Stimuli-responsive reagent system for enabling microfluidic immunoassays with biomarker purification and enrichment.

Immunoassays have been translated into microfluidic device formats, but significant challenges relating to upstream sample processing still limit their applications. Here, stimuli-responsive polymer-antibody conjugates are utilized in a microfluidic immunoassay to enable rapid biomarker purification and enrichment as well as sensitive detection. The conjugates were constructed by covalently grafting poly(N-isopropylacrylamide) (PNIPAAm), a thermally responsive polymer, to the lysine residues of anti-prostate specific antigen (PSA) Immunoglobulin G (IgG) using carbodiimide chemistry via the polymer end-carboxylate. The antibody-PNIPAAm (capture) conjugates and antibody-alkaline phosphatase (detection) conjugates formed sandwich immunocomplexes via PSA binding in 50% human plasma. The complexes were loaded into a recirculating poly(dimethylsiloxane) microreactor, equipped with micropumps and transverse flow features, for subsequent separation, enrichment, and quantification. The immunocomplexes were captured by heating the solution to 39 °C, mixed over the transverse features for 2 min, and washed with warm buffer. In one approach, the assay utilized immunocomplex solution that was contained in an 80 nL microreactor, which was loaded with solution at room temperature and subsequently heated to 39 °C. The assay took 25 min and resulted in 37 pM PSA limit of detection (LOD), which is comparable to a plate ELISA employing the same antibody pair. In another approach, the microreactor was preheated to 39 °C, and immunocomplex solution was flowed through the reactor, mixed, and washed. When the specimen volume was increased to 7.5 µL by repeating the capture process three times, the higher specimen volume led to immunocomplex enrichment within the microreactor. The resulting assay LOD was 0.5 pM, which is 2 orders of magnitude lower than the plate ELISA. Both approaches generate antigen specific signal over a clinically significant range. The sample processing capabilities and subsequent utility in a biomarker assay demonstrate the opportunity for stimuli-responsive polymer-protein conjugates in novel diagnostic technologies.

A photoinduced nanoparticle separation in microchannels via pH-sensitive surface traps.

A microfluidic surface trap was developed for capturing pH-sensitive nanoparticles via a photoinitiated proton-releasing reaction of o-nitrobenzaldehyde (o-NBA) that reduces the solution pH in microchannels. The surface trap and nanoparticles were both modified with a pH-responsive polymer-poly(N-isorpopylacylamide-co-propylacrylic acid), P(NIPAAm-co-PAA). The o-NBA-coated microchannel walls demonstrated rapid proton release upon UV light irradiation, allowing the buffered solution pH in the microchannel to decrease from 7.4 to 4.5 in 60 s. The low solution pH switched the polymer-modified surfaces to be more hydrophobic, which enabled the capture of the pH-sensitive nanobeads onto the trap. When a photomask was utilized to limit the UV irradiation to a specific channel region, we were able to restrict the particle separation to only the exposed region. Via control of the UV irradiation, this technique enables not only prompt pH changes within the channel but also the capture of target molecules at specific channel locations.

A novel peptide probe for imaging and targeted delivery of liposomal doxorubicin to lung tumor.

Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Here we have screened a phage-displayed peptide library to identify peptides that selectively bind to lung tumor cells. Evaluation of individual phage clones after screening revealed that a phage clone displaying the CSNIDARAC peptide bound to H460 lung tumor cells at higher extent than other phage clones. The synthetic CSNIDARAC peptide strongly bound to H460 cells and was efficiently internalized into the cells, while little binding of a control peptide was seen. It also preferentially bound to other lung tumor cell lines as compared to cells of different tumor types. In vivo imaging of lung tumor was achieved by homing of fluorescence dye-labeled CSNIDARAC peptide to the tumor after intravenous injection into mice. Ex vivo imaging and microscopic analysis of isolated organs further demonstrated the targeting of CSNIDARAC peptide to tumor. The CSNIDARAC peptide-targeted and doxorubicin-loaded liposomes inhibited the tumor growth more efficiently than untargeted liposomes or free doxorubicin. In vivo imaging of fluorescence dye-labeled liposomes demonstrated selective homing of the CSNIDARAC-liposomes to tumor. In the same context, higher levels of doxorubicin and apoptosis in tumor tissue were observed when treated with the targeted liposomes than untargeted liposomes or free doxorubicin. These results suggest that the CSNIDARAC peptide is a promising targeting probe that is able to direct imaging agents and therapeutics to lung tumor.

