This article was supported by Award Number R01GM087227 from the National Institute of General Medical Sciences, the Norman Ackerman Advanced Research Program, and the Robert A. Welch foundation (Grant A-1769).

1. SPPS: CK(TMR)TATG (fTAT) Synthesis
Note: dfTAT is produced in two steps: synthesis of the monomer fTAT by solid phase peptide synthesis followed by disulfide bond dimerization to form dfTAT.
<ol>
	<li>Swell 500 mg of rink amide MBHA resin in dimethylformamide (DMF) in a standard 50 ml SPPS vessel for 1 hr.</li>
	<li>Perform Fmoc deprotection by incubating the Fmoc protected resin in a 20% piperidine solution (20% piperidine in DMF, 10 ml (0.30 mmol). Carry out deprotection steps twice (1 x 5 min and 1 x 15 min) with a washing step using DMF (the washing step includes rinsing the resin multiple times with a total volume of approximately 150 ml of DMF and removing the solvent by vacuum filtration) in between reactions.</li>
	<li>Synthesize CK(TMR)RKKRRQRRRG (fTAT) on the rink amide MBHA resin. Use the following Fmoc protected amino acids: Fmoc-Lys(Mtt)-OH (only on N-terminus), Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Cys(Trt)-OH. Carry out the reaction using N2 (20 PSI) to agitate the reaction. Perform all reactions at RT.
	<ol>
		<li>Carry out each amino acid coupling reaction for 4 hr. For each coupling, use the Fmoc protected amino acid (1.2 mmol), N,N,N &#8242; ,N &#8242; -Tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (0.44 g, 1.1 mmol), and diisopropylethylamine (DIEA) (0.51 ml, 3.0 mmol) dissolved in DMF. Following the 4 hr coupling, wash the resin with DMF and perform the Fmoc deprotection step as described in step 1.2.</li>
		<li>Repeat until the linear peptide chain of fTAT (Linear peptide chain: CKRKKRRQRRRG no TMR) was synthesized.</li>
		<li>Then wash resin thoroughly using first DMF and following that with dichloromethane (DCM). Rinse the resin multiple times with a total volume of approximately 150 ml of DMF or DCM and remove the solvent by vacuum filtration.</li>
		<li>Use MALDI-TOF to confirm that the peptide obtained has the correct molecular weight.
		<ol>
			<li>Make a matrix mix by adding 1 mg of matrix to a solution composed of 50 &#181;l of 0.01% TFA in water solution and 50 &#181;l of acetonitrile.</li>
			<li>To confirm linear peptide chain mass, use &#945;-Cyano-4-hydroxycinnamic acid. Run an analytical HPLC of the crude peptide and collect 50 &#181;l from the top of the pure peak.</li>
			<li>To prepare the sample to plate on the MALDI plate: Add 8 &#181;l of the peptide to 2 &#181;l of the matrix solution and place on the MALDI plate to air dry or on a 37 &#176;C heat plate. 
			Note: Arginine is known to be an amino acid difficult to couple in SPPS. Successful coupling is established by performing a Kaiser test14 (e.g., ninhydrin test). This test allows for the detection of free amines that might remain uncoupled on the resin. In case a blue color is detected (indicative of the presence of free amines), coupling steps are repeated.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For CK(-NH-TMR)TATG (fTAT), cleave the Mtt protecting group at the -amino group of Lys on CK(-NH-Mtt)TATG with a solution composed of 1% trifluoroacetic acid (TFA) and 2% triisopropylsilane (TIS) in DCM.
	<ol>
		<li>As the removal of MTT protecting group would result in the appearance of a yellow color, incubate the resin with 20 ml of the above solution for 5 min, repeat this until no yellow color is observed. Wash the resin with DCM and DMF in between. Additionally since TIS is a scavenger that would scavenge the MTT, once no yellow color appears in the solution in the presence of MTT.</li>
