Cabozantinib Unlocks Efficient In Vivo Targeted Delivery of Neutrophil-Loaded Nanoparticles into Murine Prostate Tumors.

A major barrier to the successful application of nanotechnology for cancer treatment is the suboptimal delivery of therapeutic payloads to metastatic tumor deposits. We previously discovered that cabozantinib, a tyrosine kinase inhibitor, triggers neutrophil-mediated anticancer innate immunity, resulting in tumor regression in an aggressive PTEN/p53-deficient genetically engineered murine model of advanced prostate cancer. Here, we specifically investigated the potential of cabozantinib-induced neutrophil activation and recruitment to enhance delivery of BSA-coated polymeric nanoparticles (BSA-NPs) into murine PTEN/p53-deficient prostate tumors. On the basis of the observation that BSA coating of NPs enhanced association and internalization by activated neutrophils by approximately 6-fold in vitro, relative to uncoated NPs, we systemically injected BSA-coated, dye-loaded NPs into prostate-specific PTEN/p53-deficient mice that were pretreated with cabozantinib. Flow cytometric analysis revealed an approximately 4-fold increase of neutrophil-associated BSA-NPs and an approximately 32-fold increase in mean fluorescent dye uptake following 3 days of cabozantinib/BSA-NP administration, relative to BSA-NP alone. Strikingly, neutrophil depletion with Ly6G antibody abolished dye-loaded BSA-NP accumulation within tumors to baseline levels, demonstrating targeted neutrophil-mediated intratumoral NP delivery. Furthermore, we observed an approximately 13-fold decrease in accumulation of BSA-NPs in the liver, relative to uncoated NPs, post-cabozantinib treatment, suggesting that BSA coating of NPs can significantly enhance cabozantinib-induced, neutrophil-mediated targeted intratumoral drug delivery, while mitigating off-target toxicity. Collectively, we demonstrate a novel targeted nano-immunotherapeutic strategy for enhanced intratumoral delivery of BSA-NPs, with translational potential to significantly augment therapeutic indices of cancer medicines, thereby overcoming current pharmacologic barriers commonly encountered in preclinical/early-phase drug development.

A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue.

The precision of the delivery of therapeutics to the desired injection site by syringes and hollow needles typically depends on the operator. Here, we introduce a highly sensitive, completely mechanical and cost-effective injector for targeting tissue reliably and precisely. As the operator pushes the syringe plunger, the injector senses the loss-of-resistance on encountering a softer tissue or a cavity, stops advancing the needle and delivers the payload. We demonstrate that the injector can reliably deliver liquids to the suprachoroidal space-a challenging injection site that provides access to the back of the eye-for a wide range of eye sizes, scleral thicknesses and intraocular pressures, and target sites relevant for epidural injections, subcutaneous injections and intraperitoneal access. The design of this simple and effective injector can be adapted for a broad variety of clinical applications.

Cabozantinib Eradicates Advanced Murine Prostate Cancer by Activating Antitumor Innate Immunity.

Several kinase inhibitors that target aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in cancer clinical trial patients across several malignancies. Here, we show that cabozantinib rapidly eradicates invasive, poorly differentiated PTEN/p53-deficient murine prostate cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells, including CXCL12 and HMGB1, resulting in robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor plerixafor. Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anticancer innate immune response, resulting in tumor clearance.Significance: This study is the first to demonstrate that a tyrosine kinase inhibitor can activate neutrophil-mediated antitumor innate immunity, resulting in invasive cancer clearance. Cancer Discov; 7(7); 750-65. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.

Nanotechnology-Based Approaches for Guiding Neural Regeneration.

