 25% drug by weight, whereas some effects were observed at lower drug loading levels.
Differential scanning calorimetry (DSC) analysis indicated that the paclitaxel was at least partially miscible with the poly(butyl acrylate) phase of those block copolymers. The copolymers were also evaluated for sterilization stability by exposing both the copolymer alone and copolymer/paclitaxel coated stents to e-beam radiation at doses of 1-3 times the nominal dose used for medical device sterilization (25 kGy). It was found that the copolymers containing blocks bearing quaternary carbons within the polymer backbone were less stable to the radiation and showed a decrease in molecular weight as determined by GPC. Conversely, those block copolymers without quaternary carbons showed no significant change in molecular weight when exposed to 3 times the standard radiation dose. There was no significant change in drug release profile from any of the acrylate-based copolymers after exposure to 75 kGy of e-beam radiation, and this was attributed to the inherent radiation stability of the poly(butyl acrylate) center block.
CRP methods can be used in biomedical applications to tune the degradation rates of bioresponsive materials, and to control chain size, uniformity, shape, composition, polymer microstructure and end groups. The creation of well defined 3D architectures, tailored surface multi-valency allows for the creation of synthetic water-soluble macromolecules and new supramolecular systems such as polymeric nanogels. The following schematic illustrates the degradation of a nanogel containing distributed degradable links along the copolymer backbone and at crosslinking branching units.
Stable biodegradable poly(oligo(ethylene oxide) monomethyl ether methacrylate), (POEOMA) nanogels, cross-linked with disulfide linkages were prepared by conducting ATRP in a cyclohexane inverse miniemulsion copolymerization conducted in the presence of a disulfide-functionalized dimethacrylate crosslinker.(38-39) These nanogels could be used for targeted drug delivery scaffolds for biomedical applications since they can be degraded into lower molecular weight polymers to release the encapsulated (bio)molecules.
These nanogels can be degraded to individual polymeric chains using a reducing agent, including glutathione tripeptide, which is commonly found in cells at mM concentrations, to release the encapsulated biomolecules. These materials were proven to be nontoxic to cells and are biocompatible.
The nanogels consist of a uniform three-dimensional network, since they are prepared by a controlled polymerization process, and are capable of entrapping drugs, proteins, and nucleic acids and delivering them to specific targets.These nanogels can be degraded to individual polymeric chains using a reducing agent (including glutathione tripeptide, which is commonly found in cells at mM concentrations), to release the encapsulated biomolecules. The encapsulation and controlled release of florescent dyes and doxorubicin (Dox), an anticancer drug, has been reported.(40) Results obtained from optical fluorescence microscope images and live/dead cytotoxicity assays of HeLa cancer cells suggested that the released Dox molecules penetrated cell membranes and therefore could suppress the growth of cancer cells
Furthermore, OH-functionalized nanogels were prepared to demonstrate facile applicability toward bioconjugation with biotin. The number of biotin molecules in each nanogel was determined to be 142,000 and the formation of bioconjugates of nanogels with avidin was confirmed using optical fluorescence microscopy. The hydroxy-functionalized poly(oligo(ethylene oxide) monomethyl ether methacrylate) (HO-POEOMA) was prepared by AGET ATRP of OEOMA initiated by 2-hydroxyethyl 2-bromoisobutyrate in water or in an inverse miniemulsion of water/cyclohexane at ambient temperature. The HO-POEOMA was then further functionalized with biotin, pyrene, and GRGDS peptide.(41) In addition, ATRP and click chemistry offered an efficient route for the synthesis of telechelic di-biotin polymers. These general methods can be applied to the formation of different functional materials conjugated with proteins, dyes, nucleic acids, and drugs.
The procedure was also used to entrapped rhodamine isothiocyanate-dextran (RITC-Dx) as a model for water-soluble bio-macromolecular drugs.(42) UV-Vis spectroscopy was used to characterize the extent of incorporation of RITC-Dx into the nanogels. The loading efficiency of RITC-Dx into the nanogels exceeded 80%. These nanogels were degraded into polymeric solutions in a reducing environment to release the encapsulated carbohydrate drugs. The released carbohydrate biomolecules specifically interacted with Concanavalin A in water, suggesting that the biodegradable nanogels could be used as carriers to deliver carbohydrate drugs that can be released upon degradation to bind to pathogens based on lectins.
