Jason M. Criscione

Label-free biomarker detection from whole blood.

Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of ionic screening. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection.1 A microfluidic purification chip captures multiple biomarkers simultaneously from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 uL sample of whole blood in less than 20 minutes.

Combination delivery of TGF-β inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy

The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-β (TGF-β), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-β inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8(+) T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype.

Particulate vaccines are emerging promising technologies for the creation of tunable prophylactics against a wide variety of conditions. Vesicular and solid biodegradable polymer platforms, exemplified by liposomes and polyesters, respectively, are two of the most ubiquitous platforms in vaccine delivery studies. Here we directly compared the efficacy of each in a long-term immunization study and in protection against a model bacterial antigen. Immunization with poly(lactide-co-glycolide) (PLGA) nanoparticles elicited prolonged antibody titers compared to liposomes and alum. The magnitude of the cellular immune response was also highest in mice vaccinated with PLGA, which also showed a higher frequency of effector-like memory T cell phenotype, leading to an effective clearance of intracellular bacteria. The difference in performance of these two common particulate platforms is shown not to be due to material differences but appears to be connected to the kinetics of antigen delivery. Thus, this study highlights the importance of sustained antigen release mediated by particulate platforms and its role in the long-term appearance of effector memory cellular response.

Self-assembly of pH-responsive fluorinated dendrimer-based particulates for drug delivery and noninvasive imaging

Dendrimers are nanoscale macromolecules with well-defined branching chemical structures. Control over the architecture and function of these structures has enabled many advances in materials science and biomedical applications. Though dendrimers are directly synthesized by iteration of simple repetitive steps, generation of the larger, more complex structures required for many biomedical applications by covalent synthetic methods has been challenging. Here we demonstrate a spontaneous self-assembly of poly(amidoamine) dendrimers into complex nanoscopic and microscopic particulates following partial fluorination of the constituent dendrimer subunits. These dense particulates exhibit a stimulus-induced response to low external pH that causes their disassembly over time, enabling controlled release of encapsulated agents. In addition, we show that these assemblies offer a sufficiently high density of fluorine spins to enable detection of their site-specific accumulation in vivo by (19)F magnetic resonance imaging ((19)F MRI). Fluorinated dendrimer-based particulates present new features and capabilities important for a wide variety of emerging biomedical applications.

Determination of Molecular Configuration by Debye Length Modulation

Silicon nanowire field effect transistors (FETs) have emerged as ultrasensitive, label-free biodetectors that operate by sensing bound surface charge. However, the ionic strength of the environment (i.e., the Debye length of the solution) dictates the effective magnitude of the surface charge. Here, we show that control of the Debye length determines the spatial extent of sensed bound surface charge on the sensor. We apply this technique to different methods of antibody immobilization, demonstrating different effective distances of induced charge from the sensor surface.

Development and application of a multimodal contrast agent for SPECT/CT hybrid imaging

Hybrid or multimodality imaging is often applied in order to take advantage of the unique and complementary strengths of individual imaging modalities. This hybrid noninvasive imaging approach can provide critical information about anatomical structure in combination with physiological function or targeted molecular signals. While recent advances in software image fusion techniques and hybrid imaging systems have enabled efficient multimodal imaging, accessing the full potential of this technique requires development of a new toolbox of multimodal contrast agents that enhance the imaging process. Toward that goal, we report the development of a hybrid probe for both single photon emission computed tomography (SPECT) and X-ray computed tomography (CT) imaging that facilitates high-sensitivity SPECT and high spatial resolution CT imaging. In this work, we report the synthesis and evaluation of a novel intravascular, multimodal dendrimer-based contrast agent for use in preclinical SPECT/CT hybrid imaging systems. This multimodal agent offers a long intravascular residence time (t(1/2) = 43 min) and sufficient contrast-to-noise for effective serial intravascular and blood pool imaging with both SPECT and CT. The colocalization of the dendritic nuclear and X-ray contrasts offers the potential to facilitate image analysis and quantification by enabling correction for SPECT attenuation and partial volume errors at specified times with the higher resolution anatomic information provided by the circulating CT contrast. This may allow absolute quantification of intramyocardial blood volume and blood flow and may enable the ability to visualize active molecular targeting following clearance from the blood.

