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 a 1:1 chloroform:methanol solution in a flask.</li>
	<li>Evaporate 9 ml of the chloroform:methanol solution using N2 gas in a fume hood. Evaporate the last 1 ml of solvent in a fume hood overnight without gas. Observe a thin lipid film on the bottom of the flask.</li>
	<li>Heat 10 ml of a 10% sucrose solution to 55 &#176;C.</li>
	<li>Pour the heated 10% sucrose solution onto the thin lipid film slowly, i.e. 1 ml/min, while gently swirling the flask. Maintain the 10% sucrose and flask with lipids at 55 &#176;C so that the lipid molecules remain in the gel-liquid phase. Rehydration results in multilamellar vesicle (MLV) formation.</li>
	<li>Allow lipids to rehydrate at 55 &#176;C for at least one hr before sizing the liposomes.</li>
	<li>Assemble an extruder per manufacturer&#39;s instructions with 1.0 &#181;m filters or use 1.0 &#181;m syringe filters for sizing.</li>
	<li>Add 1 ml of the heated liposome suspension to one of the syringes on the extruder, and pass the suspension between the two syringes 11 times. Make sure that the suspension is in the opposite syringe than the starting syringe after the 11 passes.</li>
	<li>Size the liposomes again through 0.45 &#181;m then 0.2 &#181;m filters to generate large unilamellar vesicles (LUVs). Keep the liposome suspension heated for easier sizing.</li>
	<li>Incubate 20 &#181;l of the 4 nmole liposome suspension (200 &#181;M) with 200 &#181;l of a 20 &#181;M (4 nmole total) stock siRNA solution for 10 min at room temperature.</li>
	<li>Incubate 80 &#181;l of a 500 &#181;M (40 nmole total) stock RVG-9r solution with the siRNA/liposome solution for 10 min at RT. LSPCs should be used as soon as possible but can be used up to several hours after preparation. Store LSPCs at 4 &#176;C for several hr&#160;after preparation.</li>
</ol>
2. Preparation of PALETS
<ol>
	<li>Use a 55:40:5 molar ratio of either DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine):cholesterol:DSPE-PEG or DOTAP:cholesterol:DSPE-PEG for PALETS. For a 4 nmole liposome preparation, mix 2.2 nmoles of either DSPE or DOTAP, 1.6 nmoles of cholesterol, and 0.2 nmoles of DSPE-PEG into 10 ml of a 2:1 chloroform:methanol solution in a flask.</li>
	<li>Prepare liposome suspension exactly as described in Step 1.1 and 1.2 above. Rehydrate DSPE liposomes in 10 ml of 1x&#160;PBS heated to 75 &#176;C, adding 1 ml/min. Allow lipids to rehydrate at 75 &#176;C for at least 1 hr before sizing.</li>
	<li>Size the liposome suspension exactly as described in Step 1.6-1.8.</li>
	<li>Pipet 20 &#181;l of each of the 4 nmole (200 &#181;M) liposome suspensions into single use aliquots after DOTAP and DSPE liposomes have been sized, and lyophilize for 30 min using a benchtop freeze dryer (Figure 2).</li>
	<li>Incubate 200 &#181;l of a 20 &#181;M (4 nmole total) stock siRNA solution with 1.4 &#181;l of a 26.6 &#181;M solution of protamine sulfate for 10 min at RT&#160;for siRNA that is to be encapsulated into DSPE PALETS liposomes.</li>
	<li>Rehydrate the DOTAP PALETS liposomes with 200 &#181;l of a 4 nmole stock solution of siRNA or rehydrate DSPE PALETS liposomes with 201.4 &#181;l of the siRNA/protamine solution. Incubate liposome/siRNA solution for 10 min at RT.</li>
	<li>Add 10 &#181;l of a 60 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to the liposome/siRNA solution for both DOTAP and DSPE PALETS.</li>
	<li>Add 10 &#181;l of a 150 mM N-hydroxysulfosuccinimide (sulfo-NHS) solution to the liposome/siRNA solution for both DOTAP and DSPE PALETS.</li>
	<li>Incubate the EDC and sulfo-NHS with the siRNA/liposome suspension for 2 hr at RT.</li>
	<li>Incubate 80 &#181;l of a 500 &#181;M (40 nmole total) stock RVG-9r solution with the siRNA/liposome crosslinking solution for 10 min at RT. 
	NOTE: The EDC/sulfo-NHS allows the RVG-9r to covalently bond to the PEG lipids. Use PALETS within several hr&#160;after preparation. Store PALETS at 4 &#176;C for several hr&#160;after preparation.</li>
	<li>To determine siRNA encapsulation efficiency of PALETS:
	<ol>
		<li>Measure the concentration of siRNA before and after the addition of protamine and liposomes using a spectrophotometer set at 260 nm.</li>
