Chapter 7

Chapter 7 Integrin-Targeted Stabilized Nanoparticles for an Efficient Delivery of siRNAs In Vitro and In Vivo Charudharshini Srinivasan, Dan Peer, and Motomu Shimaoka Abstract Utilizing small interfering RNAs (siRNAs) to silence disease-associated genes holds promise as a potential therapeutic strategy. However, the greatest challenge for RNAi remains the delivery of siRNA to target tissues or cells. Specifically lymphocytes are difficult to transduce by conventional methods but represent good targets for anti-inflammatory therapeutics. Integrins are an important class of cell adhesion receptors on leukocytes. Antibodies to integrins have been used to inhibit inflammatory reactions in patients. Here, we describe a strategy to deliver the siRNA cargo to leukocytes by stabilized nanoparticles surface-decorated with antibodies to integrin as targeting moieties. A detailed methodology for preparation of the integrintargeted stabilized nanoparticles (I-tsNPs) and their delivery in vitro and in vivo is discussed. Key words: Liposomes, RNAi, Leukocytes, Inflammation, Hyaluronan, Antibody, Transfection, Systemic delivery

1. Introduction Post-transcriptional gene silencing by RNA interference (RNAi) has shown great potential as a therapeutic tool in targeted suppression of disease causing genes. Several siRNA delivery vectors have been investigated for their efficiency via systemic delivery in animal models. To mention a few, non-targeted delivery vectors such as stable nucleic acid-lipid particles (SNALP) (1, 2), lipidoids (3), poly D,L-lactide-co-glycolide (PLGA) microspheres (4) have been developed for RNAi. However, cell- or tissue-specific targeted siRNA delivery is highly desirable due to improved gene silencing and lower undesirable side effects than those compared to nontargeted delivery (5–7). Some targeted siRNA delivery vectors that are recently developed are; transferrin antibody targeted cyclodextrin

Marc De Ley (ed.), Cytokine Protocols, Methods in Molecular Biology, vol. 820, DOI 10.1007/978-1-61779-439-1_7, © Springer Science+Business Media, LLC 2012

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(CDP) for Ewing sarcoma in mice (8), HIV-targeted antibody protamine conjugate for mice melanoma xenografts (9), and transferrin antibody targeted cationic liposome for pancreatic tumor xenografts in mice (10). Most of these systems have focused on delivering siRNA to liver and tumors in animal models. Efforts to investigate targeted siRNA delivery to inflammatory leukocyte are vital and hold promise for future RNAi-based therapeutics in autoimmune disease, allergy, viral infections (HIV, ebola, dengue); and blood cancers (lymphoma, leukemia, and myeloma) (6, 11–14). But the greatest challenge is the delivery of siRNA to primary leukocytes that are resistant to conventional methods of transfection based on cationic lipid and polymers. Although electroporation method in in vitro conditions and hydrodynamic injection that force siRNA into cells in vivo have shown some success (15), this may not be feasible to use systemically due to disperse distribution of the leukocytes within the human body. To effectively deliver siRNAs in leukocytes, we have exploited the cell surface adhesion molecules, integrins that mediate adhesive interactions critical for leukocyte migration to sites of inflammation (16). In particular, the integrins β2 and β7 are exclusively expressed on leukocytes (17, 18). With this approach, integrin-targeted stabilized nanoparticles (I-tsNPs) was developed in our laboratory by encapsulating siRNAs within nano-sized neutral liposomes that are selectively targeted to leukocytes via surface-attached antibodies to leukocyte integrins (19–22). In this contribution, we describe the technology for developing I-tsNPs: NPs production, surface modifications, and purification. Characterizations of the NPs for their particle size, zeta potential, antibody-binding efficiency and siRNA entrapment is also discussed. In vitro transfection in TK-1 cells and in vivo delivery in mice have been demonstrated to show the efficacy of the NPs. A model siRNA Ku70, a ubiquitously expressed gene (17) is utilized to show the gene knockdown following delivery via β7 I-tsNPs (9).

