Joseph DeSimone Image Map
Apr 172015
 
Developed in the DeSimone lab in 2004, the PRINT® technology is central to both our life science and materials science research. A powerful nano-molding technique, PRINT enables the fabrication of particles with precise control over the shape, size, composition, and surface functionality. Figures 2 and 3 below illustrate the breadth of shapes, sizes, and compositions possible for nanoparticle fabrication using PRINT.

The PRINT Process

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Figure 1. A unique liquid fluoropolymer (green) is poured on the surface of the master template (gray) and photochemically cross linked. The template is then peeled away to generate a precise mold with micro- or nanoscale cavities. A pre-particle solution (red) is distributed evenly in the mold, which then solidifies. Particles are removed from the mold by bringing the mold in contact with a harvesting film (yellow). Dissolution of the film yields free-flowing particles in solution, enabling them to be easily handled, chemically modified, and analyzed.

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Figure 2. Micron-sized PRINT particles.
print_figure3Figure 3. Nano-sized PRINT particles.
 April 17, 2015
Apr 302014
 
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Fig. 1. Ex vivo fluorescent imaging of harvested organs allows for assessment of particle biodistribution, to illustrate where particles accumulate when administered intravenously. Here we observe that 55 x 77 nm PEGylated hydrogel PRINT particles accumulate mainly in clearance organs – liver and spleen, as well as in primary flank xenograft tumors, with  minimal accumulation in the lung and kidneys.

Despite decades of research and development to improve our ability to diagnose and treat cancer in all its varied and insidious forms, cancer is poised to overtake heart disease as the number one cause of death in the United States in the coming years. Increases in life expectancy broadly will only add to the number of people afflicted with cancer, exacerbating an already intransigent health care burden. Furthermore, our standard means of treating many cancers with systemic chemotherapies takes a significant toll on the quality of life for patients. Because systemic chemotherapy exposes the entire body to toxic chemicals, with only a small portion actually reaching cancer cells, patients often experience significant side effects and poor treatment efficacy. Thus, it is thought that drug delivery devices that can target diseased tissue while sparing systemic exposure will limit the toxicity of standard chemotherapy regimens while increasing treatment efficacy.

To this end, the Desimone lab’s cancer research program aims to design novel nanoscale delivery devices to improve cancer outcomes. By using precise control of nanoparticle (NP) fabrication, our goal is to identify the optimal characteristics to design NPs that enhance chemotherapy. The Particle Replication In Non-wetting Templates (PRINT) platform has been utilized to fabricate novel cross-linked hydrogel and poly(lactide-co-glycolide) (PLGA) particles with controllable size, shape, drug loading, modulus, surface chemistry, targeting ligand density and therapeutic release kinetics using multiple triggered linkers for chemotherapeutics and siRNA. Manufacturing control over these variables enables iterative understanding of how particle designs interface with biological systems, generating actionable information that can be used to improve therapeutic outcomes for cancer.

For example, plasma circulation time of particles can be controlled by size, surface chemistry and modulus, enabling tailoring of therapies to specific cancers and sites of disease in the body. In addition, chemical conjugation strategies can be used to augment the kinetics of drug release, which limits systemic exposure and enhances the maximum tolerated dose (MTD) of chemotherapeutics. Combining these approaches using PRINT NPs has resulted in prolonged survival in multiple preclinical animal models through improved tumor accumulation and increased MTD. Further studies are being conducted to optimize surface chemistry of particles with targeting ligands to enhance tumor uptake and retention. Given the tremendous complexity of cancer, be it the molecular pathways implicated or the variability in tumor location, the modular control of drug delivery devices may enable substantial advances in cancer treatment.

 April 30, 2014
Jan 282014
 

MolecMosqIMAGE_Jan2014It is estimated that well over half a billion people worldwide are infected every year by pathogens transmitted by mosquito bites. Malaria and Dengue fever are two of the most debilitating mosquito-borne diseases, and many others wreak havoc throughout the world. Traditional approaches to mosquito control include the use of chemical sprays such as insecticides that contaminate crops, pollute waters and permeate the food chain with unexpected and unplanned off-target effects. In addition, mosquitoes can develop resistance to chemical sprays, thus greatly limiting their efficacy over time and further damaging the environment through increased use of these toxins.

The molecular mosquitocide (MM) program is a research effort in collaboration with faculty from Colorado State University (Barry Beaty) and Iowa State (Lyric Bartholomay) aimed at fundamentally changing how we control mosquito populations. By using the endogenous RNA interference (RNAi) pathway found throughout the animal kingdom, the goal of the MM program is to deliver RNA sequences via nanoparticles that target mosquito genes required for disease-transmission. Such RNA sequences may prevent mosquito reproduction or may impede mosquitoes from providing an environment suitable to host pathogens which cause human disease. These RNA sequences are both species and gene-specific, thus preventing resistance development and the panoply of off-target effects that current chemical spraying methods engender. Critically, nanoparticles shield RNA from rapid degradation and promote systemic biodistribution, two parameters integral to the eventual efficacy of the MM approach.