"Smart" diblock copolymers as templates for magnetic-core gold-shell nanoparticle synthesis.

We report a new strategy for synthesizing temperature-responsive gamma-Fe(2)O(3)-core/Au-shell nanoparticles (Au-mNPs) from diblock copolymer micelles. The amphiphilic diblock copolymer chains were synthesized using reversible addition-fragmentation chain-transfer (RAFT) with a thermally responsive "smart" poly(N-isopropylacrylamide) (pNIPAAm) block and an amine-containing poly(N,N-dimethylaminoethylacrylamide) (DMAEAm) block that acted as a reducing agent during gold shell formation. The Au-mNPs reversibly aggregated upon heating the solution above the transition temperature of pNIPAAm, resulting in a red-shifted localized surface plasmon resonance.

A helical flow, circular microreactor for separating and enriching "smart" polymer-antibody capture reagents.

We report a mechanistic study of how flow and recirculation in a microreactor can be used to optimize the capture and release of stimuli-responsive polymer-protein reagents on stimuli-responsive polymer-grafted channel surfaces. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted to polydimethylsiloxane (PDMS) channel walls, creating switchable surfaces where PNIPAAm-protein conjugates would adhere at temperatures above the lower critical solution temperature (LCST) and released below the LCST. A PNIPAAm-streptavidin conjugate that can capture biotinylated antibody-antigen targets was first characterized. The conjugate's immobilization and release were limited by mass transport to and from the functionalized PNIPAAm surface. Transport and adsorption efficiencies were dependent on the aggregate size of the PNIPAAm-streptavidin conjugate above the LCST and also were dependent on whether the conjugates were heated in the presence of the stimuli-responsive surface or pre-aggregated and then flowed across the surface. As conjugate size increased, through the addition of non-conjugated PNIPAAm, recirculation and mixing were shown to markedly improve conjugate immobilization compared to diffusion alone. Under optimized conditions of flow and reagent concentrations, approximately 60% of the streptavidin conjugate bolus could be captured at the surface and subsequently successfully released. The kinetic release profile sharpness was also strongly improved with recirculation and helical mixing. Finally, the concentration of protein-polymer conjugates could be achieved by continuous conjugate flow into the heated recirculator, allowing nearly linear enrichment of the conjugate reagent from larger volumes. This capability was shown with anti-p24 HIV monoclonal antibody reagents that were enriched over 5-fold using this protocol. These studies provide insight into the mechanism of smart polymer-protein conjugate capture and release in grafted channels and show the potential of this purification and enrichment module for processing diagnostic samples.

Mixed stimuli-responsive magnetic and gold nanoparticle system for rapid purification, enrichment, and detection of biomarkers.

A new diagnostic system for the enrichment and detection of protein biomarkers from human plasma is presented. Gold nanoparticles (AuNPs) were surface-modified with a diblock copolymer synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. The diblock copolymer contained a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) block, a cationic amine-containing block, and a semi-telechelic PEG2-biotin end group. When a mixed suspension of 23 nm pNIPAAm-modified AuNPs was heated with pNIPAAm-coated 10 nm iron oxide magnetic nanoparticles (mNPs) in human plasma, the thermally responsive pNIPAAm directed the formation of mixed AuNP/mNP aggregates that could be separated efficiently with a magnet. Model studies showed that this mixed nanoparticle system could efficiently purify and strongly enrich the model biomarker protein streptavidin in spiked human plasma. A 10 ng/mL streptavidin sample was mixed with the biotinylated pNIPAAm-modified AuNPs and magnetically separated in the mixed nanoparticle system with pNIPAAm mNPs. The aggregates were concentrated into a 50-fold smaller fluid volume at room temperature where the gold nanoparticle reagent redissolved with the streptavidin target still bound. The concentrated gold-labeled streptavidin could be subsequently analyzed directly using lateral flow immunochromatography. This rapid capture and enrichment module thus utilizes the mixed stimuli-responsive nanoparticle system to achieve concentration of a gold-labeled biomarker that can be directly analyzed using lateral flow or other rapid diagnostic strategies.

