A Stimuli-Responsive, Binary Reagent System for Rapid Isolation of Protein Biomarkers.

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab's binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ∼2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.

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.

The gut microbiota regulates acute foreign body reaction and tissue repair after biomaterial implantation.

We hypothesized that the host microbiome may influence foreign body responses following biomaterial implantation. To test this, we implanted a variety of clinically relevant biomaterials into germ-free or antibiotic-treated mice. Surprisingly, these mice displayed less fibrous tissue deposition, reduced host cell recruitment to the implant site, and differential expression of angiogenic and inflammatory markers. These observations were reversed upon fecal microbiome reconstitution, confirming a causal role of the host microbiome. In a clinically relevant disease model, microbiome-depleted mice cleared hyaluronic acid and bone marrow mononuclear cells from ischemic hind limb tissues more slowly, resulting in an improved therapeutic response. Findings were confirmed in pigs which showed reduced fibrotic responses to a variety of implanted materials. Lastly, we profiled changes in the host microbiome following material implantation, implicating several key bacteria phyla.

"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.

Bio-Inspired Amphoteric Polymer for Triggered-Release Drug Delivery on Breast Cancer Cells Based on Metal Coordination.

Nanoscale coordination polymers are promising vehicles for anticancer drug delivery because their surface composition and particle size can be tuned to exploit the enhanced permeability and retention effect, and their reversible interaction with metal cations enables triggered drug release at the tumor site. Here, we develop a novel nanoscale coordination polymer using the diblock copolymer poly(2-methacryloyloxyethyl phosphorylcholine)-block-poly(serinyl acrylate) (PMPC-b-PserA) and demonstrate its use for encapsulation of a hydrophobic drug and triggered drug release to induce breast cancer cell apoptosis in vitro. The zwitterionic PMPC block was inspired by the antifouling structure of cell membranes, and the PserA block was inspired by the amphoteric amino acids of proteins. The polymer was synthesized by reversible addition-fragmentation chain transfer polymerization, and a mixture of the polymer and FeCl3 self-assembled into nanoparticles via complexation of Fe3+ with PserA, with the hydrophilic PMPC block at the particle surface. At a molar ratio of Fe3+ to serA of 3:1, the hydrodynamic diameter of the particles was 22.2 nm. Curcumin, a natural water-insoluble polyphenol used to enhance the effects of chemotherapeutics, was encapsulated in the particles as an oil-in-water emulsion, with an encapsulation efficiency of 99.6% and a particle loading capacity of 32%. Triggered release of curcumin was achieved by adding deferoxamine, an FDA-approved Fe3+ chelating agent; curcumin release efficiency increased at higher deferoxamine concentrations and lower pH. Triggered release of curcumin induced apoptosis in human triple-negative breast cancer cells; cell viability decreased to 34.3% after 24 h of treatment with the curcumin-loaded nanoparticles and deferoxamine, versus >80% viability without deferoxamine to trigger drug release. The biocompatibility, tunable composition and size, high hydrophobic drug loading, and triggered-release capability of this nanoscale coordination polymer make it well-suited for use in anticancer drug delivery.

Conjugation of antibody with temperature-responsive polymer via in situ click reaction to enable biomarker enrichment for increased diagnostic sensitivity.

Early diagnosis of infectious diseases is one of the current prevalent challenges, especially in low and limited resource settings where simple, fast, portable, cheap, and sensitive diagnostic approaches are needed. Lateral flow immunoassay (LFIA) is a common, rapid screening assay. However, the low assay sensitivity limits the utility of LFIA for specimens with low pathogenic loads (early infection stages). Antibodies conjugated with stimulus-responsive polymers have been previously utilized to improve assay sensitivity for detection of biomarkers at low concentrations. However, the loss of antibody affinity after polymer conjugation remains a significant challenge. In this study, we developed poly(N-isopropylacrylamide-co-N-(2-hydroxyisopropyl)acrylamide-co-strained alkyne-isopropylacrylamide), a novel polymer for biomarker enrichment, by polymer conjugation after antibody-antigen recognition. We employed and promoted the click chemistry in situ, to facilitate highly specific conjugation between novel temperature-responsive polymers and antibody-antigen complexes. This method could suppress the decrease in the binding constant associated with polymer conjugation (>20-fold). The conjugation was successfully demonstrated in body fluids such as urine and saliva. We achieved >5-fold antigen enrichment via thermal precipitation by conjugating polymers to the antibodies after antigen recognition. Concentrated biomarkers resulted in improved LFIA detection. This approach can potentially be utilized to improve diagnostic tests for infectious diseases in low and limited resource settings.

