Non-viral gene delivery methods represent a potential safe and effective approach for treating myriad diseases. For many gene therapy applications, delivering multiple exogenous genes and controlling the time profile that these genes are expressed would be advantageous. Polymeric non-viral gene carriers are versatile and can be readily tailored for particular therapeutic applications, have the ability to carry multiple large genes within each particle, and can be more easily manufactured than viruses used for gene delivery. A layer-by-layer (LbL) theranostic-enabling nanoparticle was developed to incorporate two plasmid types which have differing expression time-profiles. Temporally controlling the expression of exogenous DNA enables superior control over the microenvironment and could lead to better control over differentiation pathways and cell fate.
A clinically relevant magneto-optical technique (fd-FRS, frequency-domain Faraday rotation spectroscopy) for characterizing proteins using antibody-functionalized magnetic nanoparticles (MNPs) is demonstrated. This technique distinguishes between the Faraday rotation of the solvent, iron oxide core, and functionalization layers of polyethylene glycol polymers (spacer) and model antibody-antigen complexes (anti-BSA/BSA, bovine serum albumin). A detection sensitivity of ≈10 pg mL and broad detection range of 10 pg mL ≲ c ≲ 100 µg mL are observed. Combining this technique with predictive analyte binding models quantifies (within an order of magnitude) the number of active binding sites on functionalized MNPs. Comparative enzyme-linked immunosorbent assay (ELISA) studies are conducted, reproducing the manufacturer advertised BSA ELISA detection limits from 1 ng mL ≲ c ≲ 500 ng mL . In addition to the increased sensitivity, broader detection range, and similar specificity, fd-FRS can be conducted in less than ≈30 min, compared to ≈4 h with ELISA. Thus, fd-FRS is shown to be a sensitive optical technique with potential to become an efficient diagnostic in the chemical and biomolecular sciences.
Introduction: Harnessing the diversity of sensing platforms for applications in complex environments has been an ongoing challenge in biosensing. Most techniques exhibit limitations ranging from biocompatibility, sensitivity, and signal transduction through opaque media, to longevity under non-laboratory conditions. Through multidisciplinary advances in nanoscience and medical devices, we have developed an implant for the measurement of biomarkers using a magnetic particle assay platform1,2. Exposure to target induces a switch from a dispersed to an assembled state with a corresponding change in magnetic relaxation properties allowing for robust contrast. Current applications focus on diagnostics in chronic cardiac disease and spikes in biomarker presence frequently missed by serial sampling. We address design parameters for sensing functionality, tunability, and degradation to lay the groundwork for multi-month implantation in vivo. Materials and Methods: Particle core materials provided magnetic contrast while biological functionality was tuned by surface ligands for affinity toward multivalent targets (Figure 1a). Polyclonal antibodies were conjugated to superparamagnetic iron oxide nanoparticles (20-50 nm) by maleimide-thiol chemistry. Proton Magnetic Relaxation measurements were acquired on a custom-made, single-sided, inhomogeneous field relaxometer (0.43 Tesla, 25°C, NMR MOUSE) fitted with a programmable robotic scanning stage3. A Carr-Purcell-Meiboom-Gill pulse sequence was used: TE = 0.035 ms, 5454 echoes, 15 scans, TR = 6 s. Relaxation times were determined by single exponential fit to echo peak intensities with a custom MATLAB script. Delrin diffusion devices were filled with particle colloidal suspension and sealed with 30 nm pore polycarbonate semi-permeable membranes via double-sided, pressure-sensitive adhesive (Figure 1b). Devices were exposed to target while sensor response was read every 90-120 minutes for up to 48 hrs. Degradation was measured by quantitating loss of ligand from the surface (ELISA) and changes in sensor performance after elevated temperature storage (37°C) for up to 12 weeks. Results and Discussion: Ligand valency was tuned over an order of magnitude (4-40 Antibodies/Particle) by varying bioconjugation strategies and reagents. Conjugation stability under standard storage conditions indicated a lifetime >5 months by isolating free from bound protein by centrifugation. A maximum 1% of conjugated ligand was released, minimally decreasing valency and sensor functionality. Assay performance showed prozone agglutination behavior with quantitative exposure ranging from 50-2000 ng/mL of target and contrast in <2 hrs. Device dosimeter performance was validated for a cardiac Myoglobin model. Saturation was tuned from 4 to 12 μg/mL*hrs corresponding with 8 to >24 hrs of continuous biomarker elevation at 0-500 ng/mL. 4 hr equilibration and irreversible dosimeter signal stability post-exposure for >12 hrs was also observed (Figure 1c). Biochemical and aggregation performance degradation parameters were evaluated. Over 6.5 weeks of elevated temperature storage, 1.8% of the conjugated antibody was unbound. Sensor longitudinal performance on exposure to target over 12 weeks of incubation showed a monotonically decreasing response magnitude and rate (Figure 1d). Exponential fit to sensor response decay curve predicts a 29 week “switch” (on/off) sensor lifetime for an infarction model (500 ng/mL Myo). These results indicate our sensor can be derivatized for a cardiac biomarker with usable sensitivity and saturation dosimeter response corresponding to elevations expected in a Myocardial Infarction (MI). Device performance can be tuned by matching sensor characteristics with the physiological range of target sensitivity and saturation desired. By tuning bioconjugation strategies we enhanced the performance of the biosensor by an order of magnitude over prior studies. Sensor lifetime and nanostructure degradation studies have shown the limiting performance factor is not the loss of ligand from the surface of the particles but rather the loss of binding activity as seen in elevated temperature experiments. Conclusions: This implant has the potential to broaden diagnostics in personalized medicine by addressing the hurdles of longevity and robust sensor signal stability in complex environments. In situ diagnostics offer continuous sentineling of critical biomarkers, providing a deeper understanding of local biology in dynamic, heterogeneous systems. This monitoring provides valuable timescale data to clinicians with insight into their patients’ lab results for early intervention and data collection throughout treatment. This implant has the capacity to bridge the gap between lab and clinical grade sensors, significantly improving interventional medicine by leveraging the robust and tunable nature of magnetic relaxation particle assays. References: Daniel KD, Kim GY, Vassiliou CC, et al. Implantable diagnostic device for cancer monitoring. Biosens Bioelectron. 2009;24:3252-3257. doi:10.1016/j.bios.2009.04.010 Ling Y, Pong T, Vassiliou CC, Huang PL, Cima MJ. Implantable magnetic relaxation sensors measure cumulative exposure to cardiac biomarkers. Nat Biotechnol. 2011;29(3):273-277. doi:10.1038/nbt.1780 Blumich B, Blumler P, Eidmann G, et al. The NMR-MOUSE: Construction, Excitation, and Applications. Magn Reson Imaging. 1998;16(98):479-484. Figure 1
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