Translation of intravenously administered nanomaterials to the clinic is limited due to adverse infusion reactions. While these reactions are infrequent, with up to 10% prone to experiencing infusion reactions, the reactions can be severe and life-threatening. One of the innate immune pathways, the complement activation pathway, plays a significant role in mediating this response. Nanoparticle surface properties are a relevant design feature, as they control the blood proteins the nanoparticles interact with and allow the nanoparticles to evade the immune reaction. PEGylation of nanosurfaces is critical in improving the blood circulation of nanoparticles and reducing opsonization. Our goal was to understand whether modifying the surface architecture by varying the PEG density and architecture can impact the complement response in vitro. We utilized block copolymers of poly(lactic acid)-b-poly(ethylene glycol) prepared with poly(ethylene glycol) macroinitiators of molecular weights 3400 and 5000 Da. Tracking the complement biomarker C5a, we monitored the impact of changing PEGylation of the nanoparticles. We also investigated how the changing PEG length on the nanoparticle surface impacts further strengthening the stealth properties. Lastly, we determined which cytokines change upon blood incubation with nanoparticles in vitro to understand the extent to which inflammation may occur and the crosstalk between the complement and immune responses. Increasing PEGylation reduced the generation of complement-mediated anaphylatoxin C5a in vitro, with 5000 Da PEG more effectively reducing levels of C5a generated compared to 3400 Da PEG. The insights gathered regarding the impact of PEG density and PEG chain length would be critical in developing stealth nanoparticles that do not lead to infusion reactions upon intravenous administration.

3D printing has revolutionized making tissue models, but the instruments are often quite expensive, and the approach can involve heat and/or shear forces that can damage cells. As a complement to more traditional 3D printing approaches, we looked at screen printing. Screen printing is an additive manufacturing technique used to pattern inks through screens supporting patterns onto different surfaces. It has a wide range of applications ranging from the traditional printing to printing electric circuit boards. Taking cues from this we have developed a process of screen printing live cells along with a suitable scaffold on to different surfaces to generate in vitro models. The process is not only inexpensive and simple to use, it offers a wide range of advantages like the ability to use a range of bioinks limited only by their gelation time, printing on different surfaces, and the ability to autoclave all of the major components. In this paper we present the screen assembly and the setup we used to print the cells along with the resolution and limits of features printed and the effect of the printing on the cells.

In vitro models provide a good starting point for drug screening and understanding various cellular mechanisms corresponding to different conditions. 3D cultures have drawn significant interest to mimic the in vivo microenvironment better and overcome the limitations of the 2D monolayered cultures. We previously reported a technique based on the screen printing process to pattern live mammalian cells using gelatin as the bioink. Even though gelatin is an inexpensive scaffolding material with various tissue engineering applications, it might not be the ideal hydrogel material to provide various mechanical and chemical cues to the cells. In this paper, we discuss the synthesis and characterization of two synthetic chemically cross-linked hydrogel systems based on poly­(ethylene glycol) (PEG) and poly-l-lysine (PLL) to be used as the bioink in the screen printing process. These hydrogels are suitable as the bioinks for the screen printing process and serve as the barebone materials that can be tuned mechanically and augmented chemically to create a suitable in vitro microenvironment for the cells. This paper presents the synthesis, mechanical testing, and characterization of the hydrogel systems and their applications in the screen printing process.

Near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) have shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. This paper describes the synthesis of NIR-emitting Pdots with great control and tunability of emission peak wavelength. The Pdots were prepared by doping poly[(9,9dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1′,3)-thiadiazole)] (PFBT), a semiconducting polymer commonly used as a host polymer in luminescent Pdots, with a series of chlorins and bacteriochlorins with varying functional groups. Chlorins and bacteriochlorins are ideal dopants due to their high hydrophobicity, which precludes their use as molecular probes in aqueous biological media but on the other hand prevents their leakage when doped into Pdots. Additionally, chlorins and bacteriochlorins have narrow deep red to NIR-emission bands and the wide array of synthetic modifications available for modifying their molecular structure enables tuning their emission predictably and systematically. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements show the chlorin-and bacteriochlorin-doped Pdots to be nearly spherical with an average diameter of 46 ± 12 nm. Efficient energy transfer between PFBT and the doped chlorins or bacteriochlorins decreases the PFBT donor emission to near baseline level and increases the emission of the doped dyes that serve as acceptors. The chlorin-and bacteriochlorin-doped Pdots show narrow emission bands ranging from 640 to 820 nm depending on the doped dye. The paper demonstrates the utility of the systematic chlorin and bacteriochlorin synthesis approach by preparing Pdots of varying emission peak wavelength, utilizing them to visualize multiple targets using wide-field fluorescence microscopy, binding them to secondary antibodies, and determining the binding of secondary antibody-conjugated Pdots to primary antibody-labeled receptors in plant cells. Additionally, the chlorin-and bacteriochlorin-doped Pdots show a blinking behavior that could enable their use in superresolution imaging methods like STORM.

Intravenously infusible nanoparticles to control bleeding have shown promise in rodents, but translation into preclinical models has been challenging as many of these nanoparticle approaches have resulted in infusion responses and adverse outcomes in large animal trauma models. We developed a hemostatic nanoparticle technology that was screened to avoid one component of the infusion response: complement activation. We administered these hemostatic nanoparticles, control nanoparticles, or saline volume controls in a porcine polytrauma model. While the hemostatic nanoparticles promoted clotting as marked by a decrease in prothrombin time and both the hemostatic nanoparticles and controls did not active complement, in a subset of the animals, hard thrombi were found in uninjured tissues in both the hemostatic and control nanoparticle groups. Using data science methods that allow one to work across heterogeneous data sets, we found that the presence of these thrombi correlated with changes in IL-6, INF-alpha, lymphocytes, and neutrophils. While these findings might suggest that this formulation would not be a safe one for translation for trauma, they provide guidance for developing screening tools to make nanoparticle formulations in the complex milieux of trauma as well as for therapeutic interventions more broadly. This is important as we look to translate intravenously administered nanoparticle formulations for therapies, particularly considering the vascular changes seen in a subset of patients following COVID-19. We need to understand adverse events like thrombi more completely and screen for these events early to make nanomaterials as safe and effective as possible.