Name: delivery of therapeutic sirna to the cns using cationic and anionic liposomes
Uploaded: 2016-07-23T15:00:00
Description: Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

All mice were bred and maintained at Lab Animal Resources, accredited by the Association for Assessment and Accreditation of Lab Animal Care International, in accordance with protocols approved by the Institutional Animal Care and Use Committee at Colorado State University.
1. Preparation of LSPCs
<ol>
	<li>Use a 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium-propane):cholesterol ratio for LSPCs. For a 4 nmole liposome preparation, mix 2 nmoles of DOTAP and 2 nmoles of cholesterol into 10 ml of...

<ol>
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This report describes a protocol to create two targeted delivery systems that efficiently transports siRNA to the CNS. Previous methods of delivering siRNA to the CNS included injecting siRNA/shRNA vectors directly into the brain, intravenous injection of targeted siRNA, or intravenous injection of non-targeted liposome-siRNA complexes. Injection of siRNA/shRNA vectors into the CNS does cause a decrease in target protein expression levels. However, the siRNA/shRNA does not diffuse freely through the CNS. Furthermore, the...

Prions are severe neurodegenerative diseases that affect the CNS. Prion diseases result from the misfolding of the normal cellular prion protein, PrPC, by an infectious isomer called PrPRes. These diseases affect a wide variety of species including bovine spongiform encephalopathy in cows, scrapie in sheep, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease in humans1-3. Prions cause neurodegeneration that starts with synaptic loss, and progresses to vacuolization, gliosis, neuronal loss, and plaque deposits. Eventually, resulting in the death of the animal/individual4. For decades, researchers have investigated compounds meant to slow or stop the progression of prion disease. However, researchers have not found either a successful therapy or an effective systemic delivery vehicle.
Endogenous PrPC expression is required for the development of prion diseases5. Therefore, decreasing or eliminating PrPC expression may result in a delay or amelioration of disease. Several groups created transgenic mice with reduced levels of PrPC or injected lentivectors expressing shRNA directly into murine brain tissue to investigate the role of PrPC expression levels in prion disease. These researchers found reducing the amount of neuronal PrPC resulted in halting the progressive neuropathology of prion diseases and extended the life of the animals6-9. We have reported that PrPC siRNA treatment results in clearance of PrPRes in mouse neuroblastoma cells10. These studies suggest that the use of therapies to decrease PrPC expression levels, like small interfering RNA (siRNA), which cleaves mRNA, may sufficiently delay the progression of prion diseases. However, most therapies investigated for prion diseases were delivered in ways that would not be practical in a clinical setting. Therefore, a siRNA therapy needs a systemic delivery system, which is delivered intravenously and targeted to the CNS.
Investigators have studied the use of liposomes as delivery vehicles for gene therapy products. Cationic and anionic lipids are both used in the formation of liposomes. Cationic lipids are more widely used than anionic lipids because the charge difference between the cationic lipid and the DNA/RNA allows for efficient packaging. Another advantage of cationic lipids is that they cross the cell membrane more easily than other lipids11-14. However, cationic lipids are more immunogenic than anionic lipids13,14. Therefore, researchers have started to shift from using cationic to anionic lipids in liposomes. Gene therapy products can be efficiently packaged into anionic liposomes using the positively charged peptide protamine sulfate, which condenses DNA/RNA molecules15-19. Since anionic lipids are less immunogenic than cationic lipids they may have increased circulation times, and may be more tolerated in animal models13,14. Liposomes are targeted to specific tissues using targeting peptides that are attached to the liposomes. The RVG-9r neuropeptide, which binds to nicotinic acetylcholine receptors, has been used to target siRNA and liposomes to the CNS17-20.
This report outlines a protocol to produce three siRNA delivery vehicles, and to package and deliver the siRNA to neuronal cells (Figure 1). Liposome-siRNA-peptide complexes (LSPCs) are composed of liposomes with siRNA and the RVG-9r targeting peptide electrostatically attached to the outer surface of the liposome. Peptide addressed liposome encapsulated therapeutic siRNA (PALETS) are composed of siRNA and protamine encapsulated within the liposome, with RVG-9r covalently bonded to lipid PEG groups. Using the below methods to generate LSPCs and PALETS, PrPC siRNA decreases PrPC expression up to 90% in neuronal cells, which holds tremendous promise to cure or substantially delay the onset of prion disease pathology.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DOTAP lipid</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">700000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">850715</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">880125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">AC268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:104px;">EMD Millipore</td>
			<td style="width:119px;">113351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Gas</td>
			<td style="width:104px;">AirGas</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">S5-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extruder</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.0, 0.4, and 0.2um filters</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610010, 610007, 610006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Life Technologies</td>
			<td style="width:119px;">70011-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">ICN10275205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDC</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">22980</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sulfo-NHS</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">24510</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40um Cell Strainer</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rat anti-mouse CD16/CD32 Fc block</td>
			<td style="width:104px;">BD Pharmigen</td>
			<td style="width:119px;">553141</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-PrP antibody (PRC5)</td>
			<td style="width:104px;">Proprietary - PRC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