2. Materials 2.1. I-tsNP Production

1. Multilamellar liposomes (MLL): L α-Phosphatidylcholine (PC Egg, Chicken), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE), cholesterol (Chol) (Avanti polar lipids, Inc., Alabaster, AL). Rotary evaporator (Buchi Corporation, Switzerland), Thermobarrel Lipex extruderTM (Lipex biomembranes Inc., Vancover, British Columbia, Canada) and nucleopore membranes 0.1–1 μm pore size (Nucleopore, Whatman). 20 mM Hepes buffered saline, pH 7.2 (Fluka, SigmaAldrich, Saint Louis, MO), and 1× phosphate buffered saline (PBS), pH 7.4 (Cellgro, Mediatech Inc., Manassas, VA).

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2. Hyaluronan (HA) coated nanoliposomes: Hyaluronan (HA, 751 kDa or 850 kDa, intrinsic viscosity: 14–16 dL/g, Genzyme Corp, Cambridge, MA); 400 mM 1-(3-dimethylaminopropyl)3-ethylcarbodimide hydrochloride (EDAC, Sigma-Aldrich, Saint Louis, MO); 100 mM N hydroxysuccinimide (NHS, Fluka, Sigma-Aldrich, Saint Louis, MO); 0.1 M sodium acetate buffer and 0.1 M borate buffer, pH 8.6. 3. Targeting antibody at 10 mg/mL concentration (FIB504 Ratanti mouse IgG2a against β7 integrin). 1 M ethanolamine hydrochloride, pH 8.5. 4. Purification of I-tsNP: Size exclusion column, sepharose CL-4B beads (Sigma-Aldrich, Saint Louis, MO). 5. Freeze drying of I-tsNPs: Alpha 1–2 LDplus lyophilizer (Christ, Osterode, Germany). 2.2. Characterization of NPs 2.2.1. Particle and Zeta Potential Analysis

Particle size and zeta potential analysis: Malvern Zetasizer nano ZSTM (Malvern Instruments Ltd., Southborough, MA), PBS 1× buffer, pH 6.7 (with 10 mM NaCl) at 20°C.

2.2.2. Binding Efficiency

FACScan flow cytometer/FACSCalibur (BD biosciences), FACS buffer (1% (v/v) FBS and 0.01% (w/v) sodium azide). TK-1 cells (ATCC, Manassas, VA); purified β7-I-tsNP fractions from the size exclusion column; positive control, FIB504 Rat- anti mouse IgG2a against β7 (10 μg/mL); isotype control, purified rat IgG2a (10 μg/ mL), secondary antibody FITC-Anti-Rat Ab IgG2a (1 μg/mL) (BD Pharmingen).

2.2.3. siRNA Entrapment Efficiency in NPs

1. Ku70 siRNAs from Dharmacon (Boulder, CO). The following four used in equimolar ratios siRNA#1: sense 5¢-GCUCUGCUCAUCAAGUGUCUGdTdT-3¢, antisense 5¢-CAGACACUUGAUGAGCAGAGCdTdT-3¢ siRNA#2: sense 5¢-UCCUUGACUUGAUGCACCUGAdTdT-3¢, antisense 5¢-UCAGGUGCAUCAAGUCAAGGAdTdT-3¢ siRNA#3: sense 5¢-ACGGAUCUGACUACUCACUCAdTdT-3¢, antisense 5¢-UGAGUGAGUAGUCAGAUCCGUdTdT-3¢ siRNA#4: sense 5¢-ACGAAUUCUAGAGCUUGACCAdTdT-3¢ antisense 5¢-UGGUCAAGCUCUAGAAUUCGUdTdT-3¢. Alternately, pre-designed ON-TARGETplus siRNA SMARTpool, Gene ID 14375 for mouse Ku70 (Dharmacon Inc., Boulder, CO) can also be used.

