Name: polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo
Uploaded: 2019-02-05T13:00:00.000+00:00
Duration: 9 min 36 s
Description: Watch this Scientific Journal Video about Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo at JoVE.com

This work was supported by the National Natural Science Foundation of China (81772308) and the National Key Research and Development Program of China (No. 2017YFA0205502).

All methods involving live animals were performed in accordance with the animal care and use guidelines of Southeast University, China.
1. Preparation of PEI-SPIONs
<ol>
	<li>Preparation of oleic acid-modified SPIONs
	<ol>
		<li>Dissolve FeCl3&#8226;6H2O and FeSO4&#8226;7H2O in water under the protection of N2.
		<ol>
			<li>Add 28 g of FeCl3&#8226;6H2O and 20 g of FeSO4&#8226;7H2O into 80 mL of deionized water in a beaker. Introduce N2 into the water through a glass conduit and stir until the solid matter has dissolved.</li>
			<li>Heat the reaction mixture to 72 &#176;C at a stirring rate of 800 rpm, followed by addition of 40 mL of ammonia water (28%). Stir for 5 min.</li>
		</ol>
		</li>
		<li>Add 9 mL of oleic acid dropwise into the above-mentioned solution and stir it at 72 &#176;C for 3 h.</li>
		<li>Cool the resulting solution to room temperature (RT). Precipitate the solution via magnetic separation.</li>
		<li>Wash the precipitate containing SPIONs 3x with absolute ethyl alcohol and, then, disperse the precipitate in 100 mL of N-hexane.</li>
	</ol>
	</li>
	<li>Preparation of dimercaptosuccinic acid-modified SPIONs
	<ol>
		<li>Add 800 mg of oleic acid (OA)-modified SPIONs dispersed in 200 mL of N-hexane, and 400 mg of dimercaptosuccinic acid (DMSA) dispersed in 200 mL of acetone, into a three-neck flask in a water bath at 60 &#176;C. 
		Note: To determine the concentration of OA-modified SPION obtained from the previous step, take a small volume (e.g. 1 mL) of the SPION dispersion, volatilize the N-hexane and weigh the resulting powder.</li>
		<li>Add 200 &#181;L of triethylamine dropwise into the above-mentioned solution with stirring at 1,000 rpm and refluxing.</li>
		<li>After 5 h of stirring and refluxing, obtain a black precipitate by magnetic separation.</li>
		<li>Disperse the hydrophilic SPIONs in deionized water homogeneously by adjusting the pH of the solution using tetramethylammonium hydroxide.</li>
	</ol>
	</li>
	<li>Preparation of PEI-SPIONs
	<ol>
		<li>Add DMSA-modified SPION colloidal solution dropwise into PEI solution (10 kDa) in a 500-mL three-neck flask under mechanical stirring at 1,000 rpm for 2 h (WFe:WPEI = 1:3). 
		Note: The charge and size of PEI-SPIONs vary depending on the ratio of WFe to WPEI. The WFe:WPEI ratio of 1:3 can be a good starting point for synthesizing PEI-SPIONs suitable for siRNA delivery.</li>
		<li>Add the resultant solution into an ultrafiltration tube having a molecular weight cutoff of 100 kDa and a content of 15 mL; then, centrifuge at 5,400 x g for 10 min until the remaining solution is 1 mL. Add deionized water to the solution to make the volume 15 mL again and repeat the above process 10x to obtain the final product. Then, filter the solution through a 0.22-&#956;m filter and store the final product at 4 &#176;C.</li>
		<li>Determine the Fe concentration of PEI-SPIONs by the colorimetric method using phenanthroline17. Dilute PEI-SPIONs with deionized sterile water to a concentration of 1 mg Fe/mL and store it at 4 &#176;C.</li>
		<li>Dilute 10 &#956;L of the PEI-SPION solution (1 mg Fe/mL) to 1 mL with deionized water; then, test its hydrodynamic size and zeta potential by a dynamic light-scattering device. 
		Note: Prepare PEI-SPIONs in the range of about 30 - 50 nm. In this size range, the effect of the NP size on siRNA binding and cellular uptake does not appear to be significant. PEI-SPIONs bearing an average zeta potential over +37 mV can be toxic in the dose range for transfection, and a cytotoxicity assay should be performed to ensure safety. The surface charge and hydrodynamic size of the nanoparticles can be controlled within a desired range by adjusting the PEI content.</li>
	</ol>
	</li>
</ol>
2. Preparation and Agarose Gel Electrophoresis of PEI-SPION/siRNA NPs
<ol>
	<li>Dilute siRNA with RNase-free water to yield a final concentration of 20 &#956;M (0.26 &#956;g/&#956;L).</li>
	<li>Prepare five RNase-free microcentrifuge tubes labeled 0, 1, 2, 4, and 8. The labels represent different Fe:siRNA weight ratios. Pipet 3 &#956;L of siRNA solution to all tubes (~0.8 &#956;g of siRNA/tube).</li>
	<li>Add 0, 0.8, 1.6, 3.2, and 6.4 &#956;g of Fe in the form of PEI-SPIONs to the tubes labeled 0, 1, 2, 4, and 8, respectively. Keep the total sample volume of each tube less than 20 &#956;L. Mix by gently pipetting up and down.</li>