Intracellular delivery of a protein antigen with an endosomal-releasing polymer enhances CD8 T-cell production and prophylactic vaccine efficacy.

Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their inability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA) was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5-fold increase in the longevity of tumor-free survival relative to controls. Mechanistically, this response was attributed to the 8-fold increase in production of ovalbumin-specific CD8+ T-lymphocytes and an 11-fold increase in production of antiovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications.

Simple fluidic system for purifying and concentrating diagnostic biomarkers using stimuli-responsive antibody conjugates and membranes.

We report a simple fluidic system that can purify and concentrate diagnostic biomarkers through the capture and triggered release of stimuli-responsive polymer-antibody conjugates at porous membranes that are grafted with the same stimuli-responsive polymer. This technique is applied here to the capture and detection of a model streptavidin antigen and subsequently to clinical ranges of the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) from spiked human plasma. The carboxyl end-groups of semi-telechelic poly(N-isopropylacrylamide) (pNIPAAm) synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization were modified with tetrafluorophenol to yield amine-reactive ester groups for conjugation to amine groups of anti-streptavidin and anti-PfHRP2 antibodies. Stimuli-responsive membranes were constructed from 1.2 µm pore-size, hydroxylated, nylon-6,6 filters (Loprodyne, from Pall Corporation). The surface hydroxyl groups on the filters were conjugated to a 2-ethylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (EMP) RAFT chain transfer agent, and the surface-grafted pNIPAAm was obtained by subsequent polymerization. The number average molecular weight (Mn) and polydispersity indices (PDI) of the surface grafts were characterized, and membranes with either 4100 and 8400 dalton pNIPAAm grafts showed greater than 80% anti-streptavidin capture efficiency. The 8400 dalton-graft membrane showed the highest release efficiency, and it was demonstrated that at 0.2 nM starting concentration the streptavidin could be concentrated approximately 40-fold by releasing into a small 50 µL volume. This concentrator system was applied to the capture and concentration of the PfHRP2 antigen, and results showed that the PfHRP2 antigen could be processed and detected at clinically relevant concentrations of this malaria biomarker.

Intracellular delivery of a proapoptotic peptide via conjugation to a RAFT synthesized endosomolytic polymer.

Peptides derived from the third B-cell lymphoma 2 (Bcl-2) homology domain (BH3) can heterodimerize with antiapoptotic Bcl-2 family members to block their activity and trigger apoptosis. Use of these peptides presents a viable anticancer approach, but delivery barriers limit the broad application of intracellular-acting peptides as clinical therapeutics. Here, a novel diblock copolymer carrier is described that confers desirable pharmaceutical properties to intracellular-acting therapeutic peptides through site-specific molecular conjugation. This polymer was prepared using reversible addition-fragmentation chain transfer (RAFT) to form a pyridyl disulfide end-functionalized, modular diblock copolymer with precisely controlled molecular weight (M(n)) and low polydispersity (PDI). The diblock polymer (M(n) 19,000 g/mol, PDI 1.27) was composed of an N-(2-hydroxypropyl) methacrylamide (HPMA) first block (M(n) 13,800 g/mol, PDI 1.13) intended to enhance water solubility and circulation time. The second polymer block was a pH-responsive composition designed to enhance endosomal escape and consisted of equimolar quantities of dimethylaminoethyl methacrylate (DMAEMA), propylacrylic acid (PAA), and butyl methacrylate (BMA). A hemolysis assay indicated that the diblock polymer undergoes a physiologically relevant pH-dependent switch from a membrane inert (1% hemolysis, pH 7.4) to a membrane disruptive (61% hemolysis, pH 5.8) conformation. Thiol-disulfide exchange reactions were found to efficiently produce reversible polymer conjugates (75 mol % peptide reactivity with polymer) with a cell-internalized proapoptotic peptide. Microscopy studies showed that peptide delivered via polymer conjugates effectively escaped endosomes and achieved diffusion into the cytosol. Peptide-polymer conjugates also produced significantly increased apoptotic activity over peptide alone in HeLa cervical carcinoma cells as found using flow cytometric measurements of mitochondrial membrane depolarization (2.5-fold increase) and cell viability tests that showed 50% cytotoxicity after 6 h of treatment with 10 muM peptide conjugate. These results indicate that this multifunctional carrier shows significant promise for proapoptotic peptide cancer therapeutics and also as a general platform for delivery of peptide drugs with intracellular targets.

pH-responsive polymeric sirna carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.

