Nuclear delivery of macromolecules: barriers and carriers

Belting, Mattias; Sandgren, Staffan; Wittrup, Anders

doi:10.1016/j.addr.2004.10.004

Cited by 190 publications

(99 citation statements)

References 195 publications

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“…PG's form complexes with CL's, leading to the release of DNA and intracellular accumulation of CL-PG complexes. This results in inhibition of DNA uptake and a significant reduction in reporter-gene expression (Belting and Petersson 1999;Belting et al 2005;Gardner et al 2007). These findings support our observation that fresh conditioned medium has a negative effect on protein expression.…”

Section: Discussionmentioning

confidence: 99%

Development of a generic transient transfection process at 100 L scale

et al. 2008

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We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L -1 IgG.

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Section: Discussionmentioning

confidence: 99%

Development of a generic transient transfection process at 100 L scale

et al. 2008

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“…In addition, both DNA and plasma membrane are negatively charged, which means that the repulsive electrostatic force could hinder delivery of naked DNA into cells as well. 7 Various gene delivery methods have been developed to circumvent the barriers. 8 In general, viral-based methods are more efficient and capable of integrating certain desired genes into the host genome for sustained gene expression.…”

Section: Introductionmentioning

confidence: 99%

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

et al. 2017

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“…Heparan sulfate proteoglycans (HSPGs), a family of proteins substituted with polysulfated heparan sulfate (HS) polysaccharides, have a key role in the endocytic uptake of a diverse set of macromolecular drugs, growth factors, and morphogens (2,5,11,12) with important implications for early developmental patterning and cancer. Here, we have capitalized on the recent finding (13) that magnetic nanoparticles can be endocytosed efficiently by conjugation to a specific, phage-derived anti-HS antibody (αHS) for the isolation and analysis of intact, endocytic vesicles.…”

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confidence: 99%

“…drug delivery | endocytosis | heparan sulfate | proteoglycan | membrane raft E ndocytosis is the process by which cells compartmentalize constituents of the plasma membrane and the extracellular space into intracellular vesicles for further sorting to specific cellular locations (1)(2)(3)(4). Endocytosis regulates signaling events involved in cell motility and cell fate determination and can be exploited by microbial intruders for infection.…”

mentioning

confidence: 99%

Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

Wittrup¹,

Zhang²,

Svensson³

et al. 2010

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

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An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.E ndocytosis is the process by which cells compartmentalize constituents of the plasma membrane and the extracellular space into intracellular vesicles for further sorting to specific cellular locations (1-4). Endocytosis regulates signaling events involved in cell motility and cell fate determination and can be exploited by microbial intruders for infection. Interestingly, the same pathways may be used for the delivery of therapeutic macromolecules (e.g., DNA, anti-sense oligonucleotides, and siRNA) in the treatment of various diseases. A more detailed understanding of endocytic mechanisms thus is a major challenge in basic cell biology with implications for viral infection, the regulation of signaling networks in cancer (3), and the development of macromolecular drugs (5).High-resolution, live-cell imaging techniques have unraveled the heterogeneity of vesicular compartments in terms of kinetic/ dynamic parameters as well as ligand specificity. In addition to the classical clathrin-dependent mechanism of endocytosis, several clathrin-independent endocytic pathways are emerging (3,4,6). Collectively, published data from several groups indicate that ligands can be taken up by multiple lipid raft-mediated pathways; however, these pathways require further definition at the molecular level. Classification schemes based on the dependence on/association with dynamin, caveolin-1, and the RhoA family GTPases Rac1, RhoA, and CDC42 have b...

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Nuclear delivery of macromolecules: barriers and carriers

Cited by 190 publications

References 195 publications

Development of a generic transient transfection process at 100 L scale

Development of a generic transient transfection process at 100 L scale

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

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