In this account, we varied PEGylation density on the surface of hydrogel PRINT nanoparticles and systematically observed the effects on protein adsorption, macrophage uptake, and circulation time. Interestingly, the density of PEGylation necessary to promote a long-circulating particle was dramatically less than what has been previously reported. Overall, our methodology provides a rapid screening technique to predict particle behavior in vivo and our results deliver further insight to what PEG density is necessary to facilitate long-circulation.

CONSPECTUS Nanotheranostics represents the next generation of medicine, fusing nanotechnology, therapeutics, and diagnostics. By integrating therapeutic and imaging agents into one nanoparticle, this new treatment strategy has the potential not only to detect and diagnose disease but also to treat and monitor the therapeutic response. This capability could have a profound impact in both the research setting as well as in a clinical setting. In the research setting, such a capability will allow research scientists to rapidly assess the performance of new therapeutics in an effort to iterate their designs for increased therapeutic index and efficacy. In the clinical setting, theranostics offers the ability to determine whether patients enrolling in clinical trials are responding, or are expected to respond, to a given therapy based on the hypothesis associated with the biological mechanisms being tested. If not, patients can be more quickly removed from the clinical trial and shifted to other therapeutic options. To be effective, these theranostic agents must be highly site specific. Optimally, they will carry relevant cargo, demonstrate controlled release of that cargo, and include imaging probes with a high signal-to-noise ratio. There are many biological barriers in the human body that challenge the efficacy of nanoparticle delivery vehicles. These barriers include, but are not limited to, the walls of blood vessels, the physical entrapment of particles in organs, and the removal of particles by phagocytic cells. The rapid clearance of circulating particles during systemic delivery is a major challenge; current research seeks to define key design parameters that govern the performance of nanocarriers, such as size, surface chemistry, elasticity, and shape. The effect of particle size and surface chemistry on in vivo biodistribution of nanocarriers has been extensively studied, and general guidelines have been established. Recently it has been documented that shape and elasticity can have a profound effect on the behavior of delivery vehicles. Thus, having the ability to independently control shape, size, matrix, surface chemistry, and modulus is crucial for designing successful delivery agents. In this Account, we describe the use of particle replication in nonwetting templates (PRINT) to fabricate shape- and size-specific microparticles and nanoparticles. A particular strength of the PRINT method is that it affords precise control over shape, size, surface chemistry, and modulus. We have demonstrated the loading of PRINT particles with chemotherapeutics, magnetic resonance contrast agents, and fluorophores. The surface properties of the PRINT particles can be easily modified with “stealth” poly(ethylene glycol) chains to increase blood circulation time, with targeting moieties for targeted delivery or with radiolabels for nuclear imaging. These particles have tremendous potential for applications in nanomedicine and diagnostics.

Extended circulation of nanoparticles in blood is essential for most clinical applications. Nanoparticles are rapidly cleared by cells of the mononuclear phagocyte system (MPS). Approaches such as grafting polyethylene glycol onto particles (PEGylation) extend circulation times; however, these particles are still cleared, and the processes involved in this clearance remain poorly understood. Here, we present an intravital microscopybased assay for the quantification of nanoparticle clearance, allowing us to determine the effect of mouse strain and immune system function on particle clearance. We demonstrate that mouse strains that are prone to Th1 immune responses clear nanoparticles at a slower rate than Th2-prone mice. Using depletion strategies, we show that both granulocytes and macrophages participate in the enhanced clearance observed in Th2-prone mice. Macrophages isolated from Th1 strains took up fewer particles in vitro than macrophages from Th2 strains. Treating macrophages from Th1 strains with cytokines to differentiate them into M2 macrophages increased the amount of particle uptake. Conversely, treating macrophages from Th2 strains with cytokines to differentiate them into M1 macrophages decreased their particle uptake. Moreover, these results were confirmed in human monocyte-derived macrophages, suggesting that global immune regulation has a significant impact on nanoparticle clearance in humans. IntroductionThe potential clinical applications of nanoparticles and nanoformulations have been investigated for more than 30 years. Nanoparticle approaches have the potential to revolutionize drug delivery by allowing for the encapsulation of drugs with poor solubility or stability in a stable carrier particle. In addition, targeting nanoparticles to specific pathological sites may allow an increased effective dose of drug at the needed site, while decreasing systemic drug exposure, and therefore side effects. However, to date, only 2 nanoformulations for cancer treatment have been approved for clinical use (liposomal doxorubicin [Doxil] and protein-bound paclitaxel [Abraxane]) (1-3). One major obstacle for the use of nanoparticles in vivo is rapid clearance by the cells of the reticuloendothelial system (RES)/mononuclear phagocyte system (MPS) (4-7). In addition to rapid clearance, variable activity of the MPS among patients leads to widely variable pharmacokinetics of nanoformulations in the clinic, reducing the efficacy of both approved and future experimental nanoformulations (8).