Synthesis of statistical copolymers containing multiple functional peptides for nucleic Acid delivery.

Our report describes RAFT copolymerization of multiple species of active peptide monomers with N-(2-hydroxypropyl(methacrylamide) (HPMA) under aqueous conditions. Resulting statistical copolymers are narrowly disperse with highly controlled molecular weight and composition. Side-chain peptide copolymers were synthesized using a DNA condensing peptide (K12), and an endosomal escape peptide (K6H5) that had been modified with an aminohexanoic linker and capped with methacrylamide vinyl on the NH2-terminus. Copolymers of HMPA-co-K12 and HPMA-co-K12-co-K6H5 efficiently condensed DNA into small particles that maintain size stability even in 150 mM salt solutions. With increasing peptide content, the peptide-based polymers demonstrated gene delivery efficiencies to HeLa cells that were comparable to branched polyethylenimine.

Dynamic bioprocessing and microfluidic transport control with smart magnetic nanoparticles in laminar-flow devices.

In the absence of applied forces, the transport of molecules and particulate reagents across laminar flowstreams in microfluidic devices is dominated by the diffusivities of the transported species. While the differential diffusional properties between smaller and larger diagnostic targets and reagents have been exploited for bioseparation and assay applications, there are limitations to methods that depend on these intrinsic size differences. Here a new strategy is described for exploiting the sharply reversible change in size and magnetophoretic mobility of "smart" magnetic nanoparticles (mNPs) to perform bioseparation and target isolation under continuous flow processing conditions. The isolated 5 nm mNPs do not exhibit significant magnetophoretic velocities, but do exhibit high magnetophoretic velocities when aggregated by the action of a pH-responsive polymer coating. A simple external magnet is used to magnetophorese the aggregated mNPs that have captured a diagnostic target from a lower pH laminar flowstream (pH 7.3) to a second higher pH flowstream (pH 8.4) that induces rapid mNP disaggregation. In this second dis-aggregated state and flowstream, the mNPs continue to flow past the magnet rather than being immobilized at the channel surface near the magnet. This stimuli-responsive reagent system has been shown to transfer 81% of a model protein target from an input flowstream to a second flowstream in a continuous flow H-filter device.

Antigen delivery with poly(propylacrylic acid) conjugation enhances MHC-1 presentation and T-cell activation.

While many infectious diseases are controlled by vaccine strategies, important limitations continue to motivate the development of better antigen delivery systems. This study focuses on the use of a pH-sensitive polymeric carrier based on poly(propylacrylic acid) (PPAA) to address the need for more potent CD8 cytotoxic T-cell (CTL) responses. An MHC-1/CD8 CTL cell model system with ovalbumin as the protein antigen was used to test whether PPAA could enhance the delivery of ovalbumin into the MHC-1 display pathway. Ovalbumin was conjugated to poly(propylacrylic acid-co-pyridyldisulfide acrylate) (PPAA-PDSA) by disulfide exchange to make reversible conjugates that could be reduced by the glutathione redox system in the cytosol of antigen presenting cells. The PPAA-PDSA ovalbumin conjugates displayed the pH-sensitive membrane disruptive properties of the parent polymer as determined by their hemolysis activities (sharply active at the endosomal pH values of 6-6.5). The polymer-ovalbumin conjugates exhibited strong 22-fold increases in the MHC-1 presentation and ovalbumin-specific CTL activation compared to free ovalbumin. No CTL activation was observed with control conjugates of ovalbumin and poly(methylacrylic acid) (PMAA) that do not display membrane disruptive activies, suggesting that it is the membrane destabilizing properties of the polymer that result in increased MHC-1 display and CTL activation. Further mechanistic studies quantitated the time course of stable intracellular localization of radiolabeled conjugates. 52% of initially internalized PPAA-conjugated ovalbumin remained in the cells after 4 h, compared to less than 10% of ovalbumin or PMAA-ovalbumin. These results showing enhanced cytosolic delivery and MHC-1 presentation for the PPAA-antigen conjugates suggest that they warrant future characterization as a CD8-enhancing vaccine delivery system.

