Name: initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting
Uploaded: 2018-03-08T12:30:00
Description: Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

This work was funded by the Cancer Research Society (Canada) and the CIMS. The authors thank Dr. Samia Ait-Mohand and Jean-Francois Beaudoin for assistance. Dr. Angel Lopez (University of South Australia) for mAb A14.

The in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Centre Hospitalier Universitaire de Sherbrooke Ethics Committee for Animal Experiments.
1. Antibody Peptide Conjugation
NOTE: ChAcNLS can be synthesized at any commercial peptide manufacturer or university-affiliated peptide synthesis service platform. The synthesis of ChAcNLS can be found in reference 34. F...

Major goals of systemic delivery of anti-cancer agents are to increase accumulation at the tumor site, and uptake within cancer cells, and decrease unwanted side effects in healthy tissues. AC targeted delivery of molecular payloads to tumor cells is a highly promising approach to treat and detect tumors. However, the lack of efficacy caused by endosome entrapment and down stream lysosomal degradation remains an important challenge. While this protocol utilizes the ChAcNLS peptide as an example for the construction of ne...

Antibody-conjugates (ACs) are biopharmaceuticals that are maturing into a transformative class of effective drugs for improving cancer treatments and for detecting tumors. Composed of a monoclonal antibody (mAb) conjugated to molecular payloads such as radioisotopes, small molecules, and biological toxins, ACs are able to deliver these payloads to cancer cells with exquisite target antigen affinity and specificity. Thus ACs have the potential to significantly reduce nonspecific toxicity and increase payload activity at the tumor site. Therapeutically, ACs transporting cytotoxic small molecules (commonly referred to as antibody-drug conjugates) have been approved for treating patients with breast cancer and Hodgkin&#39;s lymphoma who have failed conventional treatments 1,2. In addition, ACs transporting radioisotopes (commonly referred to as radioimmunoconjugates) are also in development. An AC transporting a radioisotope for imaging is approved for identifying prostate cancer metastasis 3. With many more therapeutic ACs submitted for approval 4, optimism is high for the future of ACs to improve cancer care 5.
Nonetheless, when delivering chemotherapeutics or radioisotopes, ACs have difficulty effectively accumulating these payloads inside target cells. This aspect significantly contributes in many cases in the inability of ACs to provide long-lasting disease-free survival or high contrast tumor imaging 6,7. In general, once ACs bind their target antigen they are internalized through a process known as receptor-mediated endocytosis. The ACs are then entrapped inside endosomes and trafficked to lysosomes for degradation and payload release 8. The intracellular trafficking process poses challenges for ACs to achieve a high payload specificity and efficacy against target cancer cells. For example, many antigens such as Her2 (target for therapeutic AC Trastuzumab-emtansine) can recycle up to 85% of bound antibodies in the first 30 min 9. Furthermore, once degradation occurs, released chemotherapeutics and radioisotopes can be actively exported by increased expression and/or activity of membrane associated transport proteins 10,11. Lysosome degradation also impedes the delivery of novel biological payloads such as therapeutic enzymes and oligonucleotides that can be deactivated 12,13. In essence, the cancer cell is highly effective at abrogating the necessary intracellular accumulation of payloads delivered by ACs.
This protocol describes how to implement the concept of ACs-coupled to virus-derived peptides, specifically for escaping endosome entrapment and localizing to the cell nucleus. With such sophistication to manipulate host cell systems, it is not surprising that the development of virus-derived proteins and peptides as potential biopharmaceuticals has long been ingrained in therapeutic research 14. For millions of years viruses have evolved to acquire an exceptional collection of proteins able to exploit normal physiological mammalian cell systems in order to effectively enter host cells. For viruses that are internalized via receptor-mediated endocytosis, they