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.

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.

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.

Temperature-Responsive Magnetic Nanoparticles for Enabling Affinity Separation of Extracellular Vesicles.

Small magnetic nanoparticles that have surfaces decorated with stimuli-responsive polymers can be reversibly aggregated via a stimulus, such as temperature, to enable efficient and rapid biomarker separation. To fully realize the potential of these particles, the synthesis needs to be highly reproducible and scalable to large quantity. We have developed a new synthesis for temperature-responsive magnetic nanoparticles via an in situ co-precipitation process of Fe2+/Fe3+ salts at room temperature with poly(acrylic acid)- block-poly( N-isopropylacrylamide) diblock co-polymer template, synthesized via the reversible addition-fragmentation chain-transfer polymerization method. These particles were 56% polymer by weight with a 6.5:1 Fe/COOH ratio and demonstrated remarkable stability over a 2 month period. The hydrodynamic diameter remained constant at ∼28 nm with a consistent transition temperature of 34 °C, and the magnetic particle separation efficiency at 40 °C was ≥95% over the 2 month span. These properties were maintained for all large-scale synthesis batches. To demonstrate the practical utility of the stimuli-responsive magnetic nanoparticles, the particles were incorporated into a temperature-responsive binary reagent system and efficiently separated a model protein biomarker (mouse IgG) as well as purified extracellular vesicles derived from a human biofluid, seminal plasma. The ease of using these particles will prove beneficial for various biomedical applications.

Reloadable multidrug capturing delivery system for targeted ischemic disease treatment.

Human clinical trials of protein therapy for ischemic diseases have shown disappointing outcomes so far, mainly because of the poor circulatory half-life of growth factors in circulation and their low uptake and retention by the targeted injury site. The attachment of polyethylene glycol (PEG) extends the circulatory half-lives of protein drugs but reduces their extravasation and retention at the target site. To address this issue, we have developed a drug capture system using a mixture of hyaluronic acid (HA) hydrogel and anti-PEG immunoglobulin M antibodies, which, when injected at a target body site, can capture and retain a variety of systemically injected PEGylated therapeutics at that site. Furthermore, repeated systemic injections permit "reloading" of the capture depot, allowing the use of complex multistage therapies. This study demonstrates this capture system in both murine and porcine models of critical limb ischemia. The results show that the reloadable HA/anti-PEG system has the potential to be clinically applied to patients with ischemic diseases, who require sequential administration of protein drugs for optimal outcomes.

Improving lateral-flow immunoassay (LFIA) diagnostics via biomarker enrichment for mHealth.

Optical detection technologies based on mobile devices can be utilized to enable many mHealth applications, including a reader for lateral-flow immunoassay (LFIA). However, an intrinsic challenge associated with LFIA for clinical diagnostics is the limitation in sensitivity. Therefore, rapid and simple specimen processing strategies can directly enable more sensitive LFIA by purifying and concentrating biomarkers. Here, a binary reagent system is presented for concentrating analytes from a larger volume specimen to improve the malaria LFIA's limit of detection (LOD). The biomarker enrichment process utilizes temperature-responsive gold-streptavidin conjugates, biotinylated antibodies, and temperature-responsive magnetic nanoparticles. The temperature-responsive gold colloids were synthesized by modifying the citrate-stabilized gold colloids with a diblock copolymer, containing a thermally responsive poly(N-isopropylacrylamide) (pNIPAAm) segment and a gold-binding block composed of NIPAAm-co-N,N-dimethylaminoethylacrylamide. The gold-streptavidin conjugates were synthesized by conjugating temperature-responsive gold colloids with streptavidin via covalent linkages using carbodiimide chemistry chemistry. The gold conjugates formed half-sandwiches, gold labeled biomarker, by complexing with biotinylated antibodies that were bound to Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria antigen. When a thermal stimulus was applied in conjunction with a magnetic field, the half-sandwiches and temperature-responsive magnetic nanoparticles that were both decorated with pNIPAAm formed large aggregates that were efficiently magnetically separated from human plasma. The binary reagent system was applied to a large volume (500 µL) specimen for concentrating biomarker 50-fold into a small volume and applied directly to an off-the-shelf malaria LFIA to improve the signal-to-noise ratio.

