The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro-and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 m by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.PRINT ͉ shape ͉ size ͉ surface charge

This tutorial review presents an overview of strategies for the synthesis and fabrication of organic nanomaterials, specifically those with potential for use in medical applications. Examples include liposomes, micelles, polymer-drug conjugates and dendrimers. Methods of driving shape via"bottom-up" synthetic approaches and thermodynamics and kinetics are discussed. Furthermore, methods of driving shape via"top-down" physical and engineering techniques are also explored. Finally, a novel method (referred to as PRINT) used to produce nanoparticles that are shape-specific, can contain any cargo, and can be easily modified is examined along with its potential future role in nanomedicine.

A novel method for the fabrication of polymeric particles on the order of tens of nanometers to several microns is described. This imprint lithographic technique 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, allowing the production of monodisperse, shapespecific nanoparticles from an extensive array of organic precursors. This engineered nature of particle production has a number of advantages over the construction of traditional nanoparticles such as liposomes, dendrimers, and colloidal precipitates. The gentle "top down" approach of PRINT enables the simultaneous and independent control over particle size and shape, composition, and surface functionality, and permits the loading of delicate cargos such as small organic therapeutics and biological macromolecules. Thus, this single tool serves as a comprehensive platform for the rational design and investigation of new nanocarriers in medicine, having applications ranging from therapeutics to advanced diagnostics. Preliminary in vitro and in vivo studies were conducted, demonstrating the future utility of PRINT particles as delivery vectors in nanomedicine. Monodisperse 200 nm poly(ethylene glycol)-based (PEG) particles were fabricated using PRINT methodology and characterized via scanning electron microscopy and dynamic light scattering. Incubation with HeLa cells showed very little cytotoxicity, even at high concentrations. The biodistribution and pharmacokinetics of [ 125 I]-labeled particles were studied in healthy mice following bolus tail vein administration. The particles were distributed mainly to the liver and the spleen with an apparent distribution t 1/2 of approximately 17 min followed by slow redistribution with a t 1/2 of 3.3 h. The volume of distribution for the central and peripheral compartments was found to be approximately 3 mL and 5 mL, respectively.

CONSPECTUSIn this Account, we describe the use of perfluoropolyether (PFPE)-based materials that are able to accurately mold and replicate micro-and nanosized features using traditional techniques such as embossing as well as new techniques that we developed to exploit the exceptional surface characteristics of fluorinated substrates. Because of the unique partial wetting and nonwetting characteristics of PFPEs, we were able to go beyond the usual molding and imprint lithography approaches and have created a technique called PRINT (Particle [ PRINT is a distinctive "top-down" fabrication technique capable of generating isolated particles, arrays of particles, and arrays of patterned features for a plethora of applications in both nanomedicine and materials science. A particular strength of the PRINT technology is the high-resolution molding of well-defined particles with precise control over size, shape, deformability, and surface chemistry. The level of replication obtained showcases some of the unique characteristics of PFPE molding materials. In particular, these materials arise from very low surface energy precursors with positive spreading coefficients, can be photocured at ambient temperature, and are minimally adhesive, nonswelling, and conformable. These distinctive features enable the molding of materials with unique attributes and nanometer resolution that have unprecedented scientific and technological value. For example, in nanomedicine, the use of PFPE materials with the PRINT technique allows us to design particles in which we can tailor key therapeutic parameters such as bioavailability, biodistribution, target-specific cell penetration, and controlled cargo release. Similarly, in materials science, we can fabricate optical films and lens arrays, replicate complex, naturally occurring objects such as adenovirus particles, and create 2D patterned arrays of inorganic oxides. IntroductionReplication of submicrometer features is a challenging materials problem. The past few decades have witnessed the emergence of soft lithography as an important tool for low cost pattern replication on the micrometer and nanometer scale. 1 Soft lithography uses embossing and stamping techniques with applied forces as an alternative to photolithography for the manufacture of integrated circuits and other devices with sub-50 nm feature sizes. Embossing typically involves the patterning of materials such as polymers, organics, and biological molecules into continuous arrays of patterned features using molds made from either hard materials (quartz/glass, glassy polymers) or soft elastomeric materials to generate features that form on top of an interconnecting flash layer. 1-3 The field of soft lithography has traditionally been dominated by the elastomer poly(dimethylsiloxane) (PDMS). 4,5 Despite the advantage of PDMS for use in soft lithography, it has been shown to suffer from serious drawbacks Embossing is the process of creating a three-dimensional image or design in paper and in ductile materials. It is typically acc...

Purpose To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types. Methods A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 μm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 μm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types. Results Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study. Conclusions Cylindrical 1 μm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.