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Novel Survivin-Targeted Small Interfering RNA Delivered by Nanoparticles
Author’s Accepted Manuscript A Study of Novel siRNA Targeting Survivin Delivered by Nanoparticles Chen Feng, Tianyou Wang, Yi Zhang, Kuiyao Qu, Suoqin Tang www.elsevier.com
PII: DOI: Reference:
S0002-9629(17)30429-9 http://dx.doi.org/10.1016/j.amjms.2017.08.002 AMJMS517
To appear in: The American Journal of the Medical Sciences Received date: 30 March 2017 Revised date: 31 July 2017 Accepted date: 1 August 2017 Cite this article as: Chen Feng, Tianyou Wang, Yi Zhang, Kuiyao Qu and Suoqin Tang, A Study of Novel siRNA Targeting Survivin Delivered by Nanoparticles, The American Journal of the Medical Sciences, http://dx.doi.org/10.1016/j.amjms.2017.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A study of novel siRNA targeting survivin delivered by nanoparticles Chen Feng,MD1; Tianyou Wang, MD, PhD2; Yi Zhang, MD, PhD3; Kuiyao Qu, MD1; Suoqin Tang, MD, PhD1* 1
Department of Pediatrics, Chinese PLA General Hospital, Beijing 100853, China;
2
Department of Hematology-Oncology, Beijing Children’s Hospital,Capital Medical
University, Beijing100045, China; 3
Department of Pediatrics, Beijing Tongren Hospital, Capital Medical University,
Beijing 100730, China; *
Corresponding author Suoqin Tang Department of Pediatrics, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, China Tel: 010-55499416; Fax: 010-55499407; E-mail: [email protected]
Short title：siRNA targeting survivin nanoparticles Conflict of Interest Statement：The authors declare no conflict of interest. Funding ： This research was supported by China National 863 Program (No. 2007AA021004).
1
Abstract Background: The aim of present study was to investigate antitumor effect of our novel survivin siRNA nanoliposomes on xenograft mouse models with human cervical carcinoma HeLa cells, and to evaluate pharmacokinetics. Materials and Methods: Survivin-targeted siRNA nanoliposome was prepared and transfected into xenograft mouse models. Tumor growth in mice was determined, and survivin expression was analyzed by using histological and immunohischemical staining. Furthermore, low, moderate and high dose of survivin siRNA nanoliposomes were injected in three groups, and plasma concentrations were detected at various time points by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Biodistribution of siRNA in tumor and other important organs were also determined. Results: Survivin expression was significantly downregulated by survivin siRNA delivery-mediated by nanoliposome, along with significant suppression of cell growth. Peak concentrations were obtained at 15 minutes after injection in each group, with 1,042,538.00, 6,837,099.54 and 14,631,333.15 pg/ml, respectively, and the plasma concentration decreased significantly after 24 hours. The half-time life of survivin siRNA nanoliposomes in each group was 3.60, 2.64, and 2.80 hours, respectively. The AUC values were 952,190.88, 6,800,687.79, and 13,803,680.96 h·pg/ml, and the total drug clearance were 1050.12, 441.13, and 434.67 ml/h/kg. A significant accumulation of Cy5-labeled siRNA was found in the tumor, and a nonspecific accumulation was 2
reduced significantly in lung. Conclusions: Our finding revealed that survivin suppression by siRNA may contribute to tumor inhibition through both proliferation inhibition and apoptosis promotion effect, and the pharmacokinetic characteristics serve as a fundamental role for further studies on its applicability for cancer therapy. Keywords: Survivin; siRNA; Nanoliposome; Human cervical carcinoma cells; Biodistribution; Pharmacokinetics;
INTRODUCTION Survivin is one of the most cancer-specific proteins identified up to now, and has been reported to be upregulated in almost all types of cancers.1-3 Being a member of the inhibitor of apoptosis family, survivin regulates basic cellular process including cell apoptosis and proliferation. Overexpression of survivin in various tumor cells is related to treatment resistance or poor prognosis. Survivin is currently serving as an important target for cancer treatment and its anticancer effect has been widely studied.4-8 The anticancer effects of survivin has been reported in melanoma cells,9 human colon cancer cells 10, and the human leukemia K562 cell line.11
RNA interference is a basic post-transcriptional gene technique which is widely utilized in cancer research due to its high specificity. RNA interference can down-regulate
molecules
associated
with
tumor
proliferation,
cause
the
microenvironment of tumor change and inhibit hyperplasia and invasion of tumor.12 3
Survivin-targeted small interfering RNA (siRNA) inhibits the internal environment and proliferation of tumor cells by suppressing mRNA expression through interfering protein synthesis.13,14 It has also shown a good efficacy in antiproliferative activity to cancer cells.15,16 Nano-carriers (ranging from 1 to 1000 nm) are particles in submicron size that can overcome most obstacles in therapeutic use of siRNA.17,18 Recently, liposomes have been developed to carry siRNA and have been shown to be effective in vivo.19,20 However, the anti-tumor effects of nanoliposome-mediated survivin by siRNA on human cervical cancer HeLa cells have not been fully studied.
In this study, the antitumor effect of nanoliposome-mediated survivin siRNA was investigated by establishing xenograft mouse models with human cervical cancer HeLa cells in vivo. Furthermore, the pharmacokinetics of survivin siRNA nanoliposomes was also evaluated.
