Characterization and In Vivo Efficacy of Targeted Nanoparticles for Systemic siRNA Delivery to Tumors

D.W. Bartlett and M.E. Davis

Chemical Engineering, California Institute of Technology

1200 E. California Blvd., Pasadena, CA 91125, bartlett@caltech.edu

ABSTRACT Systemic delivery of nucleic acid molecules is one of

the major hurdles limiting the application of siRNA-based therapeutics for cancer treatment. Multifunctional nanoparticles are being investigated as systemic, nonviral nucleic acid delivery systems, and here we describe the use of cyclodextrin-containing polycations (CDP) to interact with small interfering RNA (siRNA) molecules to form nanoparticles that are approximately 60-80 nm in diameter. The modular design of these nanoparticles enables modification with PEG molecules for steric stabilization and transferrin targeting ligands for uptake by tumor cells. The nanoparticles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application. These nanoparticles are able to deliver functional siRNA molecules to metastatic and subcutaneous tumors in mice after systemic administration.

Keywords: nanoparticles, siRNA, transferrin targeting, bioluminescent imaging

1 INTRODUCTION Carrier-mediated delivery using nanoparticles has

several advantages over the delivery of individual nucleic acid molecules. Encapsulation of the payload within a lipid bilayer or through electrostatic interactions is nonspecific, so these delivery vehicles can be used for generalized nucleic acid delivery. The use of a carrier enables delivery of many nucleic acid molecules per uptake event, and isolation from exposure to the systemic environment can permit the use of unmodified nucleic acids [1]. Modularly designed delivery vehicles can also take advantage of covalent or non-covalent attachment of hydrophilic polymers for steric stabilization and/or targeting ligands for cell-specific delivery, two critical features for systemic delivery [1-3]. Such modifications can affect the resulting biodistribution of delivery vehicles through passive and/or active targeting [1-5].

A successful delivery vehicle must be engineered to have the following characteristics: (i) be small enough to extravasate and exhibit adequate tissue penetration, yet avoid rapid renal clearance; (ii) minimize nonspecific interactions and opsonization while providing specific

targeting to a given cell; and (iii) protect the nucleic acid from degradation, but willingly release it upon arrival at the proper site. The synthetic delivery system based on a cyclodextrin-containing polycation (CDP) described here has demonstrated some success in delivering nucleic acid payloads that include pDNA, siRNA, and DNAzymes [1, 2, 6-10]. This delivery system is the first to be de novo designed for systemic delivery of nucleic acids and completely formulated by self-assembly [6]. Here, the physicochemical and biological characterization of the cyclodextrin-containing polycation delivery system and its formulation with nucleic acids is described along with examples of its use to deliver functional siRNA payloads to tumors in mice after systemic administration.

2 MATERIALS AND METHODS

2.1 Formulation of Nucleic Acid Nanoparticles

The chemical structure of the cyclodextrin-containing polycation is shown in Figure 1A. This short, linear polycation can be synthesized with (CDP-Im) or without (CDP) the imidazole groups on the terminal amines [6, 7]. A schematic showing particle formation using CDP-Im and nucleic acid is shown in Figure 1B; nanoparticles are formed by mixing equal volumes of CDP-Im and nucleic acid. The ratio of positive (+) charges (2 moles of positive charge per CDP-Im monomer; denoted β-CD) to negative (-) charges (1 mole of negative charge per nucleotide) is defined as the formulation charge ratio (+/-). Polyethylene glycol (PEG) molecules containing adamantane (AD) on the proximal end and either methoxy (AD-PEG) or a targeting ligand such as transferrin (AD-PEG-Tf) on the distal end can be attached to the surface of the nanoparticles via inclusion complex formation between adamantane and the β-CD molecules on the polycation backbone [2, 6, 7]. The molecular weight of the PEG chain is typically 5,000 daltons (PEG5000).