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2. Nuclease free water (Ambion Inc., Austin, TX). 3. Human recombinant Protamine (7,500 molecular weight, Abnova, TaipeiCity, Taiwan), or (Spermine, 348.18 molecular weight, Spermidine 255, Biosynth International, Inc., Itasca, IL). 4. Quant-iT RiboGreen RNA assay kit for percent entrapment efficiency (Molecular Probes, Invitrogen, Carlsbad, CA). 2.3. In Vitro Transfection of siRNA Using I-tsNPs 2.4. Ku70-siRNA Delivery In Vivo

T cell lymphoma cell line, TK-1 cells (ATCC, Manassas, VA).

1. Mice: Wild type and β7-integrin knockout mice with C57BL/6 background (Charles River Laboratories, Wilmington, MA). Mice should be maintained in a specific pathogen-free animal facility. 2. 27-gauge needle with a tuberculin syringe to inject to the tail vein of the mice. 3. Bath sonicator (Fisher Scientific) to briefly sonicate liposome suspension before injection. 4. Isolation of splenocytes: K10 medium: RPMI + 10% (v/v) FCS + supplements

70 μm sieves: Nylon sieves, BD Falcon 352350

2% (v/v) FCS: HBSS + 2% (v/v) FCS

Frosted glass slides: VWR 48312–002

RBC (red blood cell) lysis buffer: 8.3 g/L NaCl 0.001 M Tris–HCl, pH 7.5

Small Petri dishes: BD Falcon 35 3002

3. Methods 3.1. I-tsNP Production and Purification

I-tsNPs are nanometer sized hyaluronan coated neutral liposomes possessing targeting moieties on their surface (antibodies to integrin molecules on leukocytes). The preparation involves two critical processes (1) preparation of stabilized NPs by chemical conjugation of hyaluronan that coats the surface of liposomes and (2) introduction of targeting molecules (mAbs) on the surface of the stabilized NPs (Refer to Fig. 1, Schematic). 1. Prepare multilamellar liposomes (MLL), composed of phosphatidylcholine (PC), dipalmitoylphosphatidylethanolamine (DPPE), and cholesterol (Chol) at molar ratios of 3:1:1 (PC:DPPE:Chol), using conventional lipid-film hydration method (17, 18).

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Fig. 1. The schematic showing steps involved in the production of I-tsNPs. Multilamellar vesicle (MLV) prepared by rotary evaporation method is extruded to form a unilamellar vesicle (ULV). Hyaluronan is coated onto surface of liposomes by covalently binding to DPPE part of the lipids in the ULV. A monoclonal antibody (mAb against integrin) is then covalently attached to hyaluronan via an amide bond linkage forming I-tsNPs (e.g., β7 I-tsNPs). Protamine condensed siRNAs are then entrapped within the lyophilized NPs by rehydration to form a transfection complex.

Dissolve the lipids at final concentration of 40 mg/mL in ethanol (96%) by stirring for 30–45 min at 60°C. This is followed by rotary evaporation for 1–2 h at 65°C (see Note 1). 2. Hydrate the lipid film with 20 mL of 20 mM Hepes-buffered saline pH 7.4 or 1× PBS, pH 7.4 to create MLL. Thoroughly Vortex until a thin milky liposome suspension is formed. 3. Incubate the liposome suspension in a shaker (~200 rpm) at 37°C for 2 h to ensure complete mixing and homogeneity (see Note 2). 4. Extrude the resulting MLL into unilamellar nano-liposomes (ULNL) with a Thermobarrel Lipex extruder™ at 65°C under nitrogen pressures of 300–550 psi. 5. Carry out the extrusion in a stepwise manner using progressively decreasing pore-sized membranes (from 1, 0.8, 0.6, 0.4, 0.2, to 0.1 μm), with 10 cycles per pore-size. 6. ULNL are surface-modified with high molecular weight hyaluronan (HA) (751 kDa or 850 kDa intrinsic viscosity: 14–16 dL/g) as described below. 7. Dissolve 20 mg HA in 0.1 M sodium acetate buffer. Stir at 37°C for 30 min to fully dissolve HA. Pre-activate with 400 mg of EDAC, at pH 4.0 and stir for 2 h at 37°C. Centrifuge the extruded liposome suspension (ULNL) for 1–3 h in an ultracentrifuge and resuspend the pellet in 0.1 M borate buffer, pH 8.6. Combine the activated HA with the liposome suspension (ULNL) in a 1:1 volume ratio and incubate overnight at 37°C, with gentle stirring. Separate the resulting HA-ULNL from free HA by washing three times by ultra-centrifugation (1.3 × 105 g, 4°C, for 1–3 h for each wash). 8. Perform the coupling reaction of HA-modified liposomes to mAbs using an amine-coupling method. Incubate 50 μL HA-modified liposomes with 200 μL of 400 mM EDAC and 200 μL of 100 mM NHS for 20 min at room temperature with gentle stirring.

