Delivery of siRNA Using Lipid Nanoparticles Modified with Cell

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Delivery of siRNA using lipid nanoparticles modified with cell penetrating peptide Yuhuan Li, Robert J. Lee, Kongtong Yu, Ye Bi, Yuhang Qi, Yating Sun, Yujing Li, Jing Xie, and Lesheng Teng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09991 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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ACS Applied Materials & Interfaces

Delivery of siRNA using lipid nanoparticles modified with cell penetrating peptide Yuhuan Li a, Robert J. Lee a, b, Kongtong Yu a, Ye Bi a, Yuhang Qi a, Yating Sun a, Yujing Li a, Jing Xie a*, Lesheng Teng a* a

School of Life Sciences, Jilin University, Changchun, 130012, China

b

Division of Pharmaceutics, College of Pharmacy, The Ohio State University,

Columbus, Ohio 43210, USA

ABSTRACT

Clinical development of siRNA has been hindered by the lack of an effective delivery system. Here, we report the construction of a novel siRNA delivery system, sTOLP, which is based on cell penetrating peptide, oleoyl-octaarginine (OA-R8), modified multifunctional lipid nanoparticles. sTOLP nanoparticles are composed of a protamine complexed siRNA core, OA-R8, cationic and PEGylated lipids, and transferrin as a targeting ligand. sTOLP formulation was optimized and characterized in vitro and showed excellent gene silencing activity. In vivo, siRNA encapsulated in sTOLP exhibited potent tumor inhibition (61.7%), and was preferentially taken up by hepatocytes and tumor cells in HepG2-bearing nude mice without inducing 1

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immunogenicity, and hepatic and renal toxicity. Furthermore, sTOLP-loaded siRNA had greater stability in circulation than free siRNA. These data demonstrated potential utility of sTOLP-mediated siRNA delivery in cancer therapy.

KEYWORDS

Cell penetrating peptide, multifunctional, lipid nanoparticles, cancer, siRNA, drug delivery.

INTRODUCTION RNA interference (RNAi) is an emerging therapeutic modality for cancer1-2. Taking advantage of its ability to specifically knockdown the expression of specific genes involved in cellular growth and apoptosis3, small interfering RNAs (siRNAs) are being developed both for basic research and for clinical therapy of cancer4-7. Nevertheless, there are limitations on clinical utility of siRNA5, 8, including poor stability9 and low membrane permeability due to negative charge10 and large molecular size11, which present a challenge for in vivo delivery12-14. Development of an efficient, safe and stable delivery system is required for clinical application of siRNA13-15. Delivery systems based on adenovirus16, nanoparticles17-18, liposomes19-20 and polymers21-22 have been developed for siRNA delivery. Cell penetrating peptides (CPPs) have been shown to promote nucleic acid delivery23-26. From simple in vitro treatment to in vivo systemic administration, various CPPs, alone or linked to bulky 2


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cargos, have been successfully used in the delivery of nucleic acids27, proteins28, small molecules29 and imaging agents30. Well known CPPs include TAT31, transportan32, MAP33 and polyarginines34-35, most notably R836. It has been reported that linking CPPs to a hydrophobic moiety can enhance their cell penetration efficiency37-38. Further efforts are warranted to enable its clinical translation39-41.

Lipid nanoparticles (LNPs) are among the most investigated nanocarriers. Advantages of LNPs include biocompatibility, biodegradability, and low toxicity and reduced immunogenicity. Combining CPPs with LNPs can potentially enhance their utility as delivery vehicles for siRNA.

Here, we report synthesis and evaluation of multifunctional LNPs which have customizable packaging layers based on rational design (Figure 1). The core was composed of electrostatic complexes between protamine, a polycationic peptide, and siRNA against survivin. Protamine stabilizes siRNA and the surplus in positive charge facilitates lipid encapsulation. Egg PC (ePC), cholesterol (Chol) and cationic lipid were incorporated into a supported bilayer. Asymmetric distributed cationic lipid could mediate ion-pair formation with oppositely charged lipids in the target membrane, thus promote endosomal escape of siRNA. An amphiphilic CPP, OA-R8, was incorporated into LNPs. A PEG layer and transferrin (Tf) targeting ligand constituted the outermost layer, which were designed to increase circulation time and target tumor cells in vivo. The multifunctional LNPs were characterized both in vitro and in vivo.