		<li>Add an additional solution with 1% TFA in DCM (no TIS) to the resin to insure no more MTT is removed and the solution remains clear.</li>
	</ol>
	</li>
	<li>Dissolve the three components: Carboxytetramethylrhodamine (TMR), HBTU, and DIEA (4, 3.9 and 10 equivalence in respect to the amount of resin (in mg)) in DMF and add this mixture to the resin and carry out the reaction O/N using dry N2 to provide agitation.</li>
	<li>Following Fmoc-deprotection and amino acid conjugation, wash the resin with DCM and allow it to dry. For complete cleavage of the peptide from the resin, add a solution containing 92.5% TFA, 2.5% H2O, 2.5%, TIS, and 2.5% ethanedithiol (EDT) (total volume = 4 ml) to the peptidyl-resin for 3 hr at RT to achieve global deprotection and cleavage from the resin.
	<ol>
		<li>Precipitate the crude peptide products using cold anhydrous ethyl ether (Et2O) by draining the solution from step 1.6 into 40 ml of Et2O, spin this down at 4 &#176;C and 4,000 x g for 20 - 25 min. Repeat this step to allow washing of the precipitate with cold anhydrous Et2O.</li>
		<li>Resuspend the precipitates in water (5 ml or until precipitate is completely dissolved) and lyophilize. Then resuspend the products obtained in 0.1% aqueous TFA/acetonitrile.</li>
	</ol>
	</li>
	<li>Perform HPLC analysis with an analytical C18 column (5 &#181;m, 4 x 150 mm) to analyze each peptide. Use a flow rate of 1 ml/min, and detection at 214 nm and 550 nm.</li>
	<li>Perform semi-preparative HPLC on a C18 10 x 250 mm column for peptide purification. Use a flow rate of 4 ml/min, and detection at 214 nm and 550 nm. For all runs, use linear gradients of 0.1% aqueous TFA (solvent A) and 90% acetonitrile, 9.9% water, and 0.1% TFA (solvent B).</li>
	<li>Confirm the correct identity of the peptides by MALDI-TOF according to manufacturer&#8217;s protocol: fTAT, expected mass: 2,041.17, observed mass: 2040.66. DEAC-K9, expected mass: 1,412.97, observed mass: 1,415.59. Use an &#945;- cyano-4-hydroxycinnamic acid matrix for the MALDI-TOF.</li>
</ol>
2. Oxidation Reaction: dfTAT Generation
<ol>
	<li>Aerate phosphate buffered saline (PBS) pH 7.4 for 1 hr (at least 5 ml for the reaction below).</li>
	<li>Dissolve fTAT (0.3 mg, 1.5 x 10-4 mmol) in aerated PBS pH 7.4 (5 ml). Make sure that the pH is between 7.0 - 7.5 after the addition of the peptide. If not add sodium hydroxide (1 M, in small increments 1 - 5 &#181;l) to bring the pH back up to 7.4.</li>
	<li>Note: Oxygen dissolved in PBS acts to oxidize the thiol groups on fTAT and form a disulfide bond.</li>
	<li>Nutate the reaction O/N to allow it to react until completion (100% yield based on HPLC analysis). Purify the product using reverse-phase HPLC and analyze by mass spectrometry (MALDI-TOF) as in step 1.9. Expected mass: 4,080.34, observed mass: 4,084.21.</li>
	<li>Lyophilize pure dfTAT (0.71 x10-4 mmol) and resuspend in 200 &#181;l water (concentration pure dfTAT = 356.3 &#181;M).</li>
</ol>
3. Measuring dfTAT Concentration
<ol>
	<li>Resuspend an aliquot of the purified dfTAT (typically 1 &#181;l depending on the amount of peptide purified) in a 149 &#181;l of 50 mM TCEP solution.</li>
	<li>Note: In this step dfTAT is reduced to its monomer counterpart fTAT to eliminate the absorbance quenching that occurs due to the close proximity of the TMR fluorophore in dfTAT (the extinction coefficient &#949; of TMR in dfTAT is reduced in comparison to the &#949; of TMR in reduced fTAT).</li>
	<li>Allow the sample to react for approximately 20 min (analytical HPLC can be used to confirm formation of fTAT).</li>
	<li>Add all the solution to the quartz cuvette and measure the absorbance at 556 nm.</li>
	<li>Using Beer&#8217;s Law ( A= &#949;cl : &#949;= 91,500 M-1 cm-1) to calculate the concentration of fTAT, determine the concentration of dfTAT, and divide [fTAT] by two.</li>