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9. Mix the EDAC-NHS-activated HA-nano-liposomes with 50 μL mAb (~500 μg of FIB504 Rat-anti mouse IgG2a against β7 integrin, 10 mg/mL in HBS or PBS, pH 7.4) and incubate for 150 min at room temperature with gentle stirring. Add 20 μL 1 M ethanolamine HCl (pH 8.5) to block the reactive residues (see Note 3). 10. Purify I-tsNPs (e.g., β7I-tsNPs) to remove uncoupled mAbs using a size exclusion column packed with sepharose CL-4B beads (Sigma-Aldrich, Saint Louis, MO) and equilibrated with HBS, pH 7.4. 11. Prepare the purified particle suspensions for lyophilization: Snap freeze 0.2 mL aliquots in a mixture of 100% (v/v) ethanol and dry ice for ~20–30 min; freeze the aliquots for 2–4 h at −80°C and lyophilize for 48 h using an alpha 1–2 LDplus lyophilizer (see Note 4). 12. Store the lyophilized particles at −80°C until further use. 3.2. I-tsNP Characterization 3.2.1. Particle and Zeta Potential Analysis 3.2.2. Binding Efficiency of NPs

Measure the nanoparticle diameter and surface charge (zeta potential) using a Malvern Zetasizer nano ZS™. Examples of particle size and surface charge of the NPs are given in Table 1.

Flow cytometry is used to confirm intact binding ability of the surface-attached antibodies. 1. Add cells (TK-1) at 0.5 × 106 cells/FACS tube. 2. Wash cells in 1 mL FACS buffer and centrifuge at 200 × g, 5 min, 4°C. 3. Resuspend the pellet with following controls and samples to a final antibody concentration of 10 μg/mL in respective tubes in 50 μL FACS buffer: (a) Cells alone as a mock control; (b) isotype control, purified rat IgG2a; (c) Positive Control, FIB504 Rat-anti mouse IgG2a against β7; and (d) Samples, purified β7-I-tsNP fractions. 4. Incubate on ice for 30 min.

Table 1 Particle size and zeta potential measurements Particle

Diameter

Zeta potential

IgG sNP

127 ± 13 nm

−18.5 ± 1.2 mV

β7 I-tsNP

139 ± 21 nm

−23.7 ± 2.6 mV

All measurements were done in 1× PBS, pH 6.7 (with 10 mM NaCl) at 20°C in a Zetasizer nano ZS, Malvern. Data presented as an average ± SD from n = 4 independent experiments

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β7 I-tsNP

Cell Number

IgG sNP

101

102

103

β7 expression Fig. 2. Binding efficiency of NPs by FACS. Comparison of control nanoparticles (isotype IgG NPs) and integrin-targeted NPs (β7 I-tsNPs) shows higher binding of β7 I-tsNPs in TK-1 cells that express high levels of β7 integrin. This demonstrates the specificity of the I-tsNPs to integrins expressed on leukocytes for targeted delivery.