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Figure 1.Schematic representation of the steps involved in the synthesis of the multifunctional LNP.

Results and Discussion Formulation optimization of multifunctional LNPs with a supported bilayer

A series of LNPs with varying compositions were synthesized. ePC and Chol were incorporated into the LNPs as helper lipids and three different cationic lipids were used in preparing LNPs combined with OA-R8. The compositions of LNPs are shown in Table 1. The LNPs did not contain a PEG-lipid and had a fixed molar ratio of helper lipids. The average particle size and surface charge of the LNPs were determined by dynamic light scattering on a particle size analyzer.

Table 1. Compositions and characteristics of LNP formulations. Formulation

Components

Particle size (nm) 4


PDI

Zeta potential (mV)

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1

OA-R8/ePC/Chol=45/18/35

202.2±16.4

0.306

24.7±1.3

2

DODMA/OA-R8/ePC/Chol=5/40/18/35

172.8±11.2

0.177

30.3±0.8

3


143.1±10.6

0.247

32.2±1.4

4


98.8±8.2

0.107

38.5±1.2

5


105.3±7.6

0.159

33.4±1.4

6


123.0±11.7

0.171

28.1±0.9

7

DODMA/ePC/Chol=45/18/35

107.8±10.3

0.265

8.8±0.4

8

DOTMA/ePC/Chol=45/18/35

120.3±11.4

0.215

36.6±0.9

9

DOTMA/OA-R8/ePC/Chol=20/25/18/35

99.3±10.2

0.261

31.8±1.2

10

DOTAP/ePC/Chol=45/18/35

116.1±11.5

0.228

30.6±0.3

11

DOTAP/OA-R8/ePC/Chol=20/25/18/35

108.4±10.7

0.238

18.6±2.2

PDI: polydispersity index. Values shown are mean±SD.

The LNPs containing OA-R8 and helper lipids only (Formulation 1) had larger mean particle size and polydispersity index (202.2±16.4 nm, 0.306) than cationic LNPs containing DODMA (Formulation 7) (107.8±10.3 nm, 0.265), DOTMA (Formulation 8) (120.3±11.4 nm, 0.215), or DOTAP (Formulation 10) (116.1±11.5 nm, 0.228). In DODMA based formulations (Formulations 2~7), when the molar ratio of DODMA and OA-R8 was 20/25 (Formulation 4), LNPs had the smallest particle size and highest zeta potential (98.8±8.2 nm, 38.5±1.2mV). These results showed tight packing of LNPs with DODMA and OA-R8 at this ratio. The positive zeta potential implied the presence of cationic groups from the cationic lipid and R8 on the particle surfaces. At the same time, zeta potential of the LNPs based on DODMA (Formulation 4) was greater than 5



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those of the LNPs based on DOTMA or DOTAP, and this can facilitate cellular uptake based on increased electrostatic interactions with the cellular membrane42.

LNP uptake and viability of cancer cells treated by the LNP formulations were determined (Figure 2). LNPs containing FAM-labeled siRNA were added to HepG2 cells and the cellular uptake of FAM-siRNA was determined by flow cytometry at 4 h post-addition. As shown in Figure 2A, the mean fluorescence intensity of FAM-siRNA delivered by LNPs was significantly greater than those of saline-treated control and naked siRNA. The fluorescence intensity of HepG2 cells treated with LNPs containing OA-R8 was stronger than those treated with LNPs without OA-R8 (Formulations 2~6 vs 7). Among the LNPs based on DODMA, inclusion of OA-R8 (5~25%) was found to enhance cellular uptake and cells treated with Formulation 4 had the greatest mean fluorescence intensity. Interestingly, the fluorescence intensity decreased with OA-R8 content of >25% in the LNPs. This could be because of structural changes in LNPs at high OA-R8 content. However, the introduction of OA-R8 did not have a significant effect on the cellular uptake of DOTMA and DOTAP based LNPs (Formulation 9 vs 8 and 11 vs 10).