	<li>Incubate the mixtures at RT for 30 min to allow PEI-SPION/siRNA complex formation. During this period, make a 3% agarose gel with high-purity agarose. 
	Note: Additional PEI-SPION/siRNA complexes with other Fe:siRNA ratios (e.g., 5 or 6) can be prepared and tested.</li>
	<li>Add 1 &#956;L of 6x DNA-loading buffer per 5-&#956;L sample and mix cautiously. Load all samples and run electrophoresis at 5 V/cm until the bromophenol blue migrates as far as two-thirds of the length of the gel. Stain the gel with ethidium bromide (EB) for 15 - 20 min. 
	Note: Freshly prepared electrophoresis buffer and EB solution should be used.</li>
	<li>Visualize siRNA bands under a UV imaging system. Check the Fe:siRNA ratios at which siRNA forms complexes with PEI-SPIONs and, as a result, the bands representing free siRNA are retarded or not detectable.</li>
</ol>
3. Transfection of RAW264.7 Macrophages In Vitro
<ol>
	<li>Culture mouse macrophage-like RAW264.7 cells in a 10-cm dish using DMEM complete medium containing 10% fetal bovine serum (FBS) per 100 U/mL penicillin per 100 &#956;g/mL streptomycin at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>One day prior to the transfection, aspirate medium from the cells and rinse them with phosphate-buffered saline (pH 7.4). Add 1 mL of 0.25% trypsin to the 10-cm dish. Trypsinize RAW264.7 cells for about 5 - 10 min at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>When the majority of the cells have detached (after 5 - 10 min), add 5 mL of DMEM complete medium to the dish to inactivate the trypsin. Pipet up and down to disperse cell clusters into single cells.</li>
	<li>Transfer the cell suspension to a sterile 15-mL conical tube. Centrifuge it at 300 x g for 3 min at RT. Remove the supernatant.</li>
	<li>Resuspend the cells with 5 mL of fresh DMEM complete medium and count the cells.</li>
	<li>Plate 9 x 104 cells per well in a 6-well plate with 2 mL of complete DMEM medium and incubate at 37 &#176;C in a 5% CO2 incubator for about 24 h. 
	Note: If using a plate of a different size, adjust the plated cell density in proportion to the relative surface area so that the cells reach 80% confluency at the time of transfection.</li>
	<li>When cell confluency is 80%, remove the medium from the cells and replace it with 1 mL of DMEM complete medium per well. Return the plate to the incubator until PEI-SPION/siRNA complexes have been prepared and are ready for use (about 30 min).</li>
	<li>Prepare PEI-SPION/siRNA complexes: calculate the amount of PEI-SPION/siRNA complexes needed for a transfection experiment. In a 1.5-mL RNase-free microcentrifuge tube, mix an appropriate amount of PEI-SPIONs with siRNA at a given Fe:siRNA ratio. For instance, to prepare PEI-SPION/siRNA NPs containing 100 &#181;g of Fe at a Fe:siRNA ratio of 4, add 100 &#181;L (1 mg Fe/mL) of PEI-SPIONs to 96 &#956;L of siRNA (0.26 &#956;g/&#956;L), followed by mixing it gently with a micropipette. Incubate for 30 min at RT. 
	Note: Prepare a volume of PEI-SPION/siRNA complex that is 10% in excess of the total final mass to account for any incidental losses. Make PEI-SPION/siRNA complexes at low Fe:siRNA ratios, under which siRNA molecules are completely loaded onto PEI-SPIONs and, hence, small amounts of PEI-SPIONs can be used to minimize potential cytotoxicity. Pilot experiments (gel retardation assay) for the optimization of the Fe:siRNA ratio are necessary.</li>
	<li>Take out the 6-well plate from the incubator (step 3.7). Add a required volume of PEI-SPION/siRNA complex dropwise to each well and swirl the plate cautiously to ensure an even distribution. Return the plate to the incubator until the assessment of the cellular uptake or gene knockdown efficiency (1 - 3 d). 
	Note: Transfecting macrophages with PEI-SPION/siRNA at a concentration of ~15 &#956;g Fe/mL may maximize transfection efficiency while minimizing potential cytotoxicity.</li>
</ol>
4. Systemic Delivery of siRNA to Macrophages in Rats with Experimental Arthritis
<ol>
	<li>Obtain specific pathogen-free male Wistar rats that are 7 weeks old. Habituate the rats for 7 d prior to use and provide them with adequate food and water. Induce adjuvant arthritis (AA) in the rats as previously described18.</li>
	<li>Prepare PEI-SPION/siRNA complexes as described in step 3.8.</li>
	<li>Inject the PEI-SPION/siRNA NPs (0.3 mg of siRNA/kg) into the AA rats via the tail vein. Assess the cellular uptake via, for instance, flow cytometry, tissue biodistribution via, for instance, a real-time fluorescence imaging system, or therapeutic effects based on, for instance, clinical, histologic, and radiographic analyses at desired time points18. 
	Note: For cellular and tissue biodistribution studies, treat the rats with a single injection of the desired NPs; for therapeutic studies, inject the rats with the NPs to be tested 1x a week for three consecutive weeks.</li>
</ol>