The images above show a suspension of nanogels containing RITC-dextran in water before and after the addition of dithiothreitol (DTT) (a); and optical fluorescence microscopy images of nanogels loaded with RITC-dextran before (b) and after (c) degradation in water. The scale bars in b and c are 50 mm. These results demonstrate that biodegradation of nanogels can trigger the release of encapsulated molecules including RTIC-Dx and rhodamine 6G (fluorescent dyes), and Doxorubicin (Dox), an anticancer drug, as well as facilitate the removal of empty vesicles. This topic was reviewed recently.(43)
Another example of the synthesis of biodegradable materials for a specific envisioned application was the preparation of poly(N-isopropylacrylamide-co-5,6-benzo-2-methylene-1,3-dioxepane) (poly(NIPAAm-co-BMDO)) copolymer which was synthesized by a combination of ATRP and reversible addition-fragmentation chain transfer (RAFT) polymerization. The lower critical solution temperature (LCST) of poly (NIPAAm) and poly(NIPAAm-co-BMDO) copolymers were measured using UV-Vis spectroscopy to determine the effect of varying the amount of incorporated BMDO.(44) The material prepared in the paper is degradable and possesses a LCST above room temperature and below body temperature, making it a potential candidate for use as an injectable tissue engineering scaffold to enhance fracture repair. ATRP and RAFT enabled preparation of polymers with control over molecular weight, up to Mn = 50,000 g/mol and Mw/Mn < 1.2. Degradation studies were performed in basic solution and in complete Dulbecco's modified Eagle medium. The cytotoxicity of the material and its degradation products were analyzed by in vitro cell culture analyses, including cytotoxicity live/dead and CyQUANT cell proliferation assays. Crosslinked scaffolds with degradable units within the polymer backbone and at the crosslinking sites were prepared using an ester-containing diacrylate crosslinker. Furthermore, incorporation of a fraction of macromonomers with an ω-GRGDS peptide sequence improved cell attachment to the gels providing further evidence that controlled/living radical polymerization techniques allow for precise control over macromolecular structure exemplified in this paper by synthesis of materials suitable as a scaffold for tissue engineering.
The influence of the degree of methacrylation on hyaluronic acid hydrogels properties was examined.(45) The properties of hyaluronic acid (HA) hydrogels having a broad range of methacrylation were determined and increasing solubility of glycidyl methacrylate (GM) in a co-solvent mixture during the methacrylation of HA with GM was shown to produce photopolymerizable HAGM conjugates with various degree of methacrylation (DM) ranging from 14% up to 90%. Aqueous solutions of HAGM macromonomers were photocross-linked to yield hydrogels with nearly complete vinyl group conversions after 10 min exposure under ultraviolet light (UV). Hydrogels were characterized by uniaxial compression and volumetric swelling measurements. Keeping the DM constant, the shear modulus was varied from 16 kPa up to 73 kPa by varying the macromonomer concentration. However, at a given macromonomer concentration while varying the DM, similarly the shear modulus varied from 22 kPa up to 65 kPa. Preliminary in-vitro cell culture studies showed that GRGDS (Gly-Arg-Gly-Asp-Ser) modified HAGM hydrogels promoted similarly cell interaction at both low and high DMs, 32% and 60%, respectively. Densely cross-linked hydrogels with a high DM have been shown to be more mechanically robust while maintaining cytocompability and cell adhesion.