5. Wash cells with 1 mL FACS buffer, centrifuge at 200 × g, 5 min. 6. Resuspend the cell pellets and stain with secondary antibody FITC-Anti-Rat Ab IgG2a (1 μg/mL) in 50 μL FACS buffer. 7. Incubate on ice for 20–30 min. 8. Wash with 1 mL FACS buffer, centrifuge at 200 × g, 5 min, 4°C. 9. Resuspend the cell pellets in appropriate volume of FACS buffer and analyze by FACS. An example of FACS analysis is shown in Fig. 2. 3.2.3. Entrapment Efficiency of NPs

Ku70 siRNAs entrapment in I-tsNPs is described as follows: 1. Mix siRNAs with full-length recombinant protamine (1:5, siRNA:protamine molar ratio) or spermine (1:22, siRNA: spermine molar ratio) or spermidine (1:30, siRNA:spermidine molar ratio), in nuclease free water and incubate for 20 min at RT to form a complex. 2. For siRNA entrapment in I-tsNPs, rehydrate the lyophilized nanoparticles (i.e., β7 I-tsNP, IgG-sNP, or sNP; 1–2.5 mg total lipids for in vivo experiments and 10–100 μg total lipids for in vitro experiments) by adding 0.2 mL nuclease free water containing protamine- (or spermine) condensed siRNAs (1,000–3,500 pmol for in vivo experiments and 50–750 pmol for in vitro experiments).

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Table 2 Number of mAb immobilized on the surface and entrapment of siRNAs molecules siRNAs entrapmenta (# of molecules)

Encapsulation efficiency of condensed siRNA

Nanoparticles type

Mean ± SEM

Mean ± SEM

IgG sNP

3,750 ± 1,300

78 ± 10

β7 I-tsNP

4,000 ± 1,200

80 ± 12

The amount of siRNAs that was used for encapsulation was known. Upon encapsulation, a RiboGreen assay (molecular probes) was preformed to assess the amount of siRNAs that was entrapped a

3. Perform the entrapment procedure immediately before use in in vitro transfection or in vivo injection. 4. The concentrations of siRNAs and percent entrapment are determined by a Quant-iT™ RiboGreen™ RNA assay (Molecular Probes, Invitrogen, Carlsbad, CA). An example is given in Table 2. 3.3. Ku70 siRNA Delivery In Vitro in TK-1 Cells (Studied by Flow Cytometry)

1. Plate TK-1 cells in microtiter plates (24 well plate) and culture them overnight at 37°C, 5% (v/v) CO2 (2.5 × 105 cells in 400 μl media/well) without serum or antibiotics. 2. Add 50 μl/well of β7 I-tsNP entrapping siRNAs (e.g., Ku70siRNA) dropwise and shake gently. Spin down the plate at 300 × g for 5 min. Appropriate controls should be included: cells with no treatment; cells with Ku70-siRNA alone; cells with negative control siRNA (e.g., silencer firefly Luciferase siRNA or scrambled siRNA). Culture the cells for 5 h. 3. Add 50 μL of serum containing culture media (10% (v/v) FBS in RPMI) and shake gently by rocking the plate from side to side. 4. Culture cells for further 60–72 h at 37°C, 5% (v/v) CO2 and perform intracellular staining (as described below) for detection of Ku70 to confirm the effect of siRNA delivered using I-tsNP.

3.3.1. Intracellular Staining and Flow Cytometry

1. Transfer TK-1 cells to 96 well V bottom plates. 2. For intracellular staining cells, fix and permeabilize cells with Fix-and-Perm KitTM (Caltag Laboratories, Burlingame, CA).