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The authors have nothing to disclose.

Macrophages are refractory to transfect by commonly used nonviral approaches, such as electroporation, cationic liposomes, and lipid species. Here we described a reliable and efficient method to transfect macrophages with siRNA. Using the present protocol, over 90% of macrophage-like RAW 264.7 cells (Figure 2B)&#160;and rat peritoneal macrophages18 can be transfected with siRNA without significant impairment of the cell viability. This method depends on the delivery p...

Macrophages are a type of innate immune cells distributed in all body tissues, albeit in different amounts. By producing a variety of cytokines and other mediators, they play critical roles in the host defense against invading microbial pathogens, in tissue repair following injury, and in maintaining tissue homeostasis1. Due to their importance, macrophages have continuously been the subject of intensive research. However, despite its prevalence in gene regulation and function studies, siRNA-mediated gene silencing is less likely to succeed in macrophages because these cells&#8212;particularly, primary macrophages&#8212;are often difficult to transfect. This can be ascribed to a relatively high degree of toxicity associated with most well-established transfection approaches in which the cell membrane is chemically (e.g., with polymers and lipids) or physically (e.g., by electroporation and gene guns) disrupted to let siRNA molecules cross the membrane, thereby drastically reducing macrophages&#39; viability2,3. Furthermore, macrophages are dedicated phagocytes rich in degradative enzymes. These enzymes can damage the integrity of siRNA, weakening its silencing efficiency even if gene-specific siRNA has been delivered into the cell3,4. Therefore, an effective macrophage-targeted siRNA delivery system needs to protect the integrity and stability of siRNA during delivery4.
It is increasingly evident that dysfunctional macrophages are implicated in the initiation and progression of certain common clinical disorders like autoimmune diseases, atherosclerosis, and cancer. For this reason, modulating macrophage function with, for instance, siRNA, has been emerging as an attractive methodology for treating these disorders5,6,7. Although much progress has been made, a major challenge of siRNA-based treatment strategy is the poor cell specificity of systemically administered siRNA and the insufficient siRNA uptake by macrophages, which consequently lead to undesired side effects. Compared with free nucleic acid therapeutics that usually lack optimal cell selectivity and often lead to off-target adverse effects, drug-loaded nanoparticles (NPs), owing to their spontaneous propensity of being captured by the reticuloendothelial system, can be engineered for passive targeting to macrophages in vivo, allowing for improved therapeutic efficacy with minimal side effects8. Current NPs explored for the delivery of RNA molecules include inorganic nanocarriers, various liposomes, and polymers9. Among them, polyethyleneimine (PEI), a type of cationic polymers capable of binding and condensing nucleic acids into stabilized NPs, shows the highest RNA delivering capacity9,10. PEI protects nucleic acids from enzymatic and nonenzymatic degradation, mediates their transfer across the cell membrane, and promotes their intracellular release. Although initially introduced as a DNA delivery reagent, PEI was subsequently demonstrated to be an attractive platform for in vivo siRNA delivery, either locally or systemically9,10.
Superparamagnetic iron oxide nanoparticles (SPIONs) have shown great promise in biomedicine, owing to their magnetic properties, biocompatibility, comparable size to biologically important objects, high surface-area-to-volume ratio, and easily adaptable surface for bioagent attachment11. For instance, because of their potential utility as a contrast agent and rapid uptake by macrophages, SPIONs have emerged as a favorite clinical tool to image tissue macrophages12. While SPIONs have also been extensively studied as nucleic acid delivery vehicles11,13,14,15, to our knowledge, the literature contains few reports of SPIONs as a carrier for macrophage-targeted siRNA delivery. For gene delivery by SPIONs, their surface is usually coated with a layer of hydrophilic cationic polymers onto which negatively charged nucleic acids can be electrostatically attracted and tethered. Here, we present a method for synthesizing SPIONs whose surface is modified with low-molecular-weight (10 kDa), branched PEI (PEI-SPIONs). These magnetic nanoplatforms are then employed to condense siRNA, forming PEI-SPION/siRNA complexes that enable siRNA transport into the cell. We reason that spontaneous phagocytosis of SPIONs by cells of the reticuloendothelial system16, coupled with the strong ability of binding and condensing nucleic acids by PEI, renders PEI-SPIONs suitable for the efficient transport of siRNA into macrophages. The data presented here support the feasibility of PEI-SPION/siRNA-mediated gene silencing in macrophages in culture as well as in vivo.