METHODS Preparation of survivin siRNA nanoliposomes The experimental procedures in this study conform to the National Institutes of Health Guide for the Care, and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital. siRNA targeting survivin was synthesized by Biomics Biotechnologies Co., Ltd (Beijing,
China),
with
the
sense
sequence
of 4
5’-GCAUCUCUACAUUCAAGAAdTdT-3’
and
antisense
sequence
of
5’-UUCUUGAAUGUAGAGAUGCdTdT-3’. A scrambled sequence was synthesized as a negative control (NC), and was named as si-NC with the sequences as follows: sense strand: 5’- UUC UCC GAA CGU GUC ACGU-3’ and antisense strand: 5’ACG UGA CAC GUU CGG AGAA-3’. siRNA was incorporated into nanoliposomes in ethanol–water solutions by pre-formed vesicles. The liposomes were composed of 1,2-dioleoyl-3dimethrylammonio-propane 1,2-distearoyl-sn-glycero-3-phosphocholine
(Sigma
Aldrich),
(Avanti Polar Lipids, Alablaster, AL,
USA), cholesterol (Wako) and PEG-DMA (Biomics Biotechnologies Co., Ltd, Beijing, China) at the molar ratio of 40:10:40:10. The siRNA nanoliposomes were then synthesized by mixing siRNA (30% ethanol solution) and pre-formed vesicles (30% ethanol solution) in a warm bath (35-40℃) for 10 minutes, and stirred continuously. After encapsulation, concentration and sterilization, the desired final products were achieved. The average diameter of nanoliposome obtained was 70.7±29.077 nm for survivin siRNA and 64.9±26.128 nm for NC-siRNA. HeLa xenograft model construction and treatment 24 male Balb/c nude mice (18-24 g, 7-9 weeks old) were obtained from Shanghai SLAC Laboratory Animal CO. LTD (Shanghai, China). Human cervical carcinoma cell line HeLa (Biomics Biotechnologies Co., Ltd, Beijing, China) was cultured into cell suspension with trypsin and then centrifuged and counted. HeLa cells (4-6×106/0.2 ml) were subcutaneously inoculated in the upper limb of those mice, and 5
was considered the first generation of transplanted tumors. When the tumor size reached about 1.0 cm in diameter, the mice were sacrificed and subcutaneous tumors were collected. Pieces of homogeneous texture tissues (1×1×1 mm3) were cut and transplanted into subaxillary tissue of male Balb/c nude mice.
When the tumors reached a mean diameter of 3-5 mm, the mice were randomly divided into 4 groups (n=6): the SU-IT group received intratumoral injection of survivin siRNA nanoliposome (50 μg/mouse, twice a week); the SU-IV group received intravenous injection of survivin siRNA nanoliposome (3 mg/kg, twice a week); the NC-IT group received intravenous injection of NC siRNA nanoliposome (3 mg/kg, twice a week); and the DOX group received intraperitoneal injection of doxorubicin hydrochloride (2.5 mg/kg, weekly) (Shenzhen Wanle Pharmaceutical Co., Ltd., Shenzhen, China).
Tumor volume and body weight of each mouse was measured every 3 days for 31 days. The mice were sacrificed after the last treatment, and tumors were excised and weighed. The inhibition rate of survivin siRNA nanoliposomes on the tumor growth was calculated by the following formula: [1 - T tumor weight/C tumor weight] ×100%.
6
Histological, immunohistochemical and RT-qPCR analysis Tumor tissues were collected in each group for survivin expression by using Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohischemistry and hematoxylin-eosin (HE) staining analysis. Tumor tissues were fixed in 4% paraformaldehyde at 4 °C for at least 24 hours and consequently processed for paraffin embedding. The sliced sections were then stained with hematoxylin-eosin. For immunohistochemical analysis, the sections were incubated with anti-survivin antibody, followed by incubation with a second antibody. The sections were visualized with 3, 3-diaminobenzidine and counterstained with hematoxylin. Total RNA was isolated using RISOTM RNA Isolation Reagent (Biomics, USA), and SensiMixTM One-Step Kit (Quantace, Taunton, MA, USA) was used for cDNA synthesis and subsequent polymerization according to the manufacturer’s instructions. The conditions of real-time polymerase chain reaction were as following: 95 °C for 10 minutes, 95°C for 20 seconds, 58 °C for 30 seconds to anneal, and 72°C for 30 seconds followed by 40 cycles. Specific primers were used to detect survivin (forward primer, 5’-ACG ACC CCA TAG AGG AAC AT-3’; reverse primer: 5’- TCC GCA GTT TCC TCA AAT TC -3’). GAPDH was amplified as an internal control using specific primers (forward primer, 5’-GAA GGT GAA GGT CGG AGT C-3’; reverse primer: 5’- GAA GAT GGT GAT GGG ATT TC-3’).
