Subtle Changes to Polymer Structure and Degradation Mechanism Enable Highly Effective Nanoparticles for siRNA and DNA Delivery to Human Brain Cancer

Tzeng, Stephany Y.; Green, Jordan J.

doi:10.1002/adhm.201200257

Cited by 88 publications

(120 citation statements)

References 34 publications

(31 reference statements)

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“…21, 22 PBAEs are important to study as they have been demonstrated to have virus-like efficacy in certain human primary cells and low cytotoxicity. 23 Some studies investigated how physical and chemical properties of PBAEs affect gene delivery efficacy.…”

Section: Resultsmentioning

confidence: 99%

Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

2013

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Successful gene delivery with non-viral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly(β-amino ester)s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogs of PBAEs with the same base polymer poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10 – 17 mV) nanoparticles of ~ 200 nm in size. Cellular internalization of all three formulations was inhibited the most (60 – 90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells, and that small changes to the same base polymer can modulate their cellular uptake and transfection routes.

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Section: Resultsmentioning

confidence: 99%

Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

2013

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“…However, a large number of polymers also exhibited strong delivery to normal cells (SI Appendix, Table S1). Among the 137 selective polymers, the emergence of cysteamine (A17) suggests that this functional group may promote internalization through specialized pathways that reduce trafficking to lysosomes, thereby enhancing siRNA release inside of cancer cells (33,42,(46)(47)(48)(49)(50)(51). Previous reports on cysteaminefunctionalized dendrimers (48) and the cell-penetrating peptide Pep-1 (49-51) that contain terminal cysteamine groups suggest that membrane binding and uptake is dependent on this functional group.…”

Section: A Scalable Methods Enabled Synthesis Of Polyesters With Divermentioning

confidence: 99%

Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells

Yan

Liu

Xiong

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Conventional chemotherapeutics nonselectively kill all rapidly dividing cells, which produces numerous side effects. To address this challenge, we report the discovery of functional polyesters that are capable of delivering siRNA drugs selectively to lung cancer cells and not to normal lung cells. Selective polyplex nanoparticles (NPs) were identified by high-throughput library screening on a unique pair of matched cancer/normal cell lines obtained from a single patient. Selective NPs promoted rapid endocytosis into HCC4017 cancer cells, but were arrested at the membrane of HBEC30-KT normal cells during the initial transfection period. When injected into tumor xenografts in mice, cancer-selective NPs were retained in tumors for over 1 wk, whereas nonselective NPs were cleared within hours. This translated to improved siRNA-mediated cancer cell apoptosis and significant suppression of tumor growth. Selective NPs were also able to mediate gene silencing in xenograft and orthotopic tumors via i.v. injection or aerosol inhalation, respectively. Importantly, this work highlights that different cells respond differentially to the same drug carrier, an important factor that should be considered in the design and evaluation of all NP carriers. Because no targeting ligands are required, these functional polyester NPs provide an exciting alternative approach for selective drug delivery to tumor cells that may improve efficacy and reduce adverse side effects of cancer therapies.

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“…Yo-Pro-1 iodide binding assays were run similarly to previously published results, 4 where DNA and Yo-Pro-1 iodide (Thermo Fisher) were both diluted to a concentration of 1 mM in 10 mM NaCl. For pH nanosensor trials, 100% Cy5-only-labeled DNA was used to avoid fluorescence from the green channel fluorophores.…”

Section: Nanoparticle Characterizationmentioning

confidence: 99%

“…1,3 Polymeric gene delivery in particular has made extensive progress toward increased performance through rational engineering of polymer structures as well as screening of broad libraries of polymer structures, but new quantitative bioassays are required to fully understand the mechanisms by which existing nanoparticles achieve transfection. [4][5][6][7][8][9][10][11] Barriers to successful polymeric gene delivery at the level of individual cells include cellular internalization, endosomal escape, nucleic acid unpacking, and nuclear transport. 12,13 Of these barriers, overcoming endosomal escape has been specifically identified as a critical ratelimiting step in polycation nanoparticle-mediated transfection because generally only a minor fraction of endocytosed polyplexes manage to escape to the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

et al. 2017

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There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

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Subtle Changes to Polymer Structure and Degradation Mechanism Enable Highly Effective Nanoparticles for siRNA and DNA Delivery to Human Brain Cancer

Cited by 88 publications

References 34 publications

Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery

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