Functionalized poly(lactic-co-glycolic acid) enhances drug delivery and provides chemical moieties for surface engineering while preserving biocompatibility

Poly(lactic-co-glycolic acid) (PLGA) is one of the more widely used polymers for biomedical applications. Nonetheless, PLGA lacks chemical moieties that facilitate cellular interactions and surface chemistries. Furthermore, incorporation of hydrophilic molecules is often problematic. The integration of polymer functionalities would afford the opportunity to alter device characteristics, thereby enabling control over drug interactions, conjugations and cellular phenomena. In an effort to introduce amine functionalities and improve polymer versatility, we synthesized two block copolymers (PLGA-PLL 502H and PLGA-PLL 503H) composed of PLGA and poly(epsilon-carbobenzoxy-l-lysine) utilizing dicyclohexyl carbodiimide coupling. PLGA-PLL microspheres encapsulated approximately sixfold (502H) and threefold (503H) more vascular endothelial growth factor, and 41% (503H) more ciliary neurotrophic factor than their PLGA counterparts. While the amine functionalities were amenable to the delivery of large molecules and surface conjugations, they did not compromise polymer biocompatibility. With the versatile combination of properties, biocompatibility and ease of synthesis, these block copolymers have the potential for diverse utility in the fields of drug delivery and tissue engineering.

Multiplexed SOI BioFETs

Nanoscale Field Effect Transistors have emerged as a promising technology for ultrasensitive, unlabeled diagnostic applications. However, their use as quantitative sensors has been problematic because of the need for individual sensor calibration. In this work we demonstrate an internal calibration scheme for multiplexed nanoribbon field effect sensors by utilizing the initial current rates rather than end point detection. A linear response is observed consistent with initial binding kinetics. Moreover, we are able to show that top-down fabrication techniques yield reproducible device results with minimal fluctuations, enabling internal calibration.

Nanospheres delivering the EGFR TKI AG1478 promote optic nerve regeneration: the role of size for intraocular drug delivery.

Promoting nerve regeneration involves not only modulating the postinjury microenvironment but also ensuring survival of injured neurons. Sustained delivery of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been shown to promote the survival and regeneration of neurons, but systemic administration is associated with significant side effects. We fabricated poly(lactic-co-glycolic acid) (PLGA) microspheres and nanospheres containing the EGFR TKI 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) for intravitreal administration in a rat optic nerve crush injury model. Upon administration, less backflow from the injection site was observed when injecting nanospheres compared to microspheres. Two weeks after intravitreal delivery, we were able to detect microspheres and nanospheres in the vitreous using coumarin-6 fluorescence, but fewer microspheres were observed compared to the nanospheres. At four weeks only nanospheres could be detected. AG1478 microspheres and nanospheres promoted optic nerve regeneration at two weeks, and at four weeks evidence of regeneration was found only in the nanosphere-injected animals. This observation could be attributed to the ease of administration of the nanospheres versus the microspheres, which in turn led to an increased amount of spheres delivered to the vitreous in the nanosphere group compared to the microsphere group. These data provide evidence for use of PLGA nanospheres to deliver AG1478 intravitreally in a single administration to promote nerve regeneration.

Induced Clustered Nanoconfinement of Superparamagnetic Iron Oxide in Biodegradable Nanoparticles Enhances Transverse Relaxivity for Targeted Theranostics

Combined therapeutic and diagnostic agents, “theranostics” are emerging valuable tools for noninvasive imaging and drug delivery. Here, we report on a solid biodegradable multifunctional nanoparticle that combines both features.