7
Bio-distribution of survivin siRNA liposomes in vivo In comparison with invivofectamine (Invitrogen, Carlsbad, CA), a commercial transfection agent, Cy5-labeled siRNA (LNP-1), was injected to the caudal vein of mice at a dose of 3mg/kg with diethyl-nitrosamine-induced hepatocarcinogenesis. All mice were sacrificed after 6 hours, after which, the tumors, hearts, livers, lungs, spleens and kidneys were harvested. The distribution of siRNA was measured in tumor and other organs using a Caliper IVIS Lumina II (Xenogen IVIS Lumina; Caliper Life Sciences, Hopkinton, MA). Blood sample collection for pharmacokinetics The specific-pathogen-free (SPF) male mice (18-25 g, 8 weeks old) were obtained from laboratory animal center of Nantong University and were randomly divided into 3 groups (n=8). Survivin siRNA nanoliposomes at the dosage of 1, 3 and 6 mg/kg was injected into mice from Group A to C via the caudal vein, respectively. The time was recorded as “0 minutes” immediately after the injection. Blood samples (0.1ml each) in each group were collected from the eye socket vein at time points: 5 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 7 hours, 24 hours, and 30 hours. Blood samples were centrifuged for 20 minutes at 4500 r/m and stored at -80 °C. The plasma concentrations of survivin siRNA nanoliposomes were measured after single intravenous injection in the 3 groups. Non compartmental model (WinNonLin5.2.1) was utilized to calculate the pharmacokinetic parameters (Phrarsighrt Inc). 8
Calibration curve construction A series of concentrations (2.0E+07, 2.0E+06, 2.0E+05, 2.0E+04 and 2.0E+03 pg/ml) of standard working solution were prepared. Each standard working solution (10 μl) was taken and then added into 190 μl tris buffered saline solution (contained 10 μl blank plasma). The final concentrations of the calibration were 1.0E+06, 1.0E+05, 1.0E+04, 1.0E+03 and 1.0E+02 pg/ml. RNA extraction and RT-qPCR RT-qPCR was used to detect the concentration of survivin siRNA nanoliposomes in blood plasma. Blood plasma (10 μl) was added into 190 μl 0.25% Triton X-100 in phosphate buffer solution at the temperature of 95 °C in water bath for 10 minutes. Total RNA was extracted using Trizol reagent (GIBCO Company) according to the manufacturer’s instructions. Complementary DNA synthesis and subsequent polymerization was performed as a 1-step procedure using the SensiMixTM One-Step Kit (Quantace, Taunton, MA, USA). Real-time PCR was performed using RochreLigthrcycler 480 and qRT-PCR kit (Shanghai Generay Biotech Co., Ltd). The primers used were summarized as follows: forward primer, 5’-GCA UCU CUA CAU UCA AGAA-3’; reverse primer, 5’-UUC UUG AAU GUA GAG AUGC-3’. As an internal control, GAPDH was amplified with primers (forward primer: 5'-GTC GGT TGT GTA ACG CGA TGTT-3'; reverse primer: 5'-ACT CCA ACG ACT GTA CTC CAGC-3').
9
Statistical analysis All experiments were performed at least in triplicate and data were expressed as the mean ± SD. All statistical analysis was performed by using SPSS, version 17.0 (SPSS Inc. Chicago, IL). One-way analysis of variance (ANOVA) was used to compare the significance between groups. Statistical significance was determined at p<0.05 level.
RESULTS Survivin siRNA nanoliposome inhibited the growth of HeLa xenograft The inhibitory effect of survivin siRNA nanoliposomes on the volume of tumor was investigated in HeLa xenograft model, as shown in Fig.1. Compared to the NC-IV group, the tumor volume in SU-IV group was significantly decreased in 24 days and 28 days (P<0.05). From the seventh day of study, the tumor volume of DOX group was significantly reduced (P<0.05) compared with the results in NC-IV group. The weights of xenograft nude mice are shown in Table 1. There was no significant difference in the body weight among the groups. However, 1 mouse in the DOX group died during the course of the study. In addition, the average body weight in the DOX group was significantly lower than those in other groups, indicating that the DOX treatment may be more toxic. The tumor inhibition rate in the DOX group and SU-IV group were 51.81% and 30.12%, respectively. The samples of tumor tissue were collected for survivin expression analysis. As shown in Fig. 2, the expression of survivin was mainly located in the endochylema 10
and the expression level was significantly reduced after survivin siRNA nanoliposomal treatment. On the contrary, higher survivin staining was found in the NC –IV group. The levels of survivin mRNA in tumor tissues were detected by RT-qPCR (Fig.3). We found that the levels of survivin mRNA were significantly reduced after survivin siRNA treatment, both intratumorally and intravenously, compared with the NC group which was treated with nanolipsomal-si-NC complex. The levels of survivin were slightly decreased after DOX treatment without significant difference. Biodistribution of LNPs in vivo Cy5 labeled LNPs or Invivofectmine were injected into mice and examined by In Vivo Imaging Systems. Compared with Invivofectmine, accumulation of LNP-1 in tumor was significant higher than those in other organs, and also nonspecific accumulation in lung was significantly reduced (Fig.4). Pharmacokinetics of survivin siRNA liposomes In the pharmacokinetic experiments, the calibration curve of survivin siRNA nanoliposome in plasma showed a good linear relationship in the range of 100~1,000,000 pg/ml (for plasma), the correlation coefficients were ≥0.9883 (Fig. 5). Concentration-time curves in group A ~ C were shown in Fig.6. The peak concentrations at 15 minutes in group A, B and C were1, 042,538.00, 6,837,099.54 and 14,631,333.15 pg/ml, respectively. The plasma concentrations were found to be stable at the time point of 3 hours.