The mammalian brain is a phenomenal piece of "organic machinery" that has fascinated scientists and clinicians for centuries. The intricate network of tens of billions of neurons dispersed in a mixture of chemical and biochemical constituents gives rise to thoughts, feelings, memories, and life as we know it. In turn, subtle imbalances or damage to this system can cause severe complications in physical, motor, psychological, and cognitive function. Moreover, the inevitable loss of nerve tissue caused by degenerative diseases and traumatic injuries is particularly devastating because of the limited regenerative capabilities of the central nervous system (i.e., the brain and spinal cord). Among current approaches, stem-cell-based regenerative medicine has shown the greatest promise toward repairing and regenerating destroyed neural tissue. However, establishing controlled and reliable methodologies to guide stem cell differentiation into specialized neural cells of interest (e.g., neurons and oligodendrocytes) has been a prevailing challenge in the field. In this Account, we summarize the nanotechnology-based approaches our group has recently developed to guide stem-cell-based neural regeneration. We focus on three overarching strategies that were adopted to selectively control this process. First, soluble microenvironmental factors play a critical role in directing the fate of stem cells. Multiple factors have been developed in the form of small-molecule drugs, biochemical analogues, and DNA/RNA-based vectors to direct neural differentiation. However, the delivery of these factors with high transfection efficiency and minimal cytotoxicity has been challenging, especially to sensitive cell lines such as stem cells. In our first approach, we designed nanoparticle-based systems for the efficient delivery of such soluble factors to control neural differentiation. Our nanoparticles, comprising either organic or inorganic elements, were biocompatible and offered multifunctional capabilities such as imaging and delivery. Moving from the soluble microenvironment in which cells are immersed to the underlying surface, cells can sense and consequently respond to the physical microenvironment in which they reside. For instance, changes in cell adhesion, shape, and spreading are key cellular responses to surface properties of the underlying substrate. In our second approach, we modulated the surface chemistry of two-dimensional substrates to control neural stem cell morphology and the resulting differentiation process. Patterned surfaces consisting of immobilized extracellular matrix (ECM) proteins and/or nanomaterials were generated and utilized to guide neuronal differentiation and polarization. In our third approach, building on the above-mentioned approaches, we further tuned the cell-ECM interactions by introducing nanotopographical features in the form of nanoparticle films or nanofiber scaffolds. Besides providing a three-dimensional surface topography, our unique nanoscaffolds were observed to enhance gene delivery, facilitate axonal alignment, and selectively control differentiation into neural cell lines of interest. Overall, nanotechnology-based approaches offer the precise physicochemical control required to generate tools suitable for applications in neuroscience.

Application of biomaterials to advance induced pluripotent stem cell research and therapy.

Derived from any somatic cell type and possessing unlimited self-renewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is a single vehicle that not only solubilizes hydrophobic small molecules in physiological solutions but also forms complexes with siRNA molecules, making it an attractive delivery system for controlling stem cell differentiation. Herein, we report the synthesis and application of DexAM to simultaneously deliver hydrophobic small molecules and siRNA into neural stem cells to significantly enhance their neuronal differentiation.

Axonal alignment and enhanced neuronal differentiation of neural stem cells on graphene-nanoparticle hybrid structures.

Human neural stem cells (hNSCs) cultured on graphene-nanoparticle hybrid structures show a unique behavior wherein the axons from the differentiating hNSCs show enhanced growth and alignment. We show that the axonal alignment is primarily due to the presence of graphene and the underlying nanoparticle monolayer causes enhanced neuronal differentiation of the hNSCs, thus having great implications of these hybrid-nanostructures for neuro-regenerative medicine.

Nanotopography-mediated reverse uptake for siRNA delivery into neural stem cells to enhance neuronal differentiation.

RNA interference (RNAi) for controlling gene expression levels using siRNA or miRNA is emerging as an important tool in stem cell biology. However, the conventional methods used to deliver siRNA into stem cells result in significant cytotoxicity and undesirable side-effects. To this end, we have developed a nanotopography-mediated reverse uptake (NanoRU) delivery platform to demonstrate a simple and efficient technique for delivering siRNA into neural stem cells (NSCs). NanoRU consists of a self-assembled silica nanoparticle monolayer coated with extracellular matrix proteins and the desired siRNA. We use this technique to efficiently deliver siRNA against the transcription factor SOX9, which acts as a switch between neuronal and glial fate of NSCs. The knockdown of SOX9 enhanced the neuronal differentiation and decreased the glial differentiation of the NSCs. Our NanoRU platform demonstrates a novel application and the importance of nanotopography-mediated siRNA delivery into stem cells as an effective method for genetic manipulation.

Label-free polypeptide-based enzyme detection using a graphene-nanoparticle hybrid sensor.

A graphene-nanoparticle (NP) hybrid biosensor that utilizes an electrical hysteresis change to detect the enzymatic activity and concentration of Carboxypeptidase B was developed. The results indicate that the novel graphene-NP hybrid biosensor, utilizing electrical hysteresis, has the ability to detect concentrations of targeted enzyme on the micromolar scale. Furthermore, to the knowledge of the authors, this is the first demonstration of a graphene-based biosensor that utilizes a hysteresis change resulting from metallic NPs assembled on a graphene surface.

Nanotechnology for regenerative medicine: nanomaterials for stem cell imaging.

Although stem cells hold great potential for the treatment of many injuries and degenerative diseases, several obstacles must be overcome before their therapeutic application can be realized. These include the development of advanced techniques to understand and control functions of microenvironmental signals and novel methods to track and guide transplanted stem cells. The application of nanotechnology to stem cell biology would be able to address those challenges. This review details the current challenges in regenerative medicine, the current applications of nanoparticles in stem cell biology and further potential of nanotechnology approaches towards regenerative medicine, focusing mainly on magnetic nanoparticle- and quantum dot-based applications in stem cell research.