To increase the efficiency of siRNA encapsulation within anionic PALETS, the siRNA was mixed with protamine. To determine the best protamine concentration for the siRNA, the siRNA was mixed with different concentrations of protamine, from 1:1 to 2:1 (Figure 3A). There was a 60-65% siRNA encapsulation efficiency in anionic liposomes without the use of protamine. Samples with protamine:siRNA molar ratios from 1:1 to 1.5:1 (133-266 nM) had 80-90% siRNA encapsulation. Molar r...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

The overall goal of this procedure is to design a delivery system that can deliver small molecule drugs like siRNA to the brain. This method can answer key questions in the neurodegeneration field such as Is siRNA a viable therapeutic option for protein misfolding diseases? and Can we design a reliable drug delivery system to the brain?The main advantage of creating a liposomal drug delivery system is that we can deliver our siRNA intravenously or through the blood stream rather than directly through neuronal tissue which results in neuronal damage. The implications of this technique extends towards therapeutics and diagnostics of neurodegenerative diseases because this technique allows for specific delivery across the blood-brain barrier, specifically to acetylcholine-expressing neurons. Visual demonstration of this technique is critical as mouse tail vein injections are difficult to learn due to the small size of the mouse tail vein.Begin by preparing a one-to-one DOTAP to cholesterol ratio. For a foreign animal liposome preparation, mix two nanomoles of DOTAP and two nanomoles of cholesterol into ten milliliters of a one-to-one chloroform to methanol solution. Then, evaporate nine milliliters of the chloroform and methanol solution using nitrogen gas.Evaporate the last one milliliter of solvent in a fume hood overnight without gas. The next day, a thin lipid film should be visible on the bottom of the flask. Heat both this flask with the lipid film and ten milliliters of a ten percent sucrose solution to 55 degrees Celsius.Then, slowly pipette the heated sucrose solution onto the thin lipid film while gently swirling the flask. Next, assemble an extruder with one micron filters according to the manufacturer's instructions. Then, add one milliliter of the heated liposome suspension to one of the syringes on the extruder.Then pass the suspension between the two syringes 11 times. While keeping the liposome suspension heated for easier sizing, size the liposomes through 45 micron and a 2 micron filter To generate large, unilamellar vesicles, next add 20 microliters of the four nanomole liposome suspension to 200 microliters of a 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature. Then add 80 microliters of a 500 micromolar stock of RVG-9R targeting peptide to the siRNA liposome solution and incubate for ten minutes at room temperature.Prepare a foreign animal lipsomoe suspension by mixing 2.2 nanomoles of either DSPE or DOTAP into ten milliliters of a two-to-one chloroform to methanol solution with 1.6 nanomoles of cholesterol and 2 nanomoles of DSPE peg. Afterwards, apply nitrogen gas and when most of the solvent has been evaporated, leave the flask in the fume hood overnight. Then, slowly add ten milliliters of 1X PBS heated to 75 degrees Celsius to the thin lipid film.Allow the lipids to rehydrate at 75 degrees Celsius for at least one hour before sizing. After sizing the liposome suspension using an extruder as before, pipette 20 microliters of each of the four nanomole liposome suspensions into single use aliquots and lyophilize for 30 minutes using a bench top freeze dryer. Next, add 1.4 microliters of a 26.6 micromolar solution of protamine sulfate to 200 