The plasma concentration was significantly 11
decreased after 24 hours (22.04, 44.18 and 258.36 pg/ml, respectively) (Table 2). The pharmacokinetic parameters in each group are presented in Table 3. DISCUSSION Survivin, acting as a key suppressor of apoptosis, has been reported to be overexpressed in various cancer cells. It is related to prognosis, ionizing radiation and resistance to anti-tumor agents. Currently, siRNA targeted therapy has been widely studied in tumor treatment due to the specific and potent silencing of targeted genes. This therapeutic technique has been reported to induce apoptosis, block proliferation, and even suppress tumor formation and growth.21,22 siRNA knockdown of survivin inhibited cancer cell proliferation, enhanced apoptotic susceptibility, and decreased tumorigenicity in human xenografts.23 Despite the potential therapeutic advantages, effective delivery of siRNA to tumor cells remains a major hurdle for RNA-based cancer therapy, and the success of gene therapy is highly dependent on the delivery carrier.24,25
Besides, the major limitations of application of survivin siRNA in cancer
therapy are the instability and short half-life time. It has been reported that the half-life of siRNA in serum was only about 15 minutes.26 Therefore, an effective delivery carrier could avoid enzymatic degradation, and also could promote cellular uptake. Liposome has been applied for siRNA delivery and has shown a good effectiveness. In this study, an effective siRNA sequence targeting survivin was chosen and nanoliposomes were cooperated as a carrier to deliver our survivin-targeted siRNA. 12
Additionally, several independent studies have shown survivin siRNA can be introduced into the body by this delivery technique. The injection strategy was considered to be a key factor in the clinical cancer gene therapy. The study of Zhang et al. 23 (2010) reported that injection of survivin in local region of tumor can monitor tumor growth and survival . In our study, both the intratumoral and intravenous injections were performed in xenografts models. The results indicated that tumor weight and tumor inhibition rate were significantly different between the mice in the SU-IT and SU-IV group. Compared with the SU-IT group, the results in the SU-IV group showed a relatively high tumor inhibition rate. Besides, the treatment-related toxicity was lower in the SU-IV group. Therefore, intravenous systemic administration of survivin-targeted siRNA delivered by nanoliposome was safe and effective. Apoptosis is the precondition of cell death induced by different types of anticancer agents. Studies have revealed that survivin knockdown can induce apoptosis in tumor cells through genetic deletion, anti-sense oligonucleotides, and dominant negative inhibitors.27 . BALB/c mice were chosen to avoid the confounding factors that influencing drug plasma concentration, such as gender, age, weight, and so on. Our results showed that the peak plasma concentration was reached at 15 minutes, and the peak concentration was consistent with dose concentration. Plasma concentration in each group decreased abnormally after 30 minutes, the reason we speculated is frequent blood sampling, 13
which led to concentration dilution or the unstable drug plasma concentration in initial stage of administration. Based on the outcomes in our study, the half-life of survivin siRNA nanoliposome in each group ranged from 2 to 24 hours. The plasma concentration was extremely low at time point of 24 hours. Survivin siRNA nanoliposome seems to possess a stable and fast onset time, and can be cleared from the body within 24 hours. The residence time in vivo was safe. The bioavailability was great due to their higher area under the curve and was consistent with other studies. 28,29
The plasma concentration of survivin siRNA nanoliposome was in a stable range
within 7 hours after drug delivering. Trace amount of survivin siRNA nanoliposome could be detected around 48 hours after injection, which was suggestive to the synergies with other agents.
CONCLUSION A nanoliposome-based survivin siRNA delivery system was constructed and was demonstrated to be efficient in survivin siRNA delivery in vivo study. Survivin down-regulation could inhibit tumor growth in HeLa xenograft models, indicating that survivin suppression by siRNA may contribute to tumor inhibition through both the proliferation inhibition and apoptosis promotion effects. Furthermore, our findings reveal the preliminary pharmacokinetic characteristics for anti-survivin siRNA nanoliposomes and serve as a fundamental role for further studies on its applicability for cancer therapy in vivo.
14
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Li J, Yue M, Shi X, et al. EVALUATION OF ANTI-CANCER ACTIVITY OF SURVIVIN siRNA DELIVERED BY FOLATE RECEPTOR-TARGETED POLYETHYLENE-GLYCOL LIPOSOMES IN K562-BEARING XENOGRAFT MICE. Biomedical Engineering: Applications, Basis and Communications. 2014;26(02):1450026.
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Manning BD. Challenges and opportunities in defining the essential cancer kinome. Science signaling. 2009;2(63):pe15-pe15.
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Sellers WR. A blueprint for advancing genetics-based cancer therapy. Cell. 2011;147(1):26-31.
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Labi V, Grespi F, Baumgartner F, Villunger A. Targeting the Bcl-2-regulated apoptosis pathway by BH3 mimetics: a breakthrough in anticancer therapy? 16
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Li Q, Lozano G. Molecular pathways: targeting Mdm2 and Mdm4 in cancer therapy. Clinical Cancer Research. 2013;19(1):34-41.
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Ozpolat B, Sood AK, Lopez-Berestein G. Nanomedicine based approaches for the delivery of siRNA in cancer. J Intern Med. Jan 2010;267(1):44-53.
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Zhou J, Shum KT, Burnett JC, Rossi JJ. Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges. Pharmaceuticals (Basel). 2013;6(1):85-107.
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Wang Y, Xu Z, Guo S, et al. Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis. Mol Ther. Oct 2013;21(10):1919-1929.
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Table legend: Table 1 Inhibitory effect of survivin siRNA nanoliposome on the growth of tumor in xenograft nude mice with human cervical cancer HeLa cells. Table 2 Average concentration of survivin siRNA nanoliposome in plasma after Intravenous administration (pg/ml) at different time points in three groups. Table 3 Pharmacokinetic parameters for survivin siRNA nanoliposome of each group after single dose.
Figure Legend: Fig.1 The antitumor effect of survivin-targeted small interfering RNA nanoliposomes on the volume of tumor by different injection methods in HeLa xenografts models. Compared to the NC-IV group (intravenous injection), the SU-IV group (intravenous injection) in 24 days and 28 days P<0.05, the DOX group from the seventh day P<0.05; Fig.2 Tumor samples were harvested for histological analysis (HE staining), and immunohistochemical analysis of survivin expression following survivin-targeted small interfering RNA nanoliposomes treatment.