</ol>
4. Cellular Delivery Experiments
<ol>
	<li>Seed the cells (HeLa, HDF, etc.) in a dish at a confluency of 80 - 90% (e.g., 8-well or 24-well dish). Grow cells in an appropriate medium (e.g., DMEM supplemented with 10% FBS and Pen/Strep) until 80 - 90% confluency in a 37 &#176;C humidified atmosphere containing 5% CO2.</li>
	<li>Wash the cells three times (3X) with PBS (by adding 200 &#181;l of PBS and then removing it three times).</li>
	<li>Make a 5 &#181;M working concentration of dfTAT by diluting a stock of dfTAT (in water) in nrL-15 media (for an 8 well dish the total volume should be 200 &#181;l). A concentration of 5 &#181;M dfTAT leads to efficient delivery (high level of cytosolic delivery in more than 90% of cells present in a dish) in most cell types tested to date (Figure 3). However, lower or higher concentrations might be more adequate for cell types with a high or low propensity for penetration. 
	NOTE ABOUT MEDIA: nrL-15 is used for the delivery of dfTAT since it does not contain cysteine which could reduce the disulfide bond in dfTAT. However, our data indicate that both regular L-15 (with cysteine) and DMEM can be used as the delivery media. L-15 contains the reducing amino acid cysteine but cysteine presumably oxidizes in the media to form cystine (DMEM is formulated with cystine). dfTAT therefore remains intact in these media and delivery works at the same efficiency as that obtained with nrL15.</li>
	<li>Incubate cells with dfTAT (5 &#181;M) with or without cargo (e.g., EGFP (10 &#181;M)) and keep at 37 &#176;C for 1 hr (incubation time can be reduced but dfTAT typically requires approximately 30 to 45 min to induce endosomal leakage).</li>
	<li>Wash cells with heparin (1 mg/ml) in L-15 (3 washes are recommended) to remove dfTAT bound to the plasma membrane of cells.</li>
	<li>Incubate cells with cell-impermeable nuclear stain, e.g., Sytox Blue, Sytox Green (2 &#181;M in nrL-15), to determine whether the plasma membrane of cells is compromised (dead cells will be stained while live cells will not) according to manufacturer&#8217;s protocol.</li>
	<li>Image cells using a fluorescence microscope (100X oil immersion or 20X objective). Image dfTAT using a RFP filter (Ex = 560 &#177; 20 nm/Em = 630 &#177; 35 nm). 
	Note: Successful delivery leads to a diffuse fluorescence of dfTAT throughout the cell (assessing the delivery of protein or peptide of interest will depend on application). Staining of nucleoli by fTAT (the reduced product of dfTAT upon cytosolic entry) can be used as an indication that the fluorescence detected is intracellular. fTAT will degrade within few hours. At this point, the fluorescence of the degradation fragments will appear as punctate. This should not be confused for the punctate distribution that is also seen if dfTAT remains unsuccessfully trapped inside endosomes (this can happen if dfTAT is present at too low of a concentration).</li>
</ol>
5. Controling Concentration of MOI Delivered
<ol>
	<li>Identify the &#8220;optimal delivery&#8221; concentration required to achieve efficient cytosolic release of dfTAT in the cell type used. Perform fluorescence microscopy on cells incubated with increasing concentrations of dfTAT. The optimal dfTAT concentration is defined as the minimal concentration that leads to clear cytosolic &#8220;diffuse&#8221; TMR fluorescence in approximately 100% of cells in a culture.</li>
	<li>Vary the concentration of MOI used in co-incubation protocol while keeping the concentration of dfTAT constant (e.g., using dfTAT&#8217;s optimal delivery concentration). Changing the incubation time to less than 1 hr could also be an option to vary the amount of MOI delivered.</li>
</ol>