2.2 Atomic Force Microscopy (AFM)

Nanoparticles containing CDP-Im and siRNA (0.1 g L-1) and PEGylated nanoparticles containing CDP-Im, AD-PEG (1:1 AD-PEG:β-CD mole ratio), and siRNA (0.5 g L-1) were formulated in water at a charge ratio of 3 (+/-). 20 µL of each formulation solution were dropped on a

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freshly cleaved mica disc (Ted Pella, Inc.) and dried with pressurized air. Images were acquired with a Digital Instruments MultiMode AFM with a Nanoscope IV controller in tapping mode at a scan rate of 1 Hz using a BS Multi75 probe (BudgetSensors) with a resonant frequency of 75 kHz and a force constant of 3 N m-1. Height images were flattened and processed for visualization with the derivative matrix convolution filter using WSxM scanning probe microscopy software (Nanotec Electronica).

2.3 Injection of Mice with TC71-Luc (Luciferase Expressing) Ewing’s Sarcoma cells

TC71-Luc cells were grown in RPMI 1640 with 10% FBS and antibiotics (penicillin/streptomycin). All mice were 6-8 weeks of age at the time of injection. Each mouse was injected with 5x106 TC71-Luc cells suspended in 200 µL RPMI (without FBS or antibiotics) through the tail vein using a 27-gauge needle.

2.4 In vivo Delivery of siRNA Nanoparticles

All nanoparticles were made with siRNA and CDP-Im synthesized as described previously [6, 10]. Before addition to siRNA, CDP-Im was mixed with AD-PEG and

AD-PEG-Tf such that the total moles of AD-PEG or AD-PEG-Tf equaled the number of moles of β-CD. Tf-targeted nanoparticles contained 0.1% mass AD-PEG-Tf relative to AD-PEG. This mixture was added to an equal volume of siRNA at a charge ratio of 3 (+/-). An equal volume of 10% glucose in water was added to the resulting nanoparticles to yield a 5% glucose solution suitable for injection. Each mouse was injected with 200 µL of this nanoparticle formulation containing 50 µg siRNA per 20-g mouse (2.5 mg kg-1 siRNA).

3 RESULTS

The cyclodextrin-containing polycation shown in Figure

1A can be used to form nanoparticles with a variety of nucleic acids including plasmid DNA (pDNA), DNAzymes, and siRNA. AFM images of siRNA nanoparticles are shown in Figure 2. Depending on the concentration of the nucleic acid during formulation, the nanoparticles can be formulated to have sizes ranging from 50-150 nm in diameter [2]. However, the size of the nanoparticles is independent of the nucleic acid concentration during formulation if AD-PEG is included during formulation [2]. Multi-angle light scattering can be used to determine the molecular weight of the nanoparticles, enabling estimation of the composition of an individual nanoparticle. For example, a 70-nm nanoparticle formulated for systemic administration is estimated to

Figure 2: Atomic force microscopy (AFM) images of (A) unPEGylated and (B) PEGylated siRNA particles formulated at a charge ratio of 3 (+/-).

Scale bar = 200 nm. Figure 1: Formation of nucleic acid-containing nanoparticles using CDP-Im. (A) Schematic of the chemical structure of CDP-Im. (B) Schematic of

nanoparticle assembly.

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contain approximately 10,000 CDP polymers, 2,000 siRNA molecules, 4,000 PEG5000 molecules (~43 pmol cm-2), and 100 Tf targeting ligands; this represents a significant payload of siRNA and a large ratio of siRNA to targeting ligand (20:1). The surface density of the PEG5000 molecules is estimated to be in the brush regime, providing protection against nonspecific interactions and aggregation [11]. The nanoparticles protect the nucleic acid payload from nuclease degradation, do not aggregate at physiological salt concentrations, and cause minimal erythrocyte aggregation and complement fixation at the concentrations typically used for in vivo application [2].