19
Fig.3 Reverse transcription-quantitative polymerase chain reaction analysis of survivin expression following survivin-targeted small interfering RNA nanoliposomes treatment. Fig.4 Biodistribution of Cy5-labeled survivin-targeted small interfering RNA nanoliposomes formulated in LNPs (LNP-1). The biodistribution of survivin-targeted small interfering RNA nanoliposomes in the tumor and other important organs (such as hearts, livers, and lungs) using invivofectamine and LNP-1. Fig.5 Calibration curve of survivin siRNA nanoliposomes in plasma. Fig.6 Concentration-time curve of three groups (low, medium and high dose of survivin-targeted small interfering RNA nanoliposomes injection group) after single intravenous injection in different time points.
Table 1 Inhibitory effect of survivin siRNA nanoliposome on the growth of human cervical cancer HeLa xenograft nude mice Number of Mice Group
Body Weight (g)
Pre-treat
Post-trea
pre-treatm
post-treat
ment
tment
ent
ment
Inhibi Tumor
tion
Weight (g)
Rate (%)
NC-IV
6
6
22.2±0.75
22.6±2.40
0.83±0.12
/
DOX
6
5
22.1±1.83
21.6±1.24
0.40±0.46
51.81
SU-IT
6
6
22.3±1.29
23.6±0.58
1.08±0.36
N/A
SU-IV
6
6
21.2±1.40
21.7±2.38
0.58±0.27
30.12
20
Table 2 Average concentration of survivin siRNA nanoliposome in plasma after intravenous administration (pg/ml) at different time points in three groups. Time
Group A
Group B
Group C
(low dose:1 mg/kg)
(moderate dose:3 mg/kg)
(high dose: 6mg/kg)
5 minutes
336601.5
5674548.81
4704490.01
15 minutes
1042538.00
6837099.54
14631333.15
30 minutes*
56201.62*
645714.88*
568540.18*
45 minutes
821309.3
3920663.95
10313715.46
1 hours
18392.55
81355.16
108388.11
1.5 hours
98452.32
380561.31
1014007.80
2 hours
18694.05
465844.66
389596.55
3 hours
3674.25
4765.05
86880.87
4 hours
999.59
7259.11
64239.90
6 hours
2923.55
23813.45
7439.86
7 hours
17814.76
164815.22
359524.76
24 hours
22.04
44.18
258.36
30 hours
3.05
1.12
11.04
48 hours
16.00
5.51
12.96
Table 3 Pharmacokinetic parameters for survivin siRNA nanoliposome of each group after single dose Group A
Group B
Group C
(low dose:1 mg/kg)
(moderate dose: 3 mg/kg)
(high dose: 6 mg/kg)
t1/2 (hour)
3.60
2.64
2.80
Tmax (hour)
0.25
0.25
0.25
Cmax (pg/ml)
1042537.60
6837099.54
14631333.15
Parameters
21
AUC (h∙pg/ml)
952190.88
6800687.79
13803680.96
Vz (ml/kg)
5454.76
1682.54
1757.33
CL (ml/h/kg)
1050.12
441.13
434.67
MRT (hour)
1.70
1.97
2.10
t1/2, half-life time; Tmax, time when maximum concentration arrived; Cmax, maximum concentration; AUC, area under curve; Vz, apparent volume of distribution; CL, clearance; MRT, mean residence time.
Fig. 1
22
Fig. 2
23
Fig. 3
Fig. 4
24
Fig. 5
Fig. 6
25
PII: DOI: Reference:
S0002-9629(17)30429-9 http://dx.doi.org/10.1016/j.amjms.2017.08.002 AMJMS517
To appear in: The American Journal of the Medical Sciences Received date: 30 March 2017 Revised date: 31 July 2017 Accepted date: 1 August 2017 Cite this article as: Chen Feng, Tianyou Wang, Yi Zhang, Kuiyao Qu and Suoqin Tang, A Study of Novel siRNA Targeting Survivin Delivered by Nanoparticles, The American Journal of the Medical Sciences, http://dx.doi.org/10.1016/j.amjms.2017.08.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A study of novel siRNA targeting survivin delivered by nanoparticles Chen Feng,MD1; Tianyou Wang, MD, PhD2; Yi Zhang, MD, PhD3; Kuiyao Qu, MD1; Suoqin Tang, MD, PhD1* 1
Department of Pediatrics, Chinese PLA General Hospital, Beijing 100853, China;
2
Department of Hematology-Oncology, Beijing Children’s Hospital,Capital Medical
University, Beijing100045, China; 3
Department of Pediatrics, Beijing Tongren Hospital, Capital Medical University,
Beijing 100730, China; *
Corresponding author Suoqin Tang Department of Pediatrics, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, China Tel: 010-55499416; Fax: 010-55499407; E-mail: [email protected]
Short title：siRNA targeting survivin nanoparticles Conflict of Interest Statement：The authors declare no conflict of interest. Funding ： This research was supported by China National 863 Program (No. 2007AA021004).
