In vitro and ex vivo strategies for intracellular delivery

Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

doi:10.1038/nature19764

Cited by 710 publications

(767 citation statements)

References 99 publications

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“…However, access to the whole cytoplasm by the delivered protein remains elusive. A major hurdle in cytoplasmic protein delivery is the endosomal entrapment of the delivered cargo: nanocarrier-based delivery methods result in only a fraction of the entrapped cargo (often ~1%) escaping into the cytosol (Stewart et al ., 2016). Additionally, protease-mediated degradation and exocytosis of the remaining entrapped cargo proteins make these strategies ultimately inefficient.…”

Section: Introductionmentioning

confidence: 99%

A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles

Mout¹,

Rotello²

2017

BIO-PROTOCOL

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In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al., 2017). To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency). Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

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Section: Introductionmentioning

confidence: 99%

A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles

Mout¹,

Rotello²

2017

BIO-PROTOCOL

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“…One important factor of the abovementioned unpredictability lies on the arduous control of the retention of nanoparticles inside a specific organelle, or the difficulty to generate a controlled diffusion of the nanostructures across the entire cell. At first glance it may seem that the heat generated can be retained in a single cell compartment at lower intensities or, if the irradiation is increased, it can literally "melt" the biological membranes [2], starting the diffusion of the nanoparticles throughout the cell [15] (Fig. 1).…”

Section: You Cannot Hit What You Cannot See: Understanding Pttmentioning

confidence: 99%

“…Combined with nanoparticles, it represents a relatively novel and attractive tool that has grabbed the attention of both researchers and clinicians [1,2]. It consists of the use of nanoparticles with the ultimate goal to specifically localize in the diseased area, which upon illumination with a laser are able to convert the incoming light into heat which ultimately induce cell death of the affected area [3], similar to a nanometric scalpel.…”

Section: Introduction : H Y P E R T H E R M I a Nowadaysmentioning

confidence: 99%

The True Complexity of Photothermal Therapy: A Brief Perspective

Alfranca¹,

Cui²,

Fuente³

2017

Nano BioMed ENG

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“…9 In addition, their integration into liposomes to generate proteoliposomes holds great promise to vectorize therapeutic proteins. [10][11][12] Although various strategies are currently available for the delivery of intracellular proteins, 13 there is a lack of vectors for membrane proteins. Liposomes are safe nano-carriers that are ideal for the vectorization of not only chemical drugs but also a large number of biological molecules, including nucleic acids, peptides and proteins.…”

Section: Introductionmentioning

confidence: 99%

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

Brault¹,

Vaganay²,

Roy³

et al. 2017

IJN

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International audienceChronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22 phox , the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22 phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22 phox liposomes was estimated to be around 700 nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47 phox , p67 phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22 phox liposomes for 8 h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments

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In vitro and ex vivo strategies for intracellular delivery

Cited by 710 publications

References 99 publications

A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles

A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles

The True Complexity of Photothermal Therapy: A Brief Perspective

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

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