Jonathan O. Martinez

Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions

The therapeutic efficacy of systemic drug-delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. White blood cells of the immune system--known as leukocytes--possess all of these properties and exert their targeting ability through cellular membrane interactions. Here, we show that nanoporous silicon particles can successfully perform all these actions when they are coated with cellular membranes purified from leukocytes. These hybrid particles, called leukolike vectors, can avoid being cleared by the immune system. Furthermore, they can communicate with endothelial cells through receptor-ligand interactions, and transport and release a payload across an inflamed reconstructed endothelium. Moreover, leukolike vectors retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in a tumour.

Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

Biomimetic proteolipid vesicles for targeting inflamed tissues.

A multitude of micro- and nanoparticles have been developed to improve the delivery of systemically administered pharmaceuticals, which are subject to a number of biological barriers that limit their optimal biodistribution. Bioinspired drug-delivery carriers formulated by bottom-up or top-down strategies have emerged as an alternative approach to evade the mononuclear phagocytic system and facilitate the transport across the endothelial vessel wall. Here, we describe a method that leverages the advantages of bottom-up and top-down strategies to incorporate proteins derived from the leukocyte plasma membrane into lipid nanoparticles. The resulting proteolipid vesicles - which we refer to as leukosomes - retained the versatility and physicochemical properties typical of liposomal formulations, preferentially targeted inflamed vasculature, enabled the selective and effective delivery of dexamethasone to inflamed tissues, and reduced phlogosis in a localized model of inflammation.

Bromelain Surface Modification Increases the Diffusion of Silica Nanoparticles in the Tumor Extracellular Matrix

Tumor extracellular matrix (ECM) represents a major obstacle to the diffusion of therapeutics and drug delivery systems in cancer parenchyma. This biological barrier limits the efficacy of promising therapeutic approaches including the delivery of siRNA or agents intended for thermoablation. After extravasation due to the enhanced penetration and retention effect of tumor vasculature, typical nanotherapeutics are unable to reach the nonvascularized and anoxic regions deep within cancer parenchyma. Here, we developed a simple method to provide mesoporous silica nanoparticles (MSN) with a proteolytic surface. To this extent, we chose to conjugate MSN to Bromelain (Br–MSN), a crude enzymatic complex, purified from pineapple stems, that belongs to the peptidase papain family. This surface modification increased particle uptake in endothelial, macrophage, and cancer cell lines with minimal impact on cellular viability. Most importantly Br–MSN showed an increased ability to digest and diffuse in tumor ECM in vitro and in vivo.

Engineering multi-stage nanovectors for controlled degradation and tunable release kinetics

Nanovectors hold substantial promise in abating the off-target effects of therapeutics by providing a means to selectively accumulate payloads at the target lesion, resulting in an increase in the therapeutic index. A sophisticated understanding of the factors that govern the degradation and release dynamics of these nanovectors is imperative to achieve these ambitious goals. In this work, we elucidate the relationship that exists between variations in pore size and the impact on the degradation, loading, and release of multistage nanovectors. Larger pored vectors displayed faster degradation and higher loading of nanoparticles, while exhibiting the slowest release rate. The degradation of these particles was characterized to occur in a multi-step progression where they initially decreased in size leaving the porous core isolated, while the pores gradually increased in size. Empirical loading and release studies of nanoparticles along with diffusion modeling revealed that this prolonged release was modulated by the penetration within the porous core of the vectors regulated by their pore size.

Short and Long Term, In Vitro and In Vivo Correlations of Cellular and Tissue Responses to Mesoporous Silicon Nanovectors

The characterization of nanomaterials and their influence on and interactions with the biology of cells and tissues are still partially unknown. Multistage nanovectors based on mesoporous silicon have been extensively studied for drug delivery, thermal heating, and improved diagnostic imaging. Here, the short- and long-term changes occurring in human cells upon the internalization of mesoporous silicon nanovectors (MSV) are analyzed. Using qualitative and quantitative techniques as well as in vitro and in vivo biochemical, cellular, and functional assays, it is demonstrated that MSV do not cause any significant acute or chronic effects on cells and tissues. In vitro cell toxicity and viability are analyzed, as well as the maintenance of cell phase cycling and the architecture upon the internalization of MSV. In addition, it is evaluated whether MSV produce any pro-inflammatory responses and its biocompatibility in vivo is studied. The biodistribution of MSV is followed using longitudinal in vivo imaging and organ accumulation is assessed using quantitative elemental and fluorescent techniques. Finally, a thorough pathological analysis of collected tissues demonstrates a mild transient systemic response in the liver that dissipates upon the clearance of particles. It is proposed that future endeavors aimed at understanding the toxicology of naked drug carriers should be designed to address their impact using in vitro and in vivo short- and long-term evaluations of systemic response.