microliters of 20 micromolar stock siRNA solution and incubate for ten minutes at room temperature to condense the siRNA.Following the incubation, rehydrate DSPE palets with 201.4 microliters of siRNA protamine solution. Rehydrate the DOTAP palets with 200 microliters of a foreign animal stock solution of siRNA. Incubate each liposome siRNA solution for ten minutes at room temperature.Next, add ten microliters of a 60 millimolar EDC solution to each tube of rehydrated liposomes. Then add ten microliters of 150 millimolar sulfo-NHS solution to the liposome siRNA solution for both DOTAP and DSPE palets. Incubate for two hours at room temperature.Lastly, add 80 microliters of 500 micromolar stock of RVG-9R peptide to the siRNA liposome cross linking solution and incubate for ten minutes at room temperature. After anesthetizing a mouse using two to three percent isoflurane in oxygen cover the eyes of the mouse with apthalmic ointment and confirm an anesthetization by performing a toe pinch. Proceed only if a reaction is absent.While maintain anesthesia, place the mouse on its back or side to access the tail vein. Wipe the tail with 70 percent ethanol and then use a 26 gauge insulin syringe to inject 300 microliters of LSPCs or palets into the tail vein. Begin injecting distally and move proximally if the vein collapses or ruptures.Place the mouse in a clean cage until it maintains sternal recumbency. Monitor the animal for several hours after injection to ensure anesthesia wears off and there are no ill effects of the treatment. After allowing the LSPCs or palets to circulate for at least 24 hours and then euthanizing the mouse, dissect out half a hemisphere of the brain.Then place the half hemisphere and 2.5 milliliters of fax buffer onto a 40 micron mesh cell strainer and use the plunger of a syringe to press the tissue through the filter into a petri dish. Rinse the cell strainer and petri dish with an additional 2.5 milliliters of fax buffer. Then transfer the single cell suspension to a 15 milliliter conical tube and place on ice.Next, pipette 100 microliters of the cell suspension into micro centrifuge tube and centrifuge for five minutes at 350 times G.After discarding the supernatant, resuspend the cell pellet in one milliliter of fax buffer and centrifuge again. Then, add another milliliter of fax buffer and place the cell suspension on ice. Now, incubate the cells in 100 microliters of rat anti-mouse Fc block in fax buffer for 30 to 60 minutes on ice.After pelleting and washing as before, add 100 microliters of fluorescent primary anti-body solution and place on ice for 30 to 60 minutes. After a final wash, resuspend the cells in one mililiter of RVC lysis buffer for one minute. Then, pellet the cells.After discarding the buffer, resuspend in one milliliter of fax buffer and place on ice. The cells are now ready for fax analysis. Mice treated with siRNA delivered via LSPCs showed a significant decrease of cellular prion protein levels compared to a PBS control which shows while type cellular prion protein levels.The LSPCs treated mice showed cellular prion protein levels close to that of a prion protein knock out mouse. Across three experiments, mice treated with LSPCs showed significant decreases in cellular prion protein levels as compared to to PBS while type controls. While attempting this procedure, it's important to remember that the liposomes can be stored in the fridge for several months, but the complete LSPCs must be used within 24 hours after assembly.After its development, the generation of LSPCs or palets will pave the way for researchers to explore more therapeutic options in the field of neurodegenerative diseases.

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...