Translation of biomaterial-based nanoparticle formulations to the clinic faces significant challenges including efficacy, safety, consistency and scale-up of manufacturing, and stability during long-term storage. Continuous microfluidic fabrication of polymeric nanoparticles has the potential to alleviate the challenges associated with manufacture, while offering a scalable solution for clinical level production. Poly(beta-amino esters) (PBAE)s are a class of biodegradable cationic polymers that self-assemble with anionic plasmid DNA to form polyplex nanoparticles that have been shown to be effective for transfecting cancer cells specifically in vitro and in vivo. Here, we demonstrate the use of a microfluidic device for the continuous and scalable production of PBAE/DNA nanoparticles followed by lyophilization and long term storage that results in improved in vitro efficacy in multiple cancer cell lines compared to nanoparticles produced by bulk mixing as well as in comparison to widely used commercially available transfection reagents polyethylenimine and Lipofectamine® 2000. We further characterized the nanoparticles using nanoparticle tracking analysis (NTA) to show that microfluidic mixing resulted in fewer DNA-free polymeric nanoparticles compared to those produced by bulk mixing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1813-1825, 2017.

Intracellular delivery of nucleic acids to mammalian cells using polyplex nanoparticles (NPs) remains a challenge both in vitro and in vivo, with transfections often suffering from variable efficacy. To improve reproducibility and efficacy of transfections in vitro using a next‐generation polyplex transfection material poly(beta‐amino ester)s (PBAEs), the influence of multiple variables in the preparation of these NPs on their transfection efficacy was explored. The results indicate that even though PBAE/pDNA polyplex NPs are formed by the self‐assembly of polyelectrolytes, their transfection is not affected by the manner in which the components are mixed, facilitating self‐assembly in a single step, but timing for self‐assembly of 5–20 min is optimal. In addition, even though the biomaterials are biodegradable in water, their efficacy is not affected by up to eight freeze‐thaw cycles of the polymer. It was found that there is a greater stability of nucleic acid‐complexed polymer as a polyplex nanoparticle compared with free polymer. Finally, by exploring multiple buffer systems, it was identified that utilization of divalent cation magnesium or calcium acetate buffers at pH 5.0 is optimal for transfection using these polymeric materials, boosting transfection several folds compared with monovalent cations. Together, these results can improve the reproducibility and efficacy of PBAE and similar polyplex nanoparticle transfections and improve the robustness of using these biomaterials for bioengineering and biotechnology applications.

The rate at which antimicrobial resistance (AMR) has developed and spread throughout the world has increased in recent years, and according to the Review on Antimicrobial Resistance in 2014, it is suggested that the current rate will lead to AMR-related deaths of several million people by 2050 (Review on Antimicrobial Resistance, Tackling a Crisis for the Health and Wealth of Nations , 2014). One major reservoir of resistant bacterial populations that has been linked to outbreaks of drug-resistant bacterial infections but is not well understood is in wastewater settings, where antibiotic pollution is often present.

Safe and effective delivery of DNA to post-mitotic cells, especially highly differentiated cells, remains a challenge despite significant progress in the development of gene delivery tools. Biodegradable polymeric nanoparticles (NPs) offer an array of advantages for gene delivery over viral vectors due to improved safety, carrying capacity, ease of manufacture, and cell-type specificity. Here we demonstrate the use of a high-throughput screening (HTS) platform to synthesize and screen a library of 148 biodegradable polymeric nanoparticles, successfully identifying structures that enable efficient transfection of human pluripotent stem cell differentiated human retinal pigment epithelial (RPE) cells with minimal toxicity. These NPs can deliver plasmid DNA (pDNA) to RPE monolayers more efficiently than leading commercially available transfection reagents. Novel synthetic polymers are described that enable high efficacy non-viral gene delivery to hard-to-transfect polarized human RPE monolayers, enabling gene loss-and gain-of-function studies of cell signaling, developmental, and disease-related pathways. One new synthetic polymer in particular, 3,3′-iminobis(N,Ndimethylpropylamine)-end terminated poly(1,5-pentanediol diacrylate-co-3 amino-1-propanol) (5-3-J12), was found to form selfassembled nanoparticles when mixed with plasmid DNA that transfect a majority of these human post-mitotic cells with minimal cytotoxicity. The platform described here can be utilized as an enabling technology for gene transfer to human primary and stem cellderived cells, which are often fragile and resistant to conventional gene transfer approaches. Lay SummaryMany retinal diseases are attributable to dysregulation in gene expression or lack of expression of specific genes, allowing for the possibility of prevention or cure of these diseases by effective delivery of nucleic acids coding for the necessary gene to the retina. Delivery of nucleic acids to cells of the retina is challenging due to the non-dividing nature of most retinal cells, preventing DNA from reaching the nucleus. To overcome this barrier, we engineered and tested a library of nanoparticle formulations to identify polymers that enabled safe and effective delivery of nucleic acid cargoes to retinal pigment epithelial cells. The nanoparticle technology explored here has the potential to be utilized for therapeutic delivery of nucleic acids to retinal cells, possibly enabling treatment for otherwise untreatable retinal diseases for which a specific genetic deficit is known but no drugs are available. KeywordsRetinal pigment epithelial cell . Human stem cell . Nanoparticles . Non-viral gene therapy . Poly(beta-amino ester) . Polymer . Ophthalmology Bibhudatta Mishra and David R. Wilson contributed equally to this work.