Multiscale Patterning of a Biomimetic Scaffold Integrated with Composite Microspheres

The ideal scaffold for regenerative medicine should concurrently mimic the structure of the original tissue from the nano- up to the macroscale and recapitulate the biochemical composition of the extracellular matrix (ECM) in space and time. In this study, a multiscale approach is followed to selectively integrate different types of nanostructured composite microspheres loaded with reporter proteins, in a multi-compartment collagen scaffold. Through the preservation of the structural cues of the functionalized collagen scaffold at the nano- and microscale, its macroscopic features (pore size, porosity, and swelling) are not altered. Additionally, the spatial confinement of the microspheres allows the release of the reporter proteins in each of the layers of the scaffold. Finally, the staged and zero-order release kinetics enables the temporal biochemical patterning of the scaffold. The versatile manufacturing of each component of the scaffold results in the ability to customize it to better mimic the architecture and composition of the tissues and biological systems.

Degradation and biocompatibility of multistage nanovectors in physiological systems

The careful scrutiny of drug delivery systems is essential to evaluate and justify their potential for the clinic. Among the various studies necessary for preclinical testing, the impact of degradation is commonly overlooked. In this article, we investigate the effect of fabrication (porosity and nucleation layer) and environment (buffer and pH) factors on the degradation kinetics of multistage nanovectors (MSV) composed of porous silicon. The degradation by-products of MSV were exposed to endothelial cells and analyzed for detrimental effects on cellular internalization, architecture, proliferation, and cell cycle. Increases in porosity resulted in accelerated degradation exhibiting smaller-sized particles at comparable times. Removal of the nucleation layer (thin layer of small pores formed during the initial steps of etching) triggered a premature collapse of the entire central porous region of MSV. Variations in buffers prompted a faster degradation rate yielding smaller MSV within faster time frames, whereas increases in pH stimulated erosion of MSV and thus faster degradation. In addition, exposure to these degradation by-products provoked negligible impact on the proliferation and cell cycle phases on primary endothelial cells. In this study, we propose methods that lay the foundation for future investigations toward understanding the impact of the degradation of drug delivery platforms.

The effect of multistage nanovector targeting of VEGFR2 positive tumor endothelia on cell adhesion and local payload accumulation.

Nanovectors are a viable solution to the formulation of poorly soluble anticancer drugs. Their bioaccumulation in the tumor parenchyma is mainly achieved exploiting the enhanced permeability and retention (EPR) effect of the leaky neovasculature. In this paper we demonstrate that multistage nanovectors (MSV) exhibit rapid tumoritropic homing independent of EPR, relying on particle geometry and surface adhesion. By studying endothelial cells overexpressing vascular endothelial growth factor receptor-2 (VEGFR2), we developed MSV able to preferentially target VEGFR2 expressing tumor-associated vessels. Static and dynamic targeting revealed that MSV conjugated with anti-VEGFR2 antibodies displayed greater than a 4-fold increase in targeting efficiency towards VEGFR2 expressing cells while exhibiting minimal adherence to control cells. Additionally, VEGFR2 conjugation bestowed MSV with a significant increase in breast tumor targeting and in the delivery of a model payload while decreasing their accumulation in the liver. Surface functionalization with an anti-VEGFR2 antibody provided enhanced affinity towards the tumor vascular endothelium, which promoted enhanced adhesion and tumoritropic accumulation of a reporter molecule released by the MSV.

Multistage vector delivery of sulindac and silymarin for prevention of colon cancer

Familial adenomatous polyposis (FAP) is an inherited condition secondary to germline mutations in the APC gene, thus resulting in the formation of hundreds of colonic adenomas that eventually progress into colon cancer. Surgical removal of the colon remains the only treatment option to avoid malignancy, as long-term exposure to chemopreventive agents such as sulindac (a non-steroidal anti-inflammatory drug) and silymarin (phytoestrogen) is not feasible. Here, we have developed a multistage silicon-based drug delivery platform for sulindac and silymarin that preferentially interacts with colon cancer cells as opposed to normal intestinal mucosa. Preferential binding and internalization of these drugs into colon cancer cells was obtained using a targeting strategy against the protein meprin A, which we demonstrate is overexpressed in human colon cancer cells and in the small intestine of Apc(Min/+) mice. We propose that this delivery system could potentially be used to reduce drug-induced side effects in FAP patients, thus enabling long-term prevention of adenoma formation.