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Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.

Microfluidic devices fabricated using poly(dimethylsiloxane) (PDMS) polymer are routinely used for in vitro cell culture for a wide range of cellular assays. These assays typically involve the incubation of cultured cells with a drug molecule or a fluorescent marker while monitoring a cellular response. The accuracy of these assays depends on achieving a consistent and reproducible concentration of solute molecules in solution. However, hydrophobic therapeutic and fluorescent molecules tend to diffuse into the PDMS walls of the microfluidic devices, which reduce their concentration in solution and consequently affect the accuracy and reliability of these assays. In this paper, we quantitatively investigate the relationship between the partition coefficient (log P) of a series of markers routinely used in in vitro cellular assays including [3H]-dexamethasone, [3H]-diazepam, [14C]-mannitol, [3H]-phenytoin, and rhodamine 6G and their absorption into PDMS microfluidic channels. Our results show that the absorption of a given solute into PDMS depends on the hydrophilic/hydrophobic balance defined by its log P value. Specifically, results demonstrate that molecules with log P less than 2.47 exhibit minimal absorption (<10%) into PDMS channels whereas molecules with log P larger than 2.62 exhibit extensive absorption (>90%) into PDMS channels. Further investigations showed that TiO(2) and glass coatings of PDMS channels reduced the absorption of hydrophobic molecules (log P > 2.62) by 2- and 4.5-folds, respectively.

This manuscript describes a straightforward fabrication process for embedding Ag/AgCl electrodes within a two-layer PDMS microfluidic chip where an upper and a lower channel are separated by a semi-porous membrane. This system allows for the reliable real-time measurement of transendothelial and trans-epithelial electrical resistance (TEER), an accepted quantification of cell monolayer integrity, across cells cultured on membranes inside the microchannels using impedance spectroscopy. The technique eliminates the need for costly or specialized microelectrode fabrication, enabling commercially available wire electrodes to easily be incorporated into PDMS microsystems for measuring TEER under microfluidic environments. The capability of measuring impedance across a confluent cell monolayer is confirmed using (i) brain-derived endothelial cells (bEND.3), (ii) Madin Darby Canine Kidney Cells (MDCK-2), and mouse myoblast (C2C12) (all from ATCC, Manassas, VA). TEER values as a function of cell type and cell culture time were measured and both agree with previously published values from macro-scale culture techniques. This system opens new opportunities for conveniently resolving both trans-endothelial and trans-epithelial electrical resistance to monitor cell function in real-time in microfluidic cell cultures. KeywordsTrans-endothelial electrical resistance; Trans-epithelial electrical resistance; TEER; Blood Brain Barrier; bEND.3; Impedance Spectroscopy; Impedance Spectra; Barrier Integrity; Microfluidic Endothelium; Microfluidic Epithelium Trans-membrane electrical resistance offers a quantitative technique to measure the integrity of the tight junctions that govern solute transport across the paracellular space of endothelial SUPPORTING INFORMATION AVAILABLE: Matlab Programs for resolving TEER from Impedance Spectra (circuit_fit.m and circuit_fun.m)