An extension of the work on hyaluronic acid hydrogels provided a new method to prepare nanostructured hybrid hydrogels incorporating well-defined poly(oligo (ethylene oxide) monomethyl ether methacrylate) (POEOMA300) nanogels of sizes 110-120 nm into larger three-dimensional (3D) matrix. Such materials are suitable for drug delivery scaffolds for tissue engineering applications.(46) Rhodamine B isothiocyanate-labeled dextran (RITC-Dx) or fluorescein isothiocyanate-labeled dextran (FITC-Dx)-loaded POEOMA300 nanogels with pendant hydroxyl groups were prepared by activators generated electron transfer atom transfer radical polymerization (AGET ATRP) in cyclohexane inverse miniemulsion. Hydroxyl-containing nanogels were functionalized with methacrylated groups to generate photoreactive nanospheres. 1H NMR spectroscopy confirmed that polymerizable nanogels were successfully covalently incorporated into 3D hyaluronic acid-glycidyl methacrylate (HAGM) hydrogels after free radical photo-polymerization.
The introduction of disulfide moieties into the polymerizable groups resulted in a controlled release of nanogels from the cross-linked HAGM hydrogels in a reducing environment. The effect of gel hybridization on the macroscopic properties, swelling and mechanics, was studied and the results showed that swelling and nanogel content are independent of scaffold mechanics. In-vitro assays showed the nanostructured hybrid hydrogels were cytocompatible and the GRGDS (Gly-Arg-Gly-Asp-Ser) contained in the nanogel structure promoted cell-substrate interactions within 4 days of incubation.
These nanostructured hydrogels have potential as an artificial extracellular matrix (ECM) impermeable to low molecular weight bio-active molecules with controlled pharmaceutical release capability. Moreover, the nanogels can provide controlled drug or bio-molecule delivery, while hyaluronic acid based-hydrogels can act as a macroscopic scaffold for tissue regeneration and regulator for nanogel release.
A further extension of biocompatible nanogels for bio-engineering applications was the in situ entrapment of gold nanoparticles (AuNPs), bovine serum albumin (BSA), rhodamine B isothiocyanate-dextran (RITC-Dx), and fluoresceine isothiocyanate-dextran (FITC-Dx) using atom transfer radical polymerization (ATRP) in cyclohexane inverse miniemulsion.(48) The nanogels consisted of poly(oligo(ethylene oxide) monomethyl ether methacrylate) (POEOMA), an analog of linear poly(ethylene oxide) (PEO) which can prevent nanoparticle uptake by reticuloendothelial system that were covalently attached to a GRGDS peptide sequence for targeted delivery. The nanogels were cross-linked using poly(ethylene oxide) dimethacrylate and initiated by a hydroxyl-functionalized initiator. Cell uptake of AuNPs-loaded nanogels was verified using transmission electron microscopy (TEM). Cell internalization of BSA-loaded nanogels was confirmed using fluorescence microscopy after the addition of a fluorescently tagged antibody for BSA, and by running a western blot of the cell extracts. Confocal imaging of RITC-Dx-loaded nanogels demonstrated that the nanogels were internalized, and the location of the nanogels was confirmed by staining for actin and nuclei, as well as by following along the z-axis. Furthermore, co-localization of the RITC signal with a signal for an FITC-conjugated antibody for clathrin indicated that the nanogels enter via clathrin mediated endocytosis. The internalization of FITC-Dx-loaded nanogels with and without GRGDS into cells was quantified using flow cytometry and exceeded 95%.
These results confirmed that uniform nanogels prepared by ATRP in inverse miniemulsion are endocytosed and are applicable as drug delivery devices.
The application of ATRP to nanogels synthesis allowed for the preparation of materials with many useful features.
- First, the resulting particles preserve a high degree of halide end-functionality to enable further chain extension to form functional block copolymers of direct functionalization with bio-related molecules, such as by utilizing click reactions.
- Second, they are degradable in a reducing environment to individual polymeric chains with a relatively narrow molecular weight distribution (Mw/Mn < 1.5), indicating the formation of a uniformly cross-linked network in the individual particles. This uniform structure is anticipated to improve control over the release of encapsulated agents.
- Third, properties including swelling ratio, degradation behavior, and colloidal stability of particles prepared by ATRP are superior to those prepared by conventional free radical inverse miniemulsion polymerization.