1
Abstract Background: The aim of present study was to investigate antitumor effect of our novel survivin siRNA nanoliposomes on xenograft mouse models with human cervical carcinoma HeLa cells, and to evaluate pharmacokinetics. Materials and Methods: Survivin-targeted siRNA nanoliposome was prepared and transfected into xenograft mouse models. Tumor growth in mice was determined, and survivin expression was analyzed by using histological and immunohischemical staining. Furthermore, low, moderate and high dose of survivin siRNA nanoliposomes were injected in three groups, and plasma concentrations were detected at various time points by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Biodistribution of siRNA in tumor and other important organs were also determined. Results: Survivin expression was significantly downregulated by survivin siRNA delivery-mediated by nanoliposome, along with significant suppression of cell growth. Peak concentrations were obtained at 15 minutes after injection in each group, with 1,042,538.00, 6,837,099.54 and 14,631,333.15 pg/ml, respectively, and the plasma concentration decreased significantly after 24 hours. The half-time life of survivin siRNA nanoliposomes in each group was 3.60, 2.64, and 2.80 hours, respectively. The AUC values were 952,190.88, 6,800,687.79, and 13,803,680.96 h·pg/ml, and the total drug clearance were 1050.12, 441.13, and 434.67 ml/h/kg. A significant accumulation of Cy5-labeled siRNA was found in the tumor, and a nonspecific accumulation was 2
reduced significantly in lung. Conclusions: Our finding revealed that survivin suppression by siRNA may contribute to tumor inhibition through both proliferation inhibition and apoptosis promotion effect, and the pharmacokinetic characteristics serve as a fundamental role for further studies on its applicability for cancer therapy. Keywords: Survivin; siRNA; Nanoliposome; Human cervical carcinoma cells; Biodistribution; Pharmacokinetics;
INTRODUCTION Survivin is one of the most cancer-specific proteins identified up to now, and has been reported to be upregulated in almost all types of cancers.1-3 Being a member of the inhibitor of apoptosis family, survivin regulates basic cellular process including cell apoptosis and proliferation. Overexpression of survivin in various tumor cells is related to treatment resistance or poor prognosis. Survivin is currently serving as an important target for cancer treatment and its anticancer effect has been widely studied.4-8 The anticancer effects of survivin has been reported in melanoma cells,9 human colon cancer cells 10, and the human leukemia K562 cell line.11
RNA interference is a basic post-transcriptional gene technique which is widely utilized in cancer research due to its high specificity. RNA interference can down-regulate
molecules
associated
with
tumor
proliferation,
cause
the
microenvironment of tumor change and inhibit hyperplasia and invasion of tumor.12 3
Survivin-targeted small interfering RNA (siRNA) inhibits the internal environment and proliferation of tumor cells by suppressing mRNA expression through interfering protein synthesis.13,14 It has also shown a good efficacy in antiproliferative activity to cancer cells.15,16 Nano-carriers (ranging from 1 to 1000 nm) are particles in submicron size that can overcome most obstacles in therapeutic use of siRNA.17,18 Recently, liposomes have been developed to carry siRNA and have been shown to be effective in vivo.19,20 However, the anti-tumor effects of nanoliposome-mediated survivin by siRNA on human cervical cancer HeLa cells have not been fully studied.
In this study, the antitumor effect of nanoliposome-mediated survivin siRNA was investigated by establishing xenograft mouse models with human cervical cancer HeLa cells in vivo. Furthermore, the pharmacokinetics of survivin siRNA nanoliposomes was also evaluated.
METHODS Preparation of survivin siRNA nanoliposomes The experimental procedures in this study conform to the National Institutes of Health Guide for the Care, and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Chinese PLA General Hospital. siRNA targeting survivin was synthesized by Biomics Biotechnologies Co., Ltd (Beijing,
China),
with
the
sense
sequence
of 4
5’-GCAUCUCUACAUUCAAGAAdTdT-3’
and
antisense
sequence
of
5’-UUCUUGAAUGUAGAGAUGCdTdT-3’. A scrambled sequence was synthesized as a negative control (NC), and was named as si-NC with the sequences as follows: sense strand: 5’- UUC UCC GAA CGU GUC ACGU-3’ and antisense strand: 5’ACG UGA CAC GUU CGG AGAA-3’. siRNA was incorporated into nanoliposomes in ethanol–water solutions by pre-formed vesicles. The liposomes were composed of 1,2-dioleoyl-3dimethrylammonio-propane 1,2-distearoyl-sn-glycero-3-phosphocholine
(Sigma
Aldrich),
(Avanti Polar Lipids, Alablaster, AL,
USA), cholesterol (Wako) and PEG-DMA (Biomics Biotechnologies Co., Ltd, Beijing, China) at the molar ratio of 40:10:40:10. The siRNA nanoliposomes were then synthesized by mixing siRNA (30% ethanol solution) and pre-formed vesicles (30% ethanol solution) in a warm bath (35-40℃) for 10 minutes, and stirred continuously. After encapsulation, concentration and sterilization, the desired final products were achieved. The average diameter of nanoliposome obtained was 70.7±29.077 nm for survivin siRNA and 64.9±26.128 nm for NC-siRNA. HeLa xenograft model construction and treatment 24 male Balb/c nude mice (18-24 g, 7-9 weeks old) were obtained from Shanghai SLAC Laboratory Animal CO. LTD (Shanghai, China). Human cervical carcinoma cell line HeLa (Biomics Biotechnologies Co., Ltd, Beijing, China) was cultured into cell suspension with trypsin and then centrifuged and counted. HeLa cells (4-6×106/0.2 ml) were subcutaneously inoculated in the upper limb of those mice, and 5
was considered the first generation of transplanted tumors. When the tumor size reached about 1.0 cm in diameter, the mice were sacrificed and subcutaneous tumors were collected. Pieces of homogeneous texture tissues (1×1×1 mm3) were cut and transplanted into subaxillary tissue of male Balb/c nude mice.
When the tumors reached a mean diameter of 3-5 mm, the mice were randomly divided into 4 groups (n=6): the SU-IT group received intratumoral injection of survivin siRNA nanoliposome (50 μg/mouse, twice a week); the SU-IV group received intravenous injection of survivin siRNA nanoliposome (3 mg/kg, twice a week); the NC-IT group received intravenous injection of NC siRNA nanoliposome (3 mg/kg, twice a week); and the DOX group received intraperitoneal injection of doxorubicin hydrochloride (2.5 mg/kg, weekly) (Shenzhen Wanle Pharmaceutical Co., Ltd., Shenzhen, China).