As the enhanced permeation and retention (EPR) effect continues to be a controversial topic in nanomedicine, we sought to examine EPR as a function of nanoparticle size, tumor model, and tumor location, while also evaluating tumors for EPR mediating factors such as microvessel density, vascular permeability, lymphatics, stromal content, and tumor-associated immune cells. Tumor accumulation was evaluated for 55 × 60, 80 × 180, and 80 × 320 nm PRINT particles in four subcutaneous flank tumor models (SKOV3 human ovarian, 344SQ murine nonsmall cell lung, A549 human nonsmall cell lung, and A431 human epidermoid cancer). Each tumor model revealed specific particle accumulation trends with evident particle size dependence. Immuno-histochemistry staining revealed differences in tumor microvessel densities that correlated with overall tumor accumulation. Immunofluorescence images displayed size-mediated tumor penetration with signal from the larger particles concentrated close to the blood vessels, while signal from the smaller particle was observed throughout the tissue. Differences were also observed for the 55 × 60 nm particle tumor penetration across flank tumor models as a function of stromal content. The 55 × 60 nm particles were further evaluated in three orthotopic, metastatic tumor models (344SQ, A549, and SKOV3), revealing preferential accumulation in primary tumors and metastases over healthy tissue. Moreover, we observed higher tumor accumulation in the orthotopic lung cancer models than in the flank lung cancer models, whereas tumor accumulation was constant for both orthotopic and flank ovarian cancer models, further demonstrating the variability in the EPR effect as a function of tumor model and location.

In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle. However, in vivo, both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density. Overall, nanoparticle size and passive accumulation were the dominant factors eliciting tumor sequestration.

Novel treatment strategies, including nanomedicine, are needed for improving management of triple-negative breast cancer. Patients with triple-negative breast cancer, when considered as a group, have a worse outcome after chemotherapy than patients with breast cancers of other subtypes, a finding that reflects the intrinsically adverse prognosis associated with the disease. The aim of this study was to improve the efficacy of docetaxel by incorporation into a novel nanoparticle platform for the treatment of taxane-resistant triple-negative breast cancer. Rod-shaped nanoparticles encapsulating docetaxel were fabricated using an imprint lithography based technique referred to as Particle Replication in Nonwetting Templates (PRINT). These rod-shaped PLGA-docetaxel nanoparticles were tested in the C3(1)-T-antigen (C3Tag) genetically engineered mouse model (GEMM) of breast cancer that represents the basal-like subtype of triple-negative breast cancer and is resistant to therapeutics from the taxane family. This GEMM recapitulates the genetics of the human disease and is reflective of patient outcome and, therefore, better represents the clinical impact of new therapeutics. Pharmacokinetic analysis showed that delivery of these PLGA-docetaxel nanoparticles increased docetaxel circulation time and provided similar docetaxel exposure to tumor compared to the clinical formulation of docetaxel, Taxotere. These PLGA-docetaxel nanoparticles improved tumor growth inhibition and significantly increased median survival time. This study demonstrates the potential of nanotechnology to improve the therapeutic index of chemotherapies and rescue therapeutic efficacy to treat nonresponsive cancers.

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Vaccination is an essential public health measure for infectious disease prevention. The exposure of the immune system to vaccine formulations with the appropriate kinetics is critical for inducing protective immunity. In this work, faceted microneedle arrays were designed and fabricated utilizing a three-dimensional (3D)-printing technique called continuous liquid interface production (CLIP). The faceted microneedle design resulted in increased surface area as compared with the smooth square pyramidal design, ultimately leading to enhanced surface coating of model vaccine components (ovalbumin and CpG). Utilizing fluorescent tags and live-animal imaging, we evaluated in vivo cargo retention and bioavailability in mice as a function of route of delivery. Compared with subcutaneous bolus injection of the soluble components, microneedle transdermal delivery not only resulted in enhanced cargo retention in the skin but also improved immune cell activation in the draining lymph nodes. Furthermore, the microneedle vaccine induced a potent humoral immune response, with higher total IgG (Immunoglobulin G) and a more balanced IgG1/IgG2a repertoire and achieved dose sparing. Furthermore, it elicited T cell responses as characterized by functional cytotoxic CD8+ T cells and CD4+ T cells secreting Th1 (T helper type 1)-cytokines. Taken together, CLIP 3D–printed microneedles coated with vaccine components provide a useful platform for a noninvasive, self-applicable vaccination.