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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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—particularly, primary macrophages—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' viability^2,3. 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 cell^3,4. 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 disorders^5,6,7. 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 capacity^9,10. 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 systemically^9,10.

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 vehicles^11,13,14,15, 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.

Protokół

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

1. Preparation of oleic acid-modified SPIONs
   1. Dissolve FeCl₃•6H₂O and FeSO₄•7H₂O in water under the protection of N₂.
      1. Add 28 g of FeCl₃•6H₂O and 20 g of FeSO₄•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.
      2. Heat the reaction mixture to 72 °C at a stirring rate of 800 rpm, followed by addition of 40 mL of ammonia water (28%). Stir for 5 min.
   2. Add 9 mL of oleic acid dropwise into the above-mentioned solution and stir it at 72 °C for 3 h.
   3. Cool the resulting solution to room temperature (RT). Precipitate the solution via magnetic separation.
   4. Wash the precipitate containing SPIONs 3x with absolute ethyl alcohol and, then, disperse the precipitate in 100 mL of N-hexane.
2. Preparation of dimercaptosuccinic acid-modified SPIONs
   1. 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 °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.
   2. Add 200 µL of triethylamine dropwise into the above-mentioned solution with stirring at 1,000 rpm and refluxing.
   3. After 5 h of stirring and refluxing, obtain a black precipitate by magnetic separation.
   4. Disperse the hydrophilic SPIONs in deionized water homogeneously by adjusting the pH of the solution using tetramethylammonium hydroxide.
3. Preparation of PEI-SPIONs
   1. 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 (W_Fe:W_PEI = 1:3).
      Note: The charge and size of PEI-SPIONs vary depending on the ratio of W_Fe to W_PEI. The W_Fe:W_PEI ratio of 1:3 can be a good starting point for synthesizing PEI-SPIONs suitable for siRNA delivery.
   2. 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-μm filter and store the final product at 4 °C.
   3. 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 °C.
   4. Dilute 10 μL 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.

2. Preparation and Agarose Gel Electrophoresis of PEI-SPION/siRNA NPs

1. Dilute siRNA with RNase-free water to yield a final concentration of 20 μM (0.26 μg/μL).
2. Prepare five RNase-free microcentrifuge tubes labeled 0, 1, 2, 4, and 8. The labels represent different Fe:siRNA weight ratios. Pipet 3 μL of siRNA solution to all tubes (~0.8 μg of siRNA/tube).
3. Add 0, 0.8, 1.6, 3.2, and 6.4 μg 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 μL. Mix by gently pipetting up and down.
4. 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.
5. Add 1 μL of 6x DNA-loading buffer per 5-μL 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.
6. 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.

3. Transfection of RAW264.7 Macrophages In Vitro

1. 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 μg/mL streptomycin at 37 °C in a 5% CO₂ incubator.
2. 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 °C in a 5% CO₂ incubator.
3. 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.
4. 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.
5. Resuspend the cells with 5 mL of fresh DMEM complete medium and count the cells.
6. Plate 9 x 10⁴ cells per well in a 6-well plate with 2 mL of complete DMEM medium and incubate at 37 °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.
7. 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).
8. 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 µg of Fe at a Fe:siRNA ratio of 4, add 100 µL (1 mg Fe/mL) of PEI-SPIONs to 96 μL of siRNA (0.26 μg/μL), 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.
9. 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 μg Fe/mL may maximize transfection efficiency while minimizing potential cytotoxicity.

4. Systemic Delivery of siRNA to Macrophages in Rats with Experimental Arthritis

1. 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.
2. Prepare PEI-SPION/siRNA complexes as described in step 3.8.
3. 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.

Wyniki

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 °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 ...

Dyskusje

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) and rat peritoneal macrophages¹⁸ can be transfected with siRNA without significant impairment of the cell viability. This method depends on the delivery p...

Materiały

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