Tumor volume and body weight of each mouse was measured every 3 days for 31 days. The mice were sacrificed after the last treatment, and tumors were excised and weighed. The inhibition rate of survivin siRNA nanoliposomes on the tumor growth was calculated by the following formula: [1 - T tumor weight/C tumor weight] ×100%.
6
Histological, immunohistochemical and RT-qPCR analysis Tumor tissues were collected in each group for survivin expression by using Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohischemistry and hematoxylin-eosin (HE) staining analysis. Tumor tissues were fixed in 4% paraformaldehyde at 4 °C for at least 24 hours and consequently processed for paraffin embedding. The sliced sections were then stained with hematoxylin-eosin. For immunohistochemical analysis, the sections were incubated with anti-survivin antibody, followed by incubation with a second antibody. The sections were visualized with 3, 3-diaminobenzidine and counterstained with hematoxylin. Total RNA was isolated using RISOTM RNA Isolation Reagent (Biomics, USA), and SensiMixTM One-Step Kit (Quantace, Taunton, MA, USA) was used for cDNA synthesis and subsequent polymerization according to the manufacturer’s instructions. The conditions of real-time polymerase chain reaction were as following: 95 °C for 10 minutes, 95°C for 20 seconds, 58 °C for 30 seconds to anneal, and 72°C for 30 seconds followed by 40 cycles. Specific primers were used to detect survivin (forward primer, 5’-ACG ACC CCA TAG AGG AAC AT-3’; reverse primer: 5’- TCC GCA GTT TCC TCA AAT TC -3’). GAPDH was amplified as an internal control using specific primers (forward primer, 5’-GAA GGT GAA GGT CGG AGT C-3’; reverse primer: 5’- GAA GAT GGT GAT GGG ATT TC-3’).
7
Bio-distribution of survivin siRNA liposomes in vivo In comparison with invivofectamine (Invitrogen, Carlsbad, CA), a commercial transfection agent, Cy5-labeled siRNA (LNP-1), was injected to the caudal vein of mice at a dose of 3mg/kg with diethyl-nitrosamine-induced hepatocarcinogenesis. All mice were sacrificed after 6 hours, after which, the tumors, hearts, livers, lungs, spleens and kidneys were harvested. The distribution of siRNA was measured in tumor and other organs using a Caliper IVIS Lumina II (Xenogen IVIS Lumina; Caliper Life Sciences, Hopkinton, MA). Blood sample collection for pharmacokinetics The specific-pathogen-free (SPF) male mice (18-25 g, 8 weeks old) were obtained from laboratory animal center of Nantong University and were randomly divided into 3 groups (n=8). Survivin siRNA nanoliposomes at the dosage of 1, 3 and 6 mg/kg was injected into mice from Group A to C via the caudal vein, respectively. The time was recorded as “0 minutes” immediately after the injection. Blood samples (0.1ml each) in each group were collected from the eye socket vein at time points: 5 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 6 hours, 7 hours, 24 hours, and 30 hours. Blood samples were centrifuged for 20 minutes at 4500 r/m and stored at -80 °C. The plasma concentrations of survivin siRNA nanoliposomes were measured after single intravenous injection in the 3 groups. Non compartmental model (WinNonLin5.2.1) was utilized to calculate the pharmacokinetic parameters (Phrarsighrt Inc). 8
Calibration curve construction A series of concentrations (2.0E+07, 2.0E+06, 2.0E+05, 2.0E+04 and 2.0E+03 pg/ml) of standard working solution were prepared. Each standard working solution (10 μl) was taken and then added into 190 μl tris buffered saline solution (contained 10 μl blank plasma). The final concentrations of the calibration were 1.0E+06, 1.0E+05, 1.0E+04, 1.0E+03 and 1.0E+02 pg/ml. RNA extraction and RT-qPCR RT-qPCR was used to detect the concentration of survivin siRNA nanoliposomes in blood plasma. Blood plasma (10 μl) was added into 190 μl 0.25% Triton X-100 in phosphate buffer solution at the temperature of 95 °C in water bath for 10 minutes. Total RNA was extracted using Trizol reagent (GIBCO Company) according to the manufacturer’s instructions. Complementary DNA synthesis and subsequent polymerization was performed as a 1-step procedure using the SensiMixTM One-Step Kit (Quantace, Taunton, MA, USA). Real-time PCR was performed using RochreLigthrcycler 480 and qRT-PCR kit (Shanghai Generay Biotech Co., Ltd). The primers used were summarized as follows: forward primer, 5’-GCA UCU CUA CAU UCA AGAA-3’; reverse primer, 5’-UUC UUG AAU GUA GAG AUGC-3’. As an internal control, GAPDH was amplified with primers (forward primer: 5'-GTC GGT TGT GTA ACG CGA TGTT-3'; reverse primer: 5'-ACT CCA ACG ACT GTA CTC CAGC-3').
9
Statistical analysis All experiments were performed at least in triplicate and data were expressed as the mean ± SD. All statistical analysis was performed by using SPSS, version 17.0 (SPSS Inc. Chicago, IL). One-way analysis of variance (ANOVA) was used to compare the significance between groups. Statistical significance was determined at p<0.05 level.