End-functionalized polymers and junction-functionalized diblock copolymers via RAFT chain extension with maleimido monomers.

A new strategy is described for functionalizing the omega-terminal end of polymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization that provides spatially controlled bioconjugation sites. Traditional methods for preparing omega-functional polymers require the reduction of the RAFT chain-transfer agent to yield secondary or tertiary thiols of low reactivity or the synthesis of novel chain-transfer agents that contain reactive groups. As an additional strategy, N-substituted maleimido monomers have been used in a modified block polymerization to add a single maleimido unit onto the RAFT polymer with nearly quantitative efficiency. Unique reactive groups contained in the N-substituent are thereby added to the omega-terminal end of the polymer and are subsequently available for conjugation reactions. This technique has been demonstrated using N-(2-aminoethyl)maleimide trifluoroacetate to introduce a single primary amine to the omega-terminus of poly(dimethylaminoethyl methacrylate) and poly(N-isopropyl acrylamide) and to a specialized block copolymer for siRNA delivery. Evidence for retention of functional RAFT endgroups is provided by synthesis results where chain-extended polyDMAEMA (M(n) = 10 600 g/mol, M(w)/M(n) = 1.14) was used as a macro chain transfer agent for the polymerization of styrene, yielding a diblock polymer of low polydispersity (M(n) = 20 300 g/mol, M(w)/M(n) = 1.11). It is thus also possible to construct diblock copolymers with a bioconjugation site precisely located at the junction between the two blocks. The chain-extended polymers are functionalized with an amine-reactive fluorescent dye or folic acid at conjugation efficiencies of 86 and 94%, respectively. The versatile chain-extension technique described here offers unique opportunities for the synthesis of well-defined polymeric conjugates to molecules of biological and targeting interest.

Photo-cross-linked hydrogels from thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers containing multiple methacrylate groups: mechanical property, swelling, protein release, and cytotoxicity.

Photo-cross-linked hydrogels from thermoresponsive polymers can be used as advanced injectable biomaterials via a combination of physical interaction (in situ thermal gelation) and covalent cross-links (in situ photopolymerization). This can lead to gels with significantly enhanced mechanical properties compared to non-photo-cross-linked thermoresponsive hydrogels. Moreover, the thermally phase-separated gels have attractive advantages over non-thermoresponsive gels because thermal gelation upon injection allows easy handling and holds the shape of the gels prior to photopolymerization. In this study, water-soluble thermoresponsive copolymers containing multiple methacrylate groups were synthesized via one-step deactivation enhanced atom transfer radical polymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, M(n) = 475), poly(propylene glycol) methacrylate (PPGMA, M(n) = 375), and ethylene glycol dimethacrylate (EGDMA) and were used to form covalent cross-linked hydrogels by photopolymerization. The cross-linking density was found to have a significant influence on the mechanical and swelling properties of the photo-cross-linked gels. Release studies using lysozyme as a model protein demonstrated a sustained release profile that varied dependent on the copolymer composition, cross-linking density, and the temperature. Mouse C2C12 myoblast cells were cultured in the presence of the copolymers at concentrations up to 1 mg/mL. It was found that the majority of the cells remained viable, as assessed by Alamar Blue, lactate dehydrogenase (LDH), and Live/Dead cell viability/cytotoxicity assays. These studies demonstrate that thermoresponsive PEGMEMA-PPGMA-EGDMA copolymers offer potential as in situ photopolymerizable materials for tissue engineering and drug delivery applications through a combination of facile synthesis, enhanced mechanical properties, tunable cross-linking density, low cytotoxicity, and accessible functionality for further structure modifications.

Encapsulation and stabilization of indocyanine green within poly(styrene-alt-maleic anhydride) block-poly(styrene) micelles for near-infrared imaging.