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	<li>Didenko, V. V., Ngo, H., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+as+a+transmembrane+carrier+of+fluorescently+labeled+proteins+and+antibodies.">Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies.</a> Anal Biochem. 344, 168-173 (2005).</li><li>Takeuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+Rapid+Cytosolic+Delivery+Using+Cell-Penetrating+Peptides+Mediated+by+Pyrenebutyrate.">Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate.</a> ACS Chem Biol. 1, 299-303 (2006).</li><li>Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+carrier+for+the+delivery+of+biologically+active+proteins+into+mammalian+cells.">A peptide carrier for the delivery of biologically active proteins into mammalian cells.</a> Nat Biotech. 19, 1173-1176 (2001).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+somatic+cells+via+TAT-mediated+protein+transduction+of+recombinant+factors.">Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors.</a> Biomaterials. 33, 5047-5055 (2012).</li><li>Torchilin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+of+protein+and+peptide+therapeutics.">Intracellular delivery of protein and peptide therapeutics.</a> Drug Discov Today: Technol. 5, e95-e103 (2005).</li><li>Chakravarty, P., Qian, W., El-Sayed, M. A., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+molecules+into+cells+using+carbon+nanoparticles+activated+by+femtosecond+laser+pulses.">Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses.</a> Nature nanotechnol. 5, 607-611 (2010).</li><li>Kaczmarczyk, S. J., Sitaraman, K., Young, H. A., Hughes, S. H., Chatterjee, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+engineered+virus-like+particles.">Protein delivery using engineered virus-like particles.</a> Proc Natl Acad Sci U S A. 108, 16998-17003 (2011).</li><li>Pan, C., Lu, B., Chen, H., Bishop, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+human+fibroblasts+using+HIV-1+TAT+recombinant+proteins+OCT4,+SOX2,+KLF4+and+c-MYC.">Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC.</a> Mol Biol Rep. 37, 2117-2124 (2010).</li><li>Fittipaldi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Membrane+Lipid+Rafts+Mediate+Caveolar+Endocytosis+of+HIV-1+Tat+Fusion+Proteins.">Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins.</a> J Biol Chem. 278, 34141-34149 (2003).</li><li>Lee, Y. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+endosomolytic+reagents+by+branching+of+cell-penetrating+peptides:+tools+for+the+delivery+of+bioactive+compounds+to+live+cells+in+cis+or+trans.">Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.</a> Bioconjugate chem. 21, 2164-2167 (2010).</li><li>Walev, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc Natl Acad Sci U S A. 98, 3185-3190 (2001).</li><li>Erazo-Oliveras, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+into+live+cells+by+incubation+with+an+endosomolytic+agent.">Protein delivery into live cells by incubation with an endosomolytic agent.</a> Nat methods. 11, 861-867 (2014).</li><li>Kaiser, E., Colescott, R. L., Bossinger, C. D., Cook, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+test+for+detection+of+free+terminal+amino+groups+in+the+solid-phase+synthesis+of+peptides.">Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.</a> Anal Biochem. 34, 595-598 (1970).</li><li>Muthukrishnan, N., Johnson, G. A., Lim, J., Simanek, E. E., Pellois, J. 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The authors have nothing to disclose.