RESULTS Survivin siRNA nanoliposome inhibited the growth of HeLa xenograft The inhibitory effect of survivin siRNA nanoliposomes on the volume of tumor was investigated in HeLa xenograft model, as shown in Fig.1. Compared to the NC-IV group, the tumor volume in SU-IV group was significantly decreased in 24 days and 28 days (P<0.05). From the seventh day of study, the tumor volume of DOX group was significantly reduced (P<0.05) compared with the results in NC-IV group. The weights of xenograft nude mice are shown in Table 1. There was no significant difference in the body weight among the groups. However, 1 mouse in the DOX group died during the course of the study. In addition, the average body weight in the DOX group was significantly lower than those in other groups, indicating that the DOX treatment may be more toxic. The tumor inhibition rate in the DOX group and SU-IV group were 51.81% and 30.12%, respectively. The samples of tumor tissue were collected for survivin expression analysis. As shown in Fig. 2, the expression of survivin was mainly located in the endochylema 10
and the expression level was significantly reduced after survivin siRNA nanoliposomal treatment. On the contrary, higher survivin staining was found in the NC –IV group. The levels of survivin mRNA in tumor tissues were detected by RT-qPCR (Fig.3). We found that the levels of survivin mRNA were significantly reduced after survivin siRNA treatment, both intratumorally and intravenously, compared with the NC group which was treated with nanolipsomal-si-NC complex. The levels of survivin were slightly decreased after DOX treatment without significant difference. Biodistribution of LNPs in vivo Cy5 labeled LNPs or Invivofectmine were injected into mice and examined by In Vivo Imaging Systems. Compared with Invivofectmine, accumulation of LNP-1 in tumor was significant higher than those in other organs, and also nonspecific accumulation in lung was significantly reduced (Fig.4). Pharmacokinetics of survivin siRNA liposomes In the pharmacokinetic experiments, the calibration curve of survivin siRNA nanoliposome in plasma showed a good linear relationship in the range of 100~1,000,000 pg/ml (for plasma), the correlation coefficients were ≥0.9883 (Fig. 5). Concentration-time curves in group A ~ C were shown in Fig.6. The peak concentrations at 15 minutes in group A, B and C were1, 042,538.00, 6,837,099.54 and 14,631,333.15 pg/ml, respectively. The plasma concentrations were found to be stable at the time point of 3 hours.
The plasma concentration was significantly 11
decreased after 24 hours (22.04, 44.18 and 258.36 pg/ml, respectively) (Table 2). The pharmacokinetic parameters in each group are presented in Table 3. DISCUSSION Survivin, acting as a key suppressor of apoptosis, has been reported to be overexpressed in various cancer cells. It is related to prognosis, ionizing radiation and resistance to anti-tumor agents. Currently, siRNA targeted therapy has been widely studied in tumor treatment due to the specific and potent silencing of targeted genes. This therapeutic technique has been reported to induce apoptosis, block proliferation, and even suppress tumor formation and growth.21,22 siRNA knockdown of survivin inhibited cancer cell proliferation, enhanced apoptotic susceptibility, and decreased tumorigenicity in human xenografts.23 Despite the potential therapeutic advantages, effective delivery of siRNA to tumor cells remains a major hurdle for RNA-based cancer therapy, and the success of gene therapy is highly dependent on the delivery carrier.24,25
Besides, the major limitations of application of survivin siRNA in cancer
therapy are the instability and short half-life time. It has been reported that the half-life of siRNA in serum was only about 15 minutes.26 Therefore, an effective delivery carrier could avoid enzymatic degradation, and also could promote cellular uptake. Liposome has been applied for siRNA delivery and has shown a good effectiveness. In this study, an effective siRNA sequence targeting survivin was chosen and nanoliposomes were cooperated as a carrier to deliver our survivin-targeted siRNA. 12
Additionally, several independent studies have shown survivin siRNA can be introduced into the body by this delivery technique. The injection strategy was considered to be a key factor in the clinical cancer gene therapy. The study of Zhang et al. 23 (2010) reported that injection of survivin in local region of tumor can monitor tumor growth and survival . In our study, both the intratumoral and intravenous injections were performed in xenografts models. The results indicated that tumor weight and tumor inhibition rate were significantly different between the mice in the SU-IT and SU-IV group. Compared with the SU-IT group, the results in the SU-IV group showed a relatively high tumor inhibition rate. Besides, the treatment-related toxicity was lower in the SU-IV group. Therefore, intravenous systemic administration of survivin-targeted siRNA delivered by nanoliposome was safe and effective. Apoptosis is the precondition of cell death induced by different types of anticancer agents. Studies have revealed that survivin knockdown can induce apoptosis in tumor cells through genetic deletion, anti-sense oligonucleotides, and dominant negative inhibitors.27 . BALB/c mice were chosen to avoid the confounding factors that influencing drug plasma concentration, such as gender, age, weight, and so on. Our results showed that the peak plasma concentration was reached at 15 minutes, and the peak concentration was consistent with dose concentration. Plasma concentration in each group decreased abnormally after 30 minutes, the reason we speculated is frequent blood sampling, 13
which led to concentration dilution or the unstable drug plasma concentration in initial stage of administration. Based on the outcomes in our study, the half-life of survivin siRNA nanoliposome in each group ranged from 2 to 24 hours. The plasma concentration was extremely low at time point of 24 hours. Survivin siRNA nanoliposome seems to possess a stable and fast onset time, and can be cleared from the body within 24 hours. The residence time in vivo was safe. The bioavailability was great due to their higher area under the curve and was consistent with other studies. 28,29
The plasma concentration of survivin siRNA nanoliposome was in a stable range
within 7 hours after drug delivering. Trace amount of survivin siRNA nanoliposome could be detected around 48 hours after injection, which was suggestive to the synergies with other agents.
CONCLUSION A nanoliposome-based survivin siRNA delivery system was constructed and was