Indocyanine green (ICG) is a Federal Drug Administration-approved near-infrared imaging agent susceptible to chemical degradation, nonspecific binding to blood proteins, and rapid clearance from the body. In this study, we describe the encapsulation of ICG within polymeric micelles formed from poly(styrene-alt-maleic anhydride)-block-poly(styrene) (PSMA-b-PSTY) diblock copolymers to stabilize ICG for applications in near-infrared diagnostic imaging. In aqueous solution, the diblock copolymers self-assemble to form highly stable micelles approximately 55 nm in diameter with a critical micelle concentration (CMC) of approximately 1 mg/L. Hydrophobic ICG salts readily partition into the PSTY core of these micelles with high efficiency, and produce no change in micelle morphology or CMC. Once loaded in the micelle core, ICG is protected from aqueous and thermal degradation, with no significant decrease in fluorescence emission over 14 days at room temperature and retaining 63% of its original emission at 37 degrees C. Free ICG does not release rapidly from the micelle core, with only 11% release over 24 h. The ICG-loaded micelles do not exhibit significant cell toxicity. This system has the potential to greatly improve near-infrared imaging in breast cancer detection by increasing the stability of ICG for formulation/administration, and by providing a means to target ICG to tumor tissue.

Design and development of polymers for gene delivery.

The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.

A Combinational Effect of "Bulk" and "Surface" Shape-Memory Transitions on the Regulation of Cell Alignment.

A novel shape-memory cell culture platform has been designed that is capable of simultaneously tuning surface topography and dimensionality to manipulate cell alignment. By crosslinking poly(ε-caprolactone) (PCL) macromonomers of precisely designed nanoarchitectures, a shape-memory PCL with switching temperature near body temperature is successfully prepared. The temporary strain-fixed PCLs are prepared by processing through heating, stretching, and cooling about the switching temperature. Temporary nanowrinkles are also formed spontaneously during the strain-fixing process with magnitudes that are dependent on the applied strain. The surface features completely transform from wrinkled to smooth upon shape-memory activation over a narrow temperature range. Shape-memory activation also triggers dimensional deformation in an initial fixed strain-dependent manner. A dynamic cell-orienting study demonstrates that surface topographical changes play a dominant role in cell alignment for samples with lower fixed strain, while dimensional changes play a dominant role in cell alignment for samples with higher fixed strain. The proposed shape-memory cell culture platform will become a powerful tool to investigate the effects of spatiotemporally presented mechanostructural stimuli on cell fate.

Switchable surface traps for injectable bead-based chromatography in PDMS microfluidic channels.

We report here a reversible microchannel surface capture system for stimuli-responsive grafted bioanalytical beads. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted onto polydimethylsiloxane (PDMS) surfaces by a UV-mediated graft polymerization from a photoinitiator that was preadsorbed in the channel wall. The surface grafting density and resulting switchable hydrophilic/hydrophobic properties were controlled by varying the photo-illumination times and/or the initiator concentration. At limiting PNIPAAm-graft densities, the surfaces demonstrated minimal contact angles of 35 degrees below the lower critical solution temperature (LCST) and maximal contact angles of 82 degrees above it. These contact angles could be varied depending on the graft density. The surface grafts are spatially limited to the photo-illuminated region to define where the trap is constructed. The surface traps capture PNIPAAm-grafted nanobeads uniformly above the LCST and facilitate their rapid release as the temperature is reversed to below the LCST. This dual surface trap and injectable chromatography system could be useful in many applications, such as affinity separations, immunoassays, and enzyme bioprocesses, by providing for the controlled capture and release of chromatography beads.

The early days of PEG and PEGylation (1970s-1990s).

UNLABELLED: The idea to conjugate PEG [poly(ethylene glycol)] to a protein, i.e., to "PEGylate" a protein, was first proposed by Prof. Frank Davis (Rutgers Univ.) in the late 1960s. He wanted to make the new recombinant proteins less immunogenic in our bodies, and thereby enhance their circulation and activity lifetimes. He thought that if he could conjugate a hydrophilic polymer to the "new" protein, it might not be recognized by the immune system as a foreign molecule. This article is a contribution to the Zwitterionic Special Issue as a personal commentary tracing the story of PEGylation from its beginning with Dr. Davis through a current day post-script. STATEMENT OF SIGNIFICANCE: The author knows (or knew) personally most of the early workers in the fields of PEG, PEGylation and non-fouling surfaces, and he has also been personally active in the field since its early days.