Initial studies are clarifying the role of size, shape and charge on the biodistribution of PRINT nanoparticles in Anopheles gambiae, the mosquito species which carries and transmits the causative agent of malaria (Figure 1). Using the inherent fabrication control of PRINT, these studies will help broaden our understanding of how particle parameters augment distribution throughout individual mosquito species at different stages of life (e.g., larvae and adults) with the aim of developing more tailored and efficacious molecular mosquitocides. For example, RNA sequences targeting mosquito genes involved in egg development will likely need to target the abdomen of adult mosquitoes, whereas targeting of the feeding cycle will require nanoparticles which can deliver RNA to the head of the mosquito. The molecular mosquitocide program combines the explosion of information accrued in mosquito genetics and genomics with the delivery capabilities of PRINT nanoparticles to provide an almost unlimited number of potential target genes and sequences for RNAi-based MMs.

 January 28, 2014
Mar 182010
 
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Figure 1.
TEM Images of 1um, 500nm and 200nm particles being engulfed by HeLa cells.

The exploration and utilization of nanocarriers for the delivery of therapeutics in vivo has led to dramatic improvements in the efficacy of various therapies. Over the past few years, intense research and development of novel platforms has resulted in drug delivery vehicles such as polymeric nanoparticles, micelles, immunoconjugates, DNA-polymer conjugates, dendrimers and liposomes. Clinically, the success of these carriers has been limited by the lack of control over size, chemical composition, uniformity, cell targeting and ability to consistently load and release known amounts of cargo. A recent breakthrough from the DeSimone laboratory has led to the production of monodisperse, shape-specific particles from an extensive array of organic precursors. This particle fabrication technology, called PRINT (Particle Replication In Non-wetting Templates), takes advantage of the unique properties of elastomeric molds comprised of a low surface energy perfluoropolyether network.

Understanding the interdependent role of particle size, shape, surface, and matrix composition on the intracellular pathway will lead to a deeper knowledge of the fate of organic nanoparticles in vivo. The advent of “calibration quality” particles using PRINT allows for the elucidation of mechanisms by which organic particles of controlled size, shape, site-specific surface chemistry, tunable particle matrix composition, and tunable modulus undergo endocytosis. Obtaining knowledge about the endocytic pathway used from “calibration quality” particles should lead to crucial information required for not only enhancing specific cellular internalization, but also manipulating the intracellular location of particles, and minimizing cytotoxic effects. Once the mechanisms of internalization are established, it is then possible to use these findings to better engineer the intracellular release of specific cargos. This information, in combination with ongoing efforts to understand the biodistribution of shape controlled particles, will help to establish rules toward the rational design of nanocarriers for effective in vivo delivery of various cargos, especially those cargos that need to be internalized into cells such as siRNA and antisense oligonucleotides.

 March 18, 2010
Mar 172010
 

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Figure 1. Biodistribution of 125I-labeled 200 nm particles over 24 hours in healthy mice.

Definitive biodistribution maps that establish the interdependency of the size, shape, deformability and surface chemistry of nanoparticles in vitro and in vivo over length scales ranging from cells to tissues to the entire organism are needed by many different research communities. Environmental regulators, pulmonologists, oncologists, pharmaceutical scientists, toxicologists, cell biologists and dermatologists all need definitive answers related to particle biodistributions, particle permeability and transport using “calibration quality” particles. For example, fungal and bacterial pathogens are first and foremost recognized by their form or shape; however, the complete understanding of the role and significance of that form and shape is largely lacking. Indeed, some rod-like bacterial pathogens, including the gram-negative bacteria Salmonella, Shigella, and Yersinia and the gram-positive bacterium Listeria monocytogenes can induce their entry into non-phagocytic mammalian cells. Likewise, red blood cells and neutrophils are able to deform and undergo over 100 % strain (double in length) in order to navigate through various biological barriers that would prevent non-flexible objects from crossing. As such, nanofabricated tools (e.g. precisely defined particles) hold significant promise to provide insight into the fundamentals of cellular and biological processes. These tools can also yield essential insights into the design of effective vectors for use in nanomedicine, especially for the design of nanoparticles for use as targeted therapeutics and imaging agents. Indeed, very little is known about how the interdependency of size, shape, deformability and surface chemistry can influence the biodistribution, cell-uptake, and intra-cellular trafficking of micro- and nanoparticles. Beyond understanding the biodistribution of particles delivered via parenteral routes, particle size, shape, deformability and surface chemistry should play a very significant role for understanding the mechanisms associated with particles that are inhaled, either intentionally for use as a therapeutic or during environmental exposure. Understanding the role that mechano-biology plays as a function of size, shape and surface chemistry certainly lies at the core of how biological particles like neutrophils and red blood cells navigate their barriers. Ascertaining definitive biodistribution maps through the use of precisely defined particle probes containing appropriate imaging beacons useful for quantification will undoubtedly lead to a set of rules that will be of immense use to science and to the application of nano-carriers to improve human health, treatment and diagnosis.

 March 17, 2010