The ability of these nanoparticles to deliver functional siRNA molecules to tumors in vivo is demonstrated with the luciferase reporter system. TC71-Luc Ewing’s sarcoma cells with constitutive expression of the firefly luciferase gene were injected by tail vein into NOD/scid mice. The growth of the tumor cells was monitored by bioluminescent imaging. The pattern of engraftment sites was similar to those observed clinically in Ewing’s sarcoma patients [1]. After the tumors had grown for approximately 5-6 weeks, Tf-targeted siRNA nanoparticles were injected by tail vein on two consecutive days. The siRNA used for the initial studies was designed to target luciferase mRNA, leading to decreased levels of luciferase protein expression. The results shown in Figure 3 demonstrate that the Tf-targeted nanoparticles were able to deliver functional siRNA to the tumor after systemic administration, leading to ~90% reduction in the luciferase signal 3 days after the initial injection. Consistent with the transient nature of siRNA-mediated gene silencing and the continued growth of the tumor, the luciferase expression recovered to greater than pre-injection values by 6 days after the initial injection [12].

While knocking down the luciferase gene is a useful tool for research and development, the goal of targeted siRNA delivery involves silencing genes that can have a therapeutic benefit such as tumor growth inhibition. Specific chromosomal translocations are associated with numerous hematopoietic and solid tumors, and the translocation t(11;22) produces the chimeric EWS-FLI1 fusion gene found in 85% of patients with Ewing’s sarcoma. Therefore, the targeted nanoparticles were used to deliver an siRNA against the EWS-FLI1 fusion gene. Successful delivery of the siRNA to the TC71-Luc Ewing’s sarcoma cells should result in knockdown of the EWS-FLI1 fusion gene and cause tumor growth inhibition. To test this hypothesis, TC71-Luc cells were injected by tail vein into NOD/scid mice. Beginning with the day of the injection and continuing twice-weekly for the next 5 weeks, the mice were injected by tail vein with 5% glucose (n = 10), 2.5 mg kg-1 of naked siRNA against EWS-FLI1 (n = 10), Tf-targeted nanoparticles containing 2.5 mg kg-1 of control siRNA (n = 10), Tf-targeted nanoparticles containing 2.5 mg kg-1 of siRNA against EWS-FLI1 (n = 10), or non-targeted nanoparticles containing 2.5 mg kg-1 of siRNA against EWS-FLI1 (n = 10). Steady tumor growth was observed in all treatment groups except for the group of

mice receiving the twice-weekly injections of Tf-targeted nanoparticles containing the siRNA against EWS-FLI1 (Figure 4) [1]. These results indicate that the observed effect was sequence-specific since the control siRNA did not lead to tumor growth inhibition. Additionally, the results highlight the importance of attaching targeting

Figure 3: In vivo luciferase knockdown after two consecutive daily injections of Tf-targeted

nanoparticles containing 50 µg siRNA against luciferase.

Figure 4: Tumor growth in NOD/scid mice after injection of 5x106 TC71-Luc Ewing’s on day 0

followed by twice-weekly injections (arrows) of the treatment formulations. Circles = Tf-targeted

nanoparticles containing siRNA against EWS-FLI1, squares = Non-targeted nanoparticles containing

siRNA against EWS-FLI1, diamonds = Tf-targeted nanoparticles containing a control siRNA sequence,

triangles = naked siRNA against EWS-FLI1, and asterisks = 5% glucose.

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ligands to the surface of the nanoparticles designed for systemic delivery as the non-targeted nanoparticles were not able to achieve tumor growth inhibition.

4 CONCLUSIONS The results presented here emphasize the importance of

creating a nanoparticle that consists of multiple components that function together as a system, and control over size, surface modification, payload protection, and targeting ligand to payload ratio are key parameters to consider when designing nucleic acid delivery vehicles for in vivo systemic use. These parameters also represent some of the major advantages of nanoparticle composites for delivery of nucleic acids instead of using carrier-free delivery methods. Nucleic acid delivery vehicles can help reduce renal clearance while adding features such as stabilization against nuclease degradation, cell-specific targeting, and large payload delivery. These features make them well-suited for the systemic delivery of nucleic acids in general. The cyclodextrin-containing polycations described here have demonstrated success in delivering functional siRNA to tumors in vivo after systemic administration, achieving transient luciferase knockdown with an siRNA against the luciferase gene or achieving tumor growth inhibition with an siRNA directed against the oncogenic fusion gene EWS-FLI1. Future studies looking at the differential uptake of targeted and non-targeted nanoparticles within tumors will be critical to optimizing the design of nanoparticle carriers for systemic nucleic acid delivery.

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