are also challenged with escaping trafficking to the lysosome where the onslaught of a localized concentration of proteases can be problematic for survival. A well characterized viral-derived peptide utilized in drug delivery for escaping endosome entrapment is the human immunodeficiency virus transactivator of transcription (Tat) protein 15. Tat is able to escape endosome entrapment by sensing low-pH at which point protein conformational changes occur enabling Tat to insert itself into and disrupt the endosomal membrane 16. This results in Tat-payload conjugates able to access the cytoplasm. The second viral manipulation element related to this protocol is the approach used to deliver therapeutic genes and drugs to the nucleus 17. Viruses have evolved to successfully manipulate host cell machinery for progressing past the nuclear membrane by passing through the nuclear pore complex (NPC). Cellular macromolecules contain (or bind to proteins that contain) nuclear localization signals (NLSs) necessary for binding to nuclear transport proteins (e.g. karyopherins &#945; and &#946;), which provide the required movements through the NPC. Viruses have developed proteins to contain NLS sequences that provide them with the ability to utilize host cell transport proteins for shuttling into the nucleus 18.
Numerous ACs have previously been functionalized with Tat- and NLS-derived peptides and tested for their ability to accumulate inside cancer cells and for targeting tumors 19,20,21,22,23,24,25,26,27,28,29,30 (Table 1). Studies delivering cytotoxic payloads have demonstrated that ACs modified with virus-derived peptides are able to significantly increase cellular accumulation, cytotoxicity, and tumor killing over unmodified ACs 22,26. A common feature for this novel class of AC is their construction. Typically, peptides contain a terminal cysteine providing a free sulfhydryl group. MAbs are first reacted with a noncleavable bifunctional crosslinker containing N-hydroxysuccinimide (NHS) and maleimide groups at opposite ends. The NHS esters react with primary amines on the mAb to form amide bonds. The reacted mAb with free maleimide groups is then reacted with the sulfhydryl groups on the peptides to form a thioester bond and thus linking the peptide and mAb. Although homobifunctional crosslinkers have been used 28, heterobifunctional crosslinker are more commonly used in the construction of virus-derived peptide-ACs 22,23,26,31,32. This protocol specifically uses the crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for its ease of use and because it is used in the approved antibody-drug conjugate Trastuzumab-emtansine and in many virus-derived peptide-ACs 8,22,23,26,31,32. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the primary method for initially determining conjugation efficiency and for semi-quantifying the number of peptides per mAb. Confocal microscopy using a fluorescently-labeled secondary antibody specific to the mAb is typically the method for initially evaluating intracellular distribution properties of virus-derived peptide-modified ACs. Thus far, radioisotopes are the primary payloads delivered by virus-derived peptide-modified ACs. Radioisotopes are advantageous because radioactivity in cells is easily quantified by gamma counting. In addition, ACs that are translated into mouse models of human cancers provide researchers with the ability to evaluate tumor targeting using molecular imaging modalities such as single photon emission computed tomography and positron emission tomography (PET) 23,32,33. In general, the construction and validation testing methods primarily used by researchers provide a very good assessment of ACs modified with virus-derived peptides during the initial development stage to effectively enter and deliver the payload inside target cells and to target tumors.
Tat- and NLS-modified ACs have illuminated key areas for further improving payload delivery inside cancer cells and to tumors. With respect to NLS-modified ACs, the efficiency in intracellular accumulation can be modest 23,31,34. Inefficient intracellular accumulation is caused by continued endosomal entrapment. In vivo tumor targeting can also be diminished with both Tat- and NLS-modified ACs. The active sequences of Tat and NLS contain several positive charged residues. When attached to mAbs, the overall cationic charge can be significantly increased 35. As a consequence, the Tat- and NLS-modified ACs have increased uptake in healthy tissues and increased rapid blood clearance.