- Fourth, nanogels will enhance circulation time in the blood, because they can consist of poly(oligo(ethylene oxide) monomethyl ether methacrylate) (POEOMA), an analogue of linear poly(ethylene oxide) (PEO) that can prevent nanoparticle uptake by reticuloendothelial system (RES).
(1) Yu, W. H.; Kang, E. T.; Neoh, K. G.; Zhu, S. Journal of Physical Chemistry B 2003, 107, 10198-10205.
(2) Lee, S. B.; Koepsel, R. R.; Morley, S. W.; Matyjaszewski, K.; Sun, Y.; Russell, A. J. Biomacromolecules 2004, 5, 877-882.
(3) Huang, J.; Murata, H.; Koepsel, R. R.; Russell, A. J.; Matyjaszewski, K. Biomacromolecules 2007, 8, 1396-1399.
(4) Huang, J.; Koepsel, R. R.; Murata, H.; Wu, W.; Lee, S. B.; Kowalewski, T.; Russell, A. J.; Matyjaszewski, K. Langmuir 2008, 24, 6785-6795.
(5) Dong, H.; Huang, J.; Koepsel, R. R.; Ye, P.; Russell, A. J.; Matyjaszewski, K. Biomacromolecules 2011, 12, 1305-1311.
(6) Kusumo, A.; Bombalski, L.; Lin, Q.; Matyjaszewski, K.; Schneider, J. W.; Tilton, R. D. Langmuir 2007, 23, 4448-4454.
(7) Murata, H.; Koepsel, R. R.; Matyjaszewski, K.; Russell, A. J. Biomaterials 2007, 28, 4870-4879.
(8) Jones, M.-C.; Ranger, M.; Leroux, J.-C. Bioconjugate Chemistry 2003, 14, 774-781.
(9) Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6, 3380-3387.
(10) Nicolas, J.; Mantovani, G.; Haddleton, D. M. Macromolecular Rapid Communications 2007, 28, 1083-1111.
(11) Lutz, J.-F.; Boerner, H. G.; Weichenhan, K. Aust. J. Chem. 2007, 60, 410-413.
(12) Lutz, J.-F.; Boerner, H. G. Progress in Polymer Science 2008, 33, 1-39.
(13) Oh, J. K.; Drumright, R.; Siegwart, D. J.; Matyjaszewski, K. Prog. Polym. Sci. 2008, 33, 448-477.
(14) Oh, J. K.; Siegwart, D. J.; Lee, H.-i.; Sherwood, G.; Peteanu, L.; Hollinger, J. O.; Kataoka, K.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 5939-5945.
(15) Heredia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007, 5, 45-53.
(16) Peeler, J. C.; Woodman, B. F.; Averick, S.; Miyake-Stoner, S. J.; Stokes, A. L.; Hess, K. R.; Matyjaszewski, K.; Mehl, R. A. J. Am. Chem. Soc. 2010, 132, 13575-13577.
(17) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004-2021.
(18) Sumerlin, B. S.; Tsarevsky, N. V.; Louche, G.; Lee, R. Y.; Matyjaszewski, K. Macromolecules 2005, 38, 7540-7545.
(19) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372-15373.
(20) Le Droumaguet, B.; Mantovani, G.; Haddleton, D. M.; Velonia, K. J. Mat.Chem. 2007, 17, 1916-1922.
(21) Geng, J.; Mantovani, G.; Tao, L.; Nicolas, J.; Chen, G.; Wallis, R.; Mitchell, D. A.; Johnson, B. R. G.; Evans, S. D.; Haddleton, D. M. J. Am. Chem. Soc. 2007, 129, 15156-15163.
(22) Golas, P. L.; Matyjaszewski, K. Chemical Society Reviews 2010, 39, 1338-1354.
(23) Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 16955-16960.
(24) Broyer, R. M.; Quaker, G. M.; Maynard, H. D. J. Am. Chem. Soc. 2008, 130, 1041-1047.
