The possible "proton sponge " effect of polyethylenimine (PEI) does not include change in lysosomal pH.

Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.

Hyaluronic acid immobilized polyacrylamide nanoparticle sensors for CD44 receptor targeting and pH measurement in cells.

Our ability to design receptor-targeted nanocarriers aimed at drug release after endocytosis is limited by the current knowledge of intracellular nanoparticle (NP) trafficking. It is not clear if NP size, surface chemistry, and/or targeting of cell surface receptors changes the intracellular fate of NPs; i.e., will all NPs enter acidic compartments and eventually end up in lysosomes or are there escape mechanisms or receptor-specific signaling that can be induced to change the cellular processing of an internalized NP? To give new insight into the intracellular trafficking of NPs that target the CD44 receptor, which is overexpressed on the surface of a broad variety of cancer cells, we have synthesized an NP pH sensor system that targets CD44. We used a polyacrylamide nanoparticle matrix bearing hyaluronic acid (HA) on the surface as a CD44 targeting ligand. The HA-coated NPs were prepared by radical polymerization followed by post functionalization with sensor fluorophores and physically absorbed or chemically conjugated HA. Cell uptake studies showed significant uptake of HA-coated nanosensors in HeLa cells and no uptake under the same conditions without the HA targeting ligand. The pH distribution profile in cells was measured for nanosensors with HA, cationic, and noncharged NP surface coatings giving a clear indication of the intracellular pH environment that the different NPs experience after internalization. The pH profile of cationic nanosensors in comparison to HA conjugated nanosensors indicates that the intracellular trafficking is aimed at lysosomes regardless of whether CD44 receptor-specific or unspecific uptake is induced.

Breast cancer cells can switch between estrogen receptor alpha and ErbB signaling and combined treatment against both signaling pathways postpones development of resistance.

The majority of breast cancers are estrogen responsive, but upon progression of disease other growth promoting pathways are activated, e.g., the ErbB receptor system. The present study focuses on resistance to the pure estrogen antagonist fulvestrant and strategies to treat resistant cells or even circumvent development of resistance. Limited effects were observed when targeting EGFR and ErbB2 with the monoclonal antibodies cetuximab, trastuzumab, and pertuzumab, whereas the pan-ErbB inhibitor CI-1033 selectively inhibited growth of fulvestrant resistant cell lines. CI-1033 inhibited Erk but not Akt signaling, which as well as Erk is important for antiestrogen resistant cell growth. Accordingly, combination therapy with CI-1033 and the Akt inhibitor SH-6 or the Protein Kinase C inhibitor RO-32-0432 was applied and found superior to single agent treatment. Further, the resistant cell lines were more sensitive to CI-1033 treatment when grown in the presence of fulvestrant, as withdrawal of fulvestrant restored signaling through the estrogen receptor alpha (ERalpha), partly overcoming the growth inhibitory effects of CI-1033. Thus, the resistant cells could switch between ERalpha and ErbB signaling for growth promotion. Although parental MCF-7 cell growth primarily depends on ERalpha signaling, a heregulin-1beta induced switch to ErbB signaling rescued MCF-7 cells from the growth inhibition exerted by fulvestrant-mediated blockade of ERalpha signaling. This interplay between ERalpha and ErbB signaling could be abrogated by combined therapy targeting both receptor systems. Thus, the present study indicates that upon development of antiestrogen resistance, antiestrogen treatment should be continued in combination with signal transduction inhibitors. Further, upfront combination of endocrine therapy with pan-ErbB inhibition may postpone or even prevent development of treatment resistance.

Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant.

Seven fulvestrant resistant cell lines derived from the estrogen receptor alpha positive MCF-7 human breast cancer cell line were used to investigate the importance of epidermal growth factor receptor (ErbB1-4) signaling. We found an increase in mRNA expression of EGFR and the ErbB3/ErbB4 ligand heregulin2 (hrg2) and a decrease of ErbB4 in all resistant cell lines. Western analyses confirmed the upregulation of EGFR and hrg2 and the downregulation of ErbB4. Elevated activation of EGFR and ErbB3 was seen in all resistant cell lines and the ErbB3 activation occurred by an autocrine mechanism. ErbB4 activation was observed only in the parental MCF-7 cells. The downstream kinases pAkt and pErk were increased in five of seven and in all seven resistant cell lines, respectively. Treatment with the EGFR inhibitor gefitinib preferentially inhibited growth and reduced the S phase fraction in the resistant cell lines concomitant with inhibition of Erk and unaltered Akt activation. In concert, inhibition of Erk with U0126 preferentially reduced growth of resistant cell lines. Treatment with ErbB3 neutralizing antibodies inhibited ErbB3 activation and resulted in a modest but statistically significant growth inhibition of two resistant cell lines. These data indicate that ligand activated ErbB3 and EGFR, and Erk signaling play important roles in fulvestrant resistant cell growth. Furthermore, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Our data support that a concerted action against EGFR, ErbB2 and ErbB3 may be required to obtain complete growth suppression of fulvestrant resistant cells.

Evaluating nanoparticle sensor design for intracellular pH measurements.

Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.

Polymeric nanosensors for measuring the full dynamic pH range of endosomes and lysosomes in mammalian cells.

Polymer nanoparticle sensors have been constructed for studying pH in the endocytic pathway in mammalian cells. The pH sensors for fluorescence ratiometric measurements were prepared using inverse microemulsion polymerization with rhodamine as reference fluorophor and fluorescein and oregon green as pH sensitive dyes, which gave a dynamic pH measurement range from 4.1-7.5. Thus, the sensors cover the pH range of almost all intracellular compartments in mammalian cells. Both neutral and cationic polyacrylamide particles were synthesized where (3-acrylamidopropyl) trimethylammonium chloride was used to introduce a net positive charge in the cationic particles. It was found that the positively charged particle sensors were internalized spontaneously by HepG2 cancer cells. These new pH nanosensors are potential tools in time resolved quantification of pH in the endocytic pathway of living cells.