Our group developed a composite compound consisting of cholic acid linked to NLS (ChAcNLS; Figure 1). ChAcNLS-modified ACs are able to increase intracellular accumulation of delivered radioisotopes and improve tumor targeting compared to NLS-modified and traditional ACs 33,34. The mechanism behind cholic acid is inspired by the ability of select nonenveloped viruses that cannot rely on membrane fusion to utilize cholic acid to trigger endosome escape through the formation of ceramide. For example, porcine enteric virus recruits cholic acid that activates sphingomyelinase, which catalyzes the hydrolysis of sphingomyelin into ceramide 36,37,38. This destabilizes endosomal membrane and allows for virus escape. Thus, cholic acid is another virus-derived component that complements NLS.
As this field moves forward and future advancements occur in payload delivery by ACs modified with virus-derived peptides, it is an opportune time to provide visual demonstrations of their biochemical and functional characteristics during initial development. Here, we describe our protocol for the initial evaluation of virus-derived peptide-modified ACs for the efficient yet simple determination of intracellular accumulation and tumor targeting during early stage development. We use the commercially available mAbs 7G3 and A14 as example model systems. Procedure 1 describes the use of SDS-PAGE as a method that allows for &#8216;go/no go&#8217; decisions for constructed ACs. Procedure 2 describes a method using trypsinization allowing for improved visualization of AC intracellular distribution and accumulation. Procedure 3 describes a method for improved intracellular fractionation to accurately determine nuclear localization. In this procedure we utilize the payload 64Cu (t1/2 = 12.7 h) because it is vulnerable to cellular efflux and is a positron emitter 10. Thus, Procedure 4 describes in vivo tumor targeting characterization by PET imaging to visualize tumor uptake relative to background (i.e. nontarget healthy tissues) and determine whether the example AC can specifically and effectively target tumors. These methods are sufficient for investigators developing ACs modified with virus-derived peptides to identify candidates for further advancement.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermo Scientific</td>
			<td>22122</td>
			<td>There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 mL Centrifugal Filters</td>
			<td>EMD Millipore</td>
			<td>UFC505096</td>
			<td>There are pack sizes of 8, 24, and 96. Choose according to your needs.</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Kaleidoscope Standards</td>
			<td>BioRad</td>
			<td>1610375EDU</td>
			<td>Mulicolor recombinant proteins from 10-250 kDa.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200056</td>
			<td>100 or 500 mL volumes to choose from.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td>Thermo Scientific</td>
			<td>A-21235</td>
			<td>1-10 &#956;g/mL recommended</td>
		</tr>
		<tr>
			<td>NOTA-NHS</td>
			<td>CheMatech</td>
			<td>C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamin A/C antibody (N-18)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6215</td>
			<td></td>
		</tr>
		<tr>
			<td>Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376362</td>
			<td></td>
		</tr>
		<tr>
			<td>A14 mAb</td>
			<td>BD Biosciences</td>
			<td>555902</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4x)</td>
			<td>Thermo Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TF-1a cells</td>
			<td>ATCC</td>
			<td>ATCC CRL-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>ATCC</td>
			<td>ATCC 30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis and extraction buffer</td>
			<td>Thermo Scientific</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>AMIDE medical imaging software</td>
			<td>available at amide.sourceforge.net</td>
			<td></td>
			<td>Completely free download</td>
		</tr>
		<tr>
			<td>FluoView FV1000 Confocal Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoview Software</td>
			<td>Olympus</td>
			<td></td>
			<td>www.olympus-lifescience.com</td>
		</tr>
		<tr>
			<td>ITLC strips</td>
			<td>Biodex</td>
			<td>150-771</td>
			<td></td>
		</tr>
	</tbody>
</table>

initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

For Procedure 1, the construction of 7G3 modified with ChAcNLS using sulfo-SMCC as a crosslinker is very reliable. Typically, when loaded onto a 12% gel and analyzed by SDS-PAGE, this results in distinguishable stepwise increases in MW proportional to increasing sulfo-SMCC-to-7G3 ratios used and allows for the heavy and light chains to be individually assessed for ChAcNLS conjugation (Figure 3). 7G3 reacted at 10-, 20-, 25-, and 50-to-1 sulfo-SMCC-to-7G3 ratios followed b...

Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

The overall goal of these procedures is to evaluate the specificity and effectiveness of antibody conjugates for accumulating inside target cells and tumors during the initial development stages so that they can ultimately be used in the clinic. This method can help answer key question on the answer the conjugate fields, such as the efficiency of nuclear localization and the overall intercellular accumulation. An advantage of this technique is that via PET imaging researchers can assess the effectiveness and selectivity of candidate antibody conjugates.The implications of these techniques extend towards therapy and diagnosis of cancer, because ineffective tumor-cell accumulation is a major limitation with these applications for antibody conjugates. After synthesizing a colic-acid nuclear localization sequence antibody conjugate according to the text protocol, treat target TF1A cells with 200 nanomolar 7G3 colic-acid MLS antibody conjugate. Include cells treated with modified control monoclonal antibodies.Incubate five times 10 to the sixth antigen-positive cells with the conjugates at 37 degrees Celsius for one hour. Then remove the supernatant, and use one milliliter of ice-cold PBS to wash the cells three times. Add fresh medium, and incubate the cells at 37 degrees Celsius for an additional hour.Next, add 0.5 milliliters of PBS, containing 0.25%trypsin and EDTA, and incubate the cells at 37 degrees Celsius for up to 30 minutes. Then use 1.5 milliliters of RPMI1640 medium with 10%FBS to neutralize the trypsin. Centrifuge the cells at 500 times G for five minutes, remove the supernatant, and use 0.5 milliliters of ice-cold PBS to wash the cells three times again.Add 0.5 milliliters of 1%PFA and 1%sucrose and PBS to the cells, and place them on ice for 30 minutes to fix them. Then use 0.5 milliliters of ice-cold PBS to wash the cells three times, and centrifuge the samples at 250 times G for five minutes. Next, add 0.15%Triton X-100 and PBS to the cells, and permeablize them on ice for five minutes.Then, with ice-cold PBS, wash the cells, and repeat the centrifugation. Suspend the cells in 0.1 milliliters of PBS, containing two micrograms per milliliter of an antimirroring FC secondary polyclonal antibody conjugated to Alexa Fluor 647, and incubate the samples in the dark at room temperature for one hour. Centrifuge the cells at 250 times G for five minutes, and use 0.5 milliliters of ice-cold PBS to wash the cells three times.Then suspend the cells in 0.5 milliliters of PBS. Add 10 micrograms per milliliter of propidium iodide to the cells. Then use mounting medium to mount one times 10 to the fifth cells onto glass slides before covering the sample with a glass cover slip.Examine the cells with a Plan APO 60X oil-immersion objective, numerical aperture 1.42, on an inverted laser-scanning confocal microscope. Detect PI fluorescence using the 488-nanometer argon laser and the spectral-scanning prism set for 600 to 650 nanometers. For AF 647 fluorescence, use the 633-nanometer helium-neon laser and the spectral-scanning prism set for 650 to 700 nanometers.Collect images from PI and AF 647 sequentially, 1024 by 1024 pixels, with 2X line averaging, taken at 0.5-micron intervals through the entire cell thickness. Present images as Z projections. To analyze the cells, evaluate and record whether intracellular fluorescence in the cytoplasm is grouped and near the cell surface or diffuse and homogeneous.Also evaluate the relative fluorescence intensity per cell. After preparing and concentrating radiolabeled colic-acid NLS antibody conjugate according to the text protocol, determine the radiolabeling efficiency by applying 0.5 microliters of 0.1-molar PH5.5 sodium citrate, or ITLC eluent, to an ITLC strip. Form an autoradiograph of the strip, and obtain a digital image.Carry out densitometry to obtain the proportion of bound and free Copper-64. If free Copper-64 content is greater than 5%concentrate the sample further. For Copper-64 cellular accumulation studies, treat cells with 100 nanomolar of Copper-64-labeled A14 conjugates at 37 degrees Celsius for one, six, and 24 hours, as previously described.Treat the cells with unmodified A14 to block IL5R alphacytes and at four degrees Celsius to block receptor-mediated internalization. After washing the cells and incubating with fresh medium, as demonstrated earlier in this video, add 0.5 milliliters