Three-dimensional localization of polymer nanoparticles in cells using ToF-SIMS.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) three-dimensional (3D) depth profiling and a novel background subtraction method were used to localize polymeric nanoparticles within cells. Results showed that ToF-SIMS 3D depth profiling is capable of localizing polymer nanoparticles within HeLa cells. ToF-SIMS results compared well with optical images of cells incubated with fluorescently labeled polymer nanoparticles, with both imaging techniques demonstrating clustering of nanoparticles in punctate regions consistent with endosomal localization as anticipated based on the nanoparticle design.

Functionalized nanoparticles provide early cardioprotection after acute myocardial infarction.

Recent developments in nanotechnology have created considerable potential toward diagnosis and cancer therapy. In contrast, the use of nanotechnology in tissue repair or regeneration remains largely unexplored. We hypothesized that intramyocardial injection of insulin-like growth factor (IGF)-1-complexed poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (PLGA-IGF-1 NPs) increases IGF-1 retention, induces Akt phosphorylation, and provides early cardioprotection after acute myocardial infarction (MI). We synthesized 3 different sizes of PLGA particles (60 nm, 200 nm, and 1 µm) which were complexed with IGF-1 using electrostatic force to preserve the biological function of IGF-1. Afterward, we injected PLGA-IGF-1 NPs in the heart after MI directly. Compared with the other two larger particles, the 60 nm-sized PLGA-IGF-1 NPs carried more IGF-1 and induced more Akt phosphorylation in cultured cardiomyocytes. PLGA-IGF-1 NPs also prolonged Akt activation in cardiomyocytes up to 24h and prevented cardiomyocyte apoptosis induced by doxorubicin in a dose-dependent manner. In vivo, PLGA-IGF-1 NP treatment significantly retained more IGF-1 in the myocardium than the IGF-1 alone treatment at 2, 6, 8, and 24 h. Akt phosphorylation was detected in cardiomyocytes 24h post-MI only in hearts receiving PLGA-IGF-1 NP treatment, but not in hearts receiving injection of PBS, IGF-1 or PLGA NPs. Importantly, a single intramyocardial injection of PLGA-IGF-1 NPs was sufficient to prevent cardiomyocyte apoptosis (P<0.001), reduce infarct size (P<0.05), and improve left ventricle ejection fraction (P<0.01) 21 days after experimental MI in mice. Our results not only demonstrate the potential of nanoparticle-based technology as a new approach to treating MI, but also have significant implications for translation of this technology into clinical therapy for ischemic cardiovascular diseases.

Dual magnetic-/temperature-responsive nanoparticles for microfluidic separations and assays.

A stimuli-responsive magnetic nanoparticle system for diagnostic target capture and concentration has been developed for microfluidic lab card settings. Telechelic poly(N-isopropylacrylamide) (PNIPAAm) polymer chains were synthesized with dodecyl tails at one end and a reactive carboxylate at the opposite end by the reversible addition fragmentation transfer technique. These PNIPAAm chains self-associate into nanoscale micelles that were used as dimensional confinements to synthesize the magnetic nanoparticles. The resulting superparamagnetic nanoparticles exhibit a gamma-Fe2O3 core ( approximately 5 nm) with a layer of carboxylate-terminated PNIPAAm chains as a corona on the surface. The carboxylate group was used to functionalize the magnetic nanoparticles with biotin and subsequently with streptavidin. The functionalized magnetic nanoparticles can be reversibly aggregated in solution as the temperature is cycled through the PNIPAAm lower critical solution temperature (LCST). While the magnetophoretic mobility of the individual nanoparticles below the LCST is negligible, the aggregates formed above the LCST are large enough to respond to an applied magnetic field. The magnetic nanoparticles can associate with biotinylated targets as individual particles, and then subsequent application of a combined temperature increase and magnetic field can be used to magnetically separate the aggregated particles onto the poly(ethylene glycol)-modified polydimethylsiloxane channel walls of a microfluidic device. When the magnetic field is turned off and the temperature is reversed, the captured aggregates redisperse into the channel flow stream for further downstream processing. The dual magnetic- and temperature-responsive nanoparticles can thus be used as soluble reagents to capture diagnostic targets at a controlled time point and channel position. They can then be isolated and released after the nanoparticles have captured target molecules, overcoming the problem of low magnetophoretic mobility of the individual particle while retaining the advantages of a high surface to volume ratio and faster diffusive properties during target capture.