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Isotype control

KD1

NE

Counts

KD2

113

100

101

102 Ku70

103

NE -Ku70 expression inTK-1 cells - mock treated cells. KD1 - Knockdown of Ku70 using 100pmol-siRNA delivered viaβ7 I-tsNP inTK-1 cells KD2 - Knockdown of Ku70 using750pmol-siRNA delivered viaβ7 I-tsNP inTK-1 cells

Fig. 3. β7 I-tsNP entrapping Ku70 siRNAs induces silencing in TK-1 cells. FACS histograms show gene silencing effect of β7 I-tsNPs entrapping different concentrations of Ku70 siRNAs (100 and 750 pmol, KD1 and KD2, respectively) in comparison to isotype NPs (gray area under the curve) and mock treated cells (NE).

3. Detection of Ku70 expression performed by adding antibody to Ku70 (purified mouse anti-Ku70, Santa Cruz Biotechnology, Santa Cruz, CA) at a final concentration of 10 μg/mL, 50 μL in FACS buffer. 4. Incubate on ice for 30 min and counter stain with FITCconjugated Goat anti-mouse IgG (BD Pharmingen). 5. Wash with FACS buffer and perform FACS analysis for Ku70 expression. An example of the intracellular stain is given in Fig. 3. 3.4. Delivery of Ku70-siRNA In Vivo

1. Make groups of mice (at least 3 mice/group, preferentially 5–8 mice/group). Make sure to include a mock treated group. 2. Pre-heat mice with a lamp in order to expose their tail veins. 3. Prior to injection – sonicate the suspension for 10 min in a bath sonicator to dissolve any potential aggregates. 4. Use a 30-gauge needle with a tuberculin syringe to inject to the tail vein of the mice. 100–200 μL/mouse with 2.5 mg/kg (50 μg) siRNA entrapped in 250 μg liposomes. 5. 48 or 72 h post injection sacrifice the mice and isolate the spleen (see Note 5). 6. Make a single cell suspension from the spleen as reported in Note 5, and perform an intracellular staining with anti-Ku70 mAb (Santa Cruz) as detailed above.

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4. Notes 1. Liposome preparation: Following the evaporation of organic solvents, it is advisable to pass any inert gas like argon for 2–3 min to completely remove traces of organic solvent and prevent oxidation of lipids. 2. The liposome suspension following 2 h incubation can be stored at 4°C until extrusion procedure. But prior to extrusion, the liposomes should be pre-warmed to 37°C to enable easy extrusion process. 3. Coupling reaction: EDAC/NHS-activated HA-nanoliposomes with antibody reaction mixture can be incubated overnight at room temperature and then blocked with 20 μL of 1 M ethanolamine, pH 8.5. 4. Lyophilization of NPs: Prior to lyophilization of purified liposome fractions, the particle suspensions should be tested for antibody-binding efficiency by FACS analysis as described in Subheading 3.2.2. Select the fractions that give high binding efficiency and pool all the fractions. Aliquots (0.2 mL) of the I-tsNP suspension are added into amber glass vials prior to lyophilization. Depending on the cells that are transfected, I-tsNP fractions can be diluted before aliquots are prepared for lyophilization to give an optimal transfection or gene-silencing efficiency. 5. Isolation of splenocytes: One spleen yields approximately 108 splenocytes, of which ~10% are CD8+ and ~20% are CD4+. (a) Harvest spleens into K10 media, removing as much connective tissue as possible. (b) Place ~3 mL K10 media and the splenocytes in a small petri dish. Homogenize into a single cell suspension: ●

Using the flat top of a 5 mL syringe shaft, homogenize the splenocytes (or)

●

Using the frosted ends of two glass sides, homogenize the splenocytes.

(c) Rinse the sieve or slides with K10 media or 2% (v/v) FCS, then transfer splenocytes into a 15-mL conical tube. (d) Spin down cells for 5 min at 320 × g. (e) Aspirate off supernatant and flick cell pellet to loosen. (f) Resuspend cells in 2 mL RBC lysis buffer. (g) Incubate at 37°C for 5 min. (h) Add 10 mL 2% (v/v) FCS and spin 5 min, 320 × g.

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