of PBS containing 0.25%trypsin and EDTA, and incubate at 37 degrees Celsius for 15 minutes. Then, after neutralizing and washing the cells as before, add plasma-membrane lysis buffer to the cells, and incubate them on ice for 10 minutes.Centrifuge the plasma-membrane-lysed cells at 90 times G for five minutes. Remove the supernatant, and transfer it to a fresh tube. This represents the cytoplasmic fraction.Then use ice-cold PBS to wash the nuclei three times, and add the washes to the cytoplasmic fraction. Ensure vials containing the nuclear and cytoplasmic fractions are sealed, then place them in a gamma counter calibrated for Copper-64 in order to convert raw counts to megabecaril. Determine the quality of nuclei isolation by performing Western-Blot analysis for lamin AC, a restricted nuclear protein;and Rab-7, an abundant cytoplasmic protein, on wholesale lysate, nuclear, and cytoplasmic fractions.Treat five times 10 to the six cells with 500 microliters of radio-amino-precipitation assay, or RIPA buffer, and plasma-membrane lysis buffer containing 1%2%and 4%NP-40. Furthermore, treat the isolated nuclei with 500 microliters of RIPA buffer to obtain isolated nuclear proteins. Add four times the sample volume of cold acetone to the isolated nuclear, cytoplasmic, and wholesale proteins, and incubate for 60 minutes at minus 20 degrees Celsius.Centrifuge the samples at 13, 000 times G for 10 minutes. Decant the supernatant, being careful not to dislodge the protein pellet. Then add 100 microliters of PBS to dissolve the proteins.After performing Western Blotting as previously described, cut the membrane in half at the 40-kilodalton molecular-weight marker. Use lamin-AC-specific antibodies to probe the blot containing the higher-molecular-weight proteins, and use Rab-7-specific antibodies to probe the lower half of the membrane. In groups containing four nodskit mice, inject into the tail vein 20 to 30 micrograms of radiolabeled A14 colic-acid NLS antibody conjugate, radiolabeled A14 NLS, and radiolabeled A14.On the same day, place a cylindrical phantom containing five megabecaril of Copper-64 into the PET scanner to convert the radioactive counts per second into injected dose per gram of tissue. 48 hours later, after anesthetizing the mice according to the text protocol, rapidly transfer a mouse to a PET scanner table in the headfirst prone position with a nosecone for isoflurane. Start the PET data acquisition using a regular sampling-mode setting and an energy window of 250 to 650 kiloelectron volts.After the scan, remove the mouse from the scanner, and place it back in the cage. As shown in this figure, the arrows show the difference when evaluating antibody conjugate accumulation with and without trypsinization. In cells that are not trypsinized, intracellular accumulation and distribution is difficult to evaluate due to the over expression of the target cell's surface receptors.The arrows show the difference in intracellular accumulation and distribution of two antibody conjugate candidates when cells are properly trypsinized. Radiolabeled A14 colic-acid NLS antibody conjugate shows nanomolar affinity for IL5R alpha. With increasing concentrations of radiolabeled A14 colic-acid NLS antibody conjugate, specific binding of A14 colic-acid NLS antibody conjugate approached saturation at concentrations of 3.5 nanomolar in both HT-1376 and HT-B9 cells.Gamma counting reveals that the increase of radioactivity in the nucleus and in the cytoplasm from radiolabeled A14 colic-acid NLS antibody conjugate is specific and increases over time. HT-1376 and HT-B9 cells were incubated with plasma-membrane lysis buffer containing 1%2%or 4%NP-40. For HT-1376 and HT-B9 cells, the lysis buffer containing one or 2%NP-40 Rap-7 was not detected in the nuclear fraction, and lamin AC was not detected in the cytoplasmic fraction.PET imaging allows for the evaluation of candidate antibody conjugates for their ability to target antigen-positive tumors, red and blue arrows, and their associated biodistribution properties in comparison to the parental non-peptide-modified antibody. One can see the signal accumulation in the tumor to help select on the potential for a developed antibody conjugate. PET imaging also allows for the evaluation of candidate antibody conjugate uptake in healthy tissues such as the liver.After mastering these techniques, researchers in the field of targeted tumor delivery of molecular payloads will be able to explore additional agents where tumor-cell accumulation is hindered due to endosome entrapment.

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Research

JoVE Journal

Bioengineering

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

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 ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

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.

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 ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...