		<li>Filter the unencapsulated siRNA from the liposome/encapsulated siRNA suspension using 50 kDa centrifugal filters. Centrifuge at 1,000 x g for 20 min.</li>
		<li>Measure the concentration of unencapsulated siRNA in the filtrate and the concentration of encapsulated siRNA in the retentate using a spectrophotometer at 260 nm.</li>
	</ol>
	</li>
</ol>
3. Injecting Mice with LSPCs or PALETS
<ol>
	<li>Expose mice for 5-10 min to a heat lamp to dilate vasculature.</li>
	<li>Anesthetize mouse with a 2-3% isofluorane/oxygen flow 5-10 min before injection, and during injection. Confirm anesthetization when animal is no longer ambulant and respirations have slowed by doing a toe pinch. Place the mouse on its back or side to access the tail vein. Use vet ointment on mouse&#39;s eyes to prevent drying while under anesthesia.</li>
	<li>Wipe/spray the tail with 70% EtOH and inject 300 &#181;l of LSPCs or PALETS onto the tail vein of the mouse using a 26 G insulin syringe. Start injecting distally into the tail vein and move proximally if the vein collapses or ruptures.</li>
	<li>Place mouse in a clean cage until it maintains sternal recumbency. Do not place mouse with other cage mates until it has fully recovered.&#160;Mice should recover in 5 to 15 min.&#160; They can be placed on a heating pad or under a heat lamp to maintain body temperature if recovery takes longer.&#160; Mice should be monitored closely to safeguard against over-heating.</li>
	<li>Monitor the animal several hours after injection to ensure anesthesia wears off, and there are no ill effects of the treatment. Allow the LSPCs or PALETS to circulate for at least 24 hr.</li>
</ol>
4. Analysis of Protein Expression via Flow Cytometry
<ol>
	<li>Euthanize mice with a 20% CO2 flow rate for 15 min.</li>
	<li>Dissect out half a hemisphere of brain by cutting away the skin and skull of the mouse using scissors. Peel away half a hemisphere of brain away from the skull using tongs or the end of a pair of scissors.</li>
	<li>Press half a hemisphere of brain from each mouse through a 40 &#181;m mesh cell strainer using 2.5 ml of FACS buffer (1x&#160;PBS, 1% fetal bovine serum, 10 mM EDTA) in a Petri dish with the plunger of a syringe.</li>
	<li>Rinse the cell strainer and Petri dish with an additional 2.5 ml of FACS buffer. Put the single cell suspension in a 15 ml conical tube on ice.</li>
	<li>Pipet 100 &#181;l of the 5 ml single cell suspension into a 1.5 ml microcentrifuge tube and centrifuge for 5 min at 350 x g. Discard the supernatant and resuspend cell pellet in 1 ml of FACS buffer. Spin at 350 x g for 5 min. Repeat with another 1 ml of FACS buffer. Keep the cell suspension on ice.</li>
	<li>Incubate the cells in 100 &#181;l of 1:100 dilution of a 0.5 mg/ml rat anti-mouse Fc block in FACS buffer for 30-60 min on ice. Pellet and wash the cells as in step 4.5. Keep the cell suspension on ice.</li>
	<li>Incubate the cells in 100 &#181;l of a 20 &#181;g/ml fluorescent antibody against mouse PrPC in 7% mouse serum in FACS buffer on ice for 30-60 min. Pellet and wash the cells as in step 4.5. Keep the cell suspension on ice.</li>
	<li>Pellet and re-suspend the cells in 1 ml of RBC lysis buffer (1x&#160;PBS, 155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 1&#160;min. Pellet as in step 4.5 and resuspend the cells in 1 ml of FACS buffer. Keep the cell suspension on ice.</li>
	<li>Analyze PrPC expression using a flow cytometer as described previously10. Gate live cells from total cell population. Gate PrPC + cells from live cell population to determine MFI (median fluorescent intensity).</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

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

Research

JoVE Journal

Bioengineering

10.4K Views.  Colorado State University. 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. 

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

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 ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

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 ...