Enhancement of cellular uptake by increasing the number of encapsulated gold nanoparticles in polymeric micelles.

We apply a combination of polycaprolactone (PCL)-thiol ligand functionalization with flow-controlled microfluidic block copolymer self-assembly to produce biocompatible gold nanoparticle (GNP)-loaded micellar polymer nanoparticles (GNP-PNPs) in which GNPs are encapsulated within PCL cores surrounded by an external layer of poly(ethylene glycol) (PEG). By varying both the relative amount of block copolymer and the microfluidic flow rate, a series of GNP-PNPs are produced in which the mean number of GNPs per PNP in the < 50-nm fraction (Zave,d< 50 nm) varies between 0.1 and 1.9 while the external PEG surface is constant. Zave,d< 50 nm values are determined by statistical analysis of TEM images and compared with the results of cell uptake experiments on MDA-MB-231 cancer cells. For Zave,d< 50 nm ≤ 1 (including a control sample of individual GNPs also with a PEG surface layer), cell uptake is relatively constant, but increases sharply for Zave,d< 50 nm > 1, with a factor of 7 enhancement as Zave,d< 50 nm increases from 1 to ∼2. Enabled by the shear processing control provided by the microfluidic chip, these results provide the first evidence that cellular uptake can be enhanced specifically by increasing the number of GNPs per vector, with other parameters, including polymeric material, internal structure, and external surface chemistry, held constant. They also demonstrate a versatile platform for packaging GNPs in biocompatible polymeric carriers with flow-controlled formulation optimization for various therapeutic and diagnostic applications.

Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors.

Microfluidic manufacturing of advanced gene delivery vectors necessitates consideration of the effects of microfluidic shear forces on the structural integrity of plasmid DNA (pDNA). In this paper, we expose pDNA to variable shear forces in a two-phase, gas-liquid microfluidic reactor and apply gel electrophoresis to analyze the products of on-chip shear-induced degradation. The effects of shear rate, solvent environment, pDNA size, and copolymer complexation on shear-induced degradation are investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibits shear rate-dependent shear degradation in the microfluidic channels in a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1 w/w/w), with the extents of both supercoil isoform relaxation and complete fragmentation increasing as the maximum shear rates increase from 4 × 105 to 2 × 106 s-1. However, over the same range of shear rates, the same pDNA sample shows no evidence of microfluidic shear-induced degradation in a pure aqueous environment. Quiescent control experiments in the same mixed organic solvent prove that a combination of solvent and shear forces is involved in the observed shear-induced degradation. Furthermore, we show that shear degradation effects in mixed organic solvents can be significantly attenuated by complexation of pDNA with the block copolymer polycaprolactone-block-poly(2-vinylpyridine) prior to exposure to microfluidic shear. Finally, we demonstrate that medium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA are more sensitive to shear-induced microfluidic degradation in the mixed organic solvent environment than small pDNA, with both plasmids showing complete fragmentation even at the lowest shear rate, although we found no evidence of shear-induced damage in water for the largest investigated naked pDNA even at the highest flow rate. The resulting understanding of the interplay of the solvent and shear effects during microfluidic processing should inform microfluidic manufacturing routes to new gene therapy formulations.

Controlling Structure and Function of Polymeric Drug Delivery Nanoparticles Using Microfluidics.

We demonstrate control of multiscale structure and drug delivery function for paclitaxel (PAX)-loaded polycaprolactone-block-poly(ethylene oxide) (PCL-b-PEO) polymeric nanoparticles (PNPs) via synthesis and flow-directed shear processing in a two-phase gas-liquid microfluidic reactor. This strategy takes a page from the engineering of commodity plastics, where processing rather than polymer chemistry provides an experimental handle on properties and function. PNPs formed from copolymers with three different PCL block lengths show sizes, morphologies, and loading efficiencies that depend on both the PCL block length and the microfluidic flow rate. By varying flow rate and comparing with a conventional bulk method of PNP preparation, we show that flow-variable shear processing provides control of PNP sizes and morphologies and enables slower PAX release times (up to 2 weeks) compared to bulk-prepared PNPs. Antiproliferative effects against cultured MCF-7 breast cancer cells were greatest for PNPs formed at an intermediate flow rate, corresponding to small and low-polydispersity spheres formed uniquely at this flow condition. Formation and flow-directed nanoscale shear processing in gas-liquid microfluidic reactors provides a manufacturing platform for drug delivery PNPs that could enable more effective and selective nanomedicines through multiscale structural control.