Previous study has shown that OA-R8 produces a weak toxic effect on cancer and normal cells37, so the cytotoxicity of HepG2 cells treated by the LNPs containing OA-R8 was investigated by MTT assay. According to Figure 2B, the viability of cells treated with OA-R8 modified LNPs was reduced. In fact, the greater the density of OA-R8, the stronger the observed cytotoxicity. For multifunctional LNPs modified with OA-R8, parameters such as particle size and zeta potential affect the delivery 6


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efficiency for siRNA. LNPs in Formulation 4 had the smallest particle size and highest zeta potential. These characteristics are likely to facilitate cellular uptake. In fact, the cellular uptake data showed that Formulation 4 had the best cellular uptake. Therefore, Formulation 4 was chosen as the optimal formulation of LNPs with a supported bilayer, the molar ratio of DODMA/OA-R8/ePC/Chol was 20/25/18/35. Thus, in our study, DODMA and OA-R8 were selected as the main components of supported bilayer in all LNPs formulations, while the composition of other helper lipids was fixed. Based on these parameters, we formulated multifunctional LNPs for in vivo delivery, with addition of PEGylation and Tf to increase circulation longevity and to target Tf receptor expressing tumor cells. The particle size, surface charge, cellular uptake and cytotoxicity of the LNP formulations were evaluated.

Figure 2. Formulation optimization of the supported layer in LNPs. (A) Uptake of eleven different formulations in HepG2 cells at 4 h post-treatment. The mean fluorescence intensity of Cy3-siRNA delivered by various LNPs was measured by flow 7



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cytometry analysis. (B) Relative viability of HepG2 cells treated by 11 different LNP formulations at 48 h post addition.

Characterization of the multifunctional LNPs According to the optimal composition of the supported bilayer, four formulations of LNPs

loaded

with

siRNA

were

prepared.

The

DODMA/OA-R8/ePC/Chol/ mPEG2000-DSPE at 20/25/18/35/2.

composition

was

The formulations

synthesized include LNPs loaded with siRNA (sLP), OA-R8-modified LNPs loaded with siRNA (sOLP), Tf-modified LNPs loaded with siRNA (sTLP) and OA-R8/Tf-modified LNPs loaded with siRNA (sTOLP). Particle size and zeta potential are shown in Table 2.

Table 2. Average particle size and zeta potential of LNPs. Formulation

Particle size (nm)

PDI

Zeta potential (mV)

sLP

108.4±12.3

0.195

3.7±2.2

sOLP

125.9±16.4

0.184

7.9±3.4

sTLP

139.8±15.7

0.191

-2.5±2.1

sTOLP

150.5±14.6

0.189

5.4±2.3

Data are reported as mean±SD.

The average particle sizes of the above four formulations of LNPs were under 200 nm; sLP had the smallest average particle size. With introduction of OA-R8 and Tf, the

8


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particle size of sTLP, sOLP and sTOLP increased. The zeta potential results indicate that, the influence of PEG ligand on the surface of the LNPs was significant. For LNPs containing DODMA, the zeta potential of sLP was still low. As an exception, sTLP had a negative charge. This could be because of the presence of a targeting ligand Tf on the surface of sTLP, which was negatively charged. After the introduction of positive charged OA-R8, zeta potentials of the multifunctional LNPs were increased. sTOLP had relatively uniform particle size distribution. The average particle size was 150.5±14.6 nm, zeta potential was 5.4±2.3 mV.

The structures of the LNPs were observed under transmission electron microscopy (TEM).

The results are shown in Figure 3A. The TEM images show that the

nanoparticles had a spherical morphology and the particle sizes were relatively homogeneous.

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Figure 3. The structure and in vitro characteristics of multifunctional LNPs. (A) TEM images of the four LNP formulations. (B) Relative viability of HepG2 cells treated by multifunctional LNPs after 48 h post-treatment. (C) Uptake of LNPs in HepG2 cells. **P

Delivery of siRNA Using Lipid Nanoparticles Modified with Cell

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