(25) Kang, S. M.; Choi, I. S.; Lee, K.-B.; Kim, Y. Macromol. Res. 2009, 17, 259-264.
(26) Kang, S. M.; Lee, B. S.; Kim, W.-J.; Choi, I. S.; Kil, M.; Jung, H.-j.; Oh, E. Macromol. Res. 2009, 17, 174-180.
(27) Averick S E; Simakova A; Magenau A J D; Seong A; Woodman B F; Mehl R A; Matyjaszewski K. Polym. Chem. 2011, 2, published on web 3/24/11.
(28) Morille, M.; Passirani, C.; Vonarbourg, A.; Clavreul, A.; Benoit, J.-P. Biomaterials 2008, 29, 3477-3496.
(29) Hamilton, A. J.; Baulcombe, D. C. Science 1999, 286, 950-952.
(30) Blow, N. Nat. Methods 2009, 6, 305-309.
(31) Gao, H.; Matyjaszewski, K. Prog. Polym. Sci. 2009, 34, 317-350.
(32) Gao, H.; Matyjaszewski, K. Macromolecules 2006, 39, 3154-3160.
(33) Zhang, X.; Xia, J.; Matyjaszewski, K. Macromolecules 2000, 33, 2340-2345.
(34) Cho, H. Y.; Gao, H.; Srinivasan, A.; Hong, J.; Bencherif, S. A.; Siegwart, D. J.; Paik, H.-j.; Hollinger, J. O.; Matyjaszewski, K. Biomacromolecules 2010, 11, 2199-2203.
(35) Park, S.; Cho, H. Y.; Yoon, J. A.; Kwak, Y.; Srinivasan, A.; Hollinger, J. O.; Paik, H.-j.; Matyjaszewski, K. Biomacromolecules 2010, 11, 2647-2652.
(36) Richard, R. E.; Schwarz, M.; Ranade, S.; Chan, A. K.; Matyjaszewski, K.; Sumerlin, B. Biomacromolecules 2005, 6, 3410-3418.
(37) Richard, R. E.; Schwarz, M.; Ranade, S.; Chan, A. K.; Matyjaszewski, K.; Sumerlin, B. ACS Symposium Series 2006, 944, 234-251.
(38) Oh, J. K.; Tang, C.; Gao, H.; Tsarevsky, N. V.; Matyjaszewski, K. J. Am. Chem. Soc. 2006, 128, 5578-5584.
(39) Oh, J. K.; Perineau, F.; Matyjaszewski, K. Macromolecules 2006, 39, 8003-8010.
(40) Siegwart, D. J.; Oh, J. K.; Gao, H.; Bencherif, S. A.; Perineau, F.; Bohaty, A. K.; Hollinger, J. O.; Matyjaszewski, K. Macromol. Chem. Phys. 2008, 209, 2179-2193.
(41) Oh, J. K.; Siegwart, D. J.; Matyjaszewski, K. Biomacromolecules 2007, 8, 3326-3331.
(42) Siegwart, D. J.; Bencherif, S. A.; Srinivasan, A.; Hollinger, J. O.; Matyjaszewski, K. J. Biomed. Mater. Res., Part A 2008, 87A, 345-358.
(43) Bencherif Sidi, A.; Srinivasan, A.; Horkay, F.; Hollinger Jeffrey, O.; Matyjaszewski, K.; Washburn Newell, R. Biomaterials 2008, 29, 1739-1749.
(44) Bencherif, S. A.; Siegwart, D. J.; Srinivasan, A.; Horkay, F.; Hollinger, J. O.; Washburn, N. R.; Matyjaszewski, K. Biomaterials 2009, 30, 5270-5278.
(45) Siegwart, D. J.; Srinivasan, A.; Bencherif, S. A.; Karunanidhi, A.; Oh, J. K.; Vaidya, S.; Jin, R.; Hollinger, J. O.; Matyjaszewski, K. Biomacromolecules 2009, 10, 2300-2309.
(46) Oh, J. K.; Bencherif, S. A.; Matyjaszewski, K. Polymer 2009, 50, 4407-4423.