The cells used for dfTAT delivery should not be overly confluent (&#62;90% confluency) since this might affect the delivery efficiency. The cells should also be healthy: dead cells in the culture can release apoptotic fragments with which dfTAT can interact (e.g., DNA from degraded nuclei). This in turn can interfere with delivery efficiency and the quality of imaging. Cells should be washed thoroughly to remove FBS before adding dfTAT. BSA present in FBS can bind to dfTAT and this can lower the delivery efficie...

The delivery of proteins, peptides or cell-impermeable small molecules into live cells is often desirable in many biological or biotechnological applications (cellular imaging, functional assays, cellular reprogramming, etc.)1-4. Many delivery approaches have already been reported, including microinjection, electroporation, or use of carrier agents (e.g., cell-penetrating peptides such as TAT, lipids)5-7. Each technique typically has specific pros and cons that might make these approaches adequate for certain applications but not for others. Common issues involve poor delivery efficiencies and/or lack of control of how much material is delivered8,9, toxicity or deleterious physiological impact10,11, lack of temporal control, delivery in few cells but not in a whole population (e.g., microinjection)12, and complex chemical conjugation or formulation schemes11.
Recently, we have developed a novel delivery strategy that circumvents these limitations. This strategy relies on a peptide named dfTAT (dimer fluorescent TAT)13. dfTAT is derived from the well-know cell&#8211;penetrating peptide (CPP) TAT. dfTAT contains two disulfide bonded copies of TAT labeled with the fluorophore tetramethylrhodamine. Despite their similarities, TAT and dfTAT differ significantly in activity. TAT is typically internalized into cells by endocytosis. This CPP remains however mostly trapped inside endosomes (this usually lead to a punctate distribution of the peptide inside cells when examined by fluorescence microscopy). Like TAT, dfTAT is efficiently endocytosed by cells. However, dfTAT does not stay trapped inside endosomes. Instead, it mediates endosomal leakage in a manner that is extremely efficient. The endosomolytic activity of dfTAT can then be exploited to deliver macromolecules by a simple incubation assay.
The current understanding of the delivery process is as follows. dfTAT induces macropinocytosis. As a result, cells incubated with dfTAT take up soluble proteins, peptides, or small molecules (molecules of interest, MOI) present in media by fluid-phase endocytosis (see Figure 1). Interactions between dfTAT and MOI are not necessary as long as both entities traffic together within the endocytic pathway. As dfTAT reaches a certain threshold within the lumen of endocytic organelles, it expresses its endosomal leakage activity (the molecular details remain to be fully characterized). The content of the lumen of leaky organelles, and therefore the MOI, is then released into the cells. This approach is therefore very convenient as no conjugation or formulation schemes with MOI are required. Moreover, because dfTAT is not directly modifying a MOI, it should also not interfere with MOIs function once intracellular delivery is achieved. In addition, the concentration of dfTAT used delivery is independent of that of MOI in media. For instance, dfTAT concentration can be kept constant between different experiments to guarantee reproducible efficiencies in endosomal leakage. In contrast, concentration of MOI in media can be gradually changed to achieve desired levels of MOI delivered in cytosol.
The high endosomal leakage efficiency achieved with dfTAT is remarkably innocuous to many of the cells tested to date. This is a surprise because endocytic organelles are important component of the cells and one would expect that the dramatic leakage mediated by dfTAT would be accompanied by deleterious cellular responses. Yet, treated cells proliferate at the same rate as untreated cells and do not display any significant changes in their transcriptome. Moreover, delivery can be repeated within minutes with reproducible delivery efficiencies, indicating that cells can either tolerate or recover from the delivery process without losing their capacity for endocytosis or endosomal leakage. Subtle cellular responses might take place during dfTAT delivery and the molecular details of what these responses might be remain to be explored. Yet, by combining high efficiency, convenience of protocols, and lack of toxicity, this delivery approach should prove immediately useful in many cell-based applications. The protocols presented herein are aimed at making this technology accessible to the research community.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Heparin&#160;</td>
			<td>Sigma</td>
			<td>CAS 9041-08-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Blue</td>
			<td>Invitrogen</td>
			<td>S11348</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Green</td>
			<td>Invitrogen</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>nrL15 L-15 (&#8211;) cysteine</td>
			<td>Hyclone</td>
			<td></td>
			<td>Special order</td>
		</tr>
		<tr>
			<td>Fmoc-protected Amino acids</td>
			<td>Novabiochem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dfTAT</td>
			<td></td>
			<td></td>
			<td>Samples will be provided upon request (contact: pellois@tamu.edu)</td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted epifluorescence microscope</td>
			<td>Olympus</td>
			<td>model IXB1</td>
			<td>equipped with a heating stage maintained at 37 &#176;C and with a Rolera-MGI Plus back-illuminated electron-multiplying charge-coupled device (EMCCD) camera (Qimaging).&#160;</td>
		</tr>
		<tr>
			<td>37 &#176;C humidified, 5% CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide synthesizer or vessel to preform manual peptide synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of proteins, peptides or cell-impermeable small molecules into live cells by incubation with the endosomolytic reagent dftat

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

To assess the difference between fTAT and dfTAT, HeLa cells are incubated for 1 hr with each peptide to determine the difference in their cellular localization. The internalization of the two CPP&#8217;s was assessed using fluorescence microscopy. Figure 2 shows that fTAT (20 &#181;M) localizes in a punctate distribution. This distribution is consistent with the peptide remaining entrapped inside endosomes. In contrast, the fluorescence signal of dfTAT (5 &#181;M) displays a homogenous distribution throu...

Watch this Scientific Journal Video about Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT at JoVE.com

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

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Bioengineering

10.2K visualizações.  Texas A&M University. We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky. 

Vídeo: Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...