<table><tbody><tr><td>DMEM</td><td>Gibco</td><td>C11995500BT</td><td>Warm in 37&amp;deg;C water bath before use</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>A31608-02</td><td></td></tr><tr><td>Penicillin/streptomycin (1.5 ml)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Tetrazolium-based MTS assay kit</td><td>Promega</td><td>G3582</td><td>For cytotoxicity analysis</td></tr><tr><td>RAW 264.7 cell line</td><td>Cell Bank of Chinese Academy of Sciences, Shanghai, China</td><td>TCM13</td><td></td></tr><tr><td>Tissue culture plates (6-well)</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Tissue culture dishes (10 cm)</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>RNase-free tubes (1.5 ml)</td><td>AXYGEN</td><td>MCT-150-C</td><td></td></tr><tr><td>Centrifuge tubes (15 ml)</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Wistar rats</td><td>Shanghai Experimental Animal Center of Chinese&lt;br/&gt; Academy of Sciences</td><td></td><td></td></tr><tr><td>Bacillus Calmette&amp;ndash;Gu&amp;eacute;rin freeze-dried powder</td><td>National&lt;br/&gt; Institutes for Food and Drug Control, China</td><td></td><td>for inducing adjuvant arthritis in rats</td></tr><tr><td>siRNA</td><td>GenePharma (Shanghai, China)</td><td></td><td></td></tr><tr><td>Cy3-siRNA</td><td>RiboBio (Guangzhou, China)</td><td></td><td></td></tr><tr><td>Polyethyleneimine (10 kDa)</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>E107079</td><td></td></tr><tr><td>Ammonia water</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>A112077</td><td></td></tr><tr><td>Oleic acid</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>O108484</td><td></td></tr><tr><td>Dimethylsulfoxide</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>D103272</td><td></td></tr><tr><td>FeSO4&amp;bull;7H2O</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10012118</td><td></td></tr><tr><td>FeCl3&amp;bull;6H2O</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10011918</td><td></td></tr><tr><td>Dimercaptosuccinic acid</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>D107254</td><td></td></tr><tr><td>ultrafiltration tube</td><td>Millipore</td><td>UFC910096</td><td></td></tr><tr><td>Tetramethylammonium hydroxide solution</td><td>Aladdin Chemical Reagent Co., Ltd.</td><td>T100882</td><td></td></tr><tr><td>Particle size and zeta potential analyzer</td><td>Malvern, England</td><td>Nano ZS90</td><td></td></tr></tbody></table>

polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

The size and zeta potential of PEI-SPIONs prepared with this protocol were in the range of 29 - 48 nm (polydispersity index: 0.12 - 0.23) and 30 - 48 mV, respectively. They were stable in water at 4 &#176;C for over 12 months without obvious aggregation. To evaluate their siRNA binding ability, PEI-SPIONs were mixed with siRNA at various Fe:siRNA weight ratios. Figure 1 shows that when the Fe:siRNA weight ratio reaches 4 and above, the band of free siRNA was ...

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Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

polyethyleneimine-coated-iron-oxide-nanoparticles-as-vehicle-for

Research

JoVE Journal

Medicine

8.6K Views.  Southeast University. Deze maatregel kan helpen bij het beantwoorden van belangrijke vragen. Op het gebied van humanologie. Dat antwoord in de functie van macrofagen onder fysiologische eind aan pathologische voorwaarden.Het belangrijkste voordeel van deze techniek is dat polyethyleenimine gecoat efficiënt en vitro kan zonder in te leveren op cel. Onder optimale doses. De implicaties van deze techniek breiden zich uit tot therapie en diagnose van vele chronische ontstekingsstoornissen en kankers.Waa...

Video: Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Cancer Research

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 ...

Bioengineering

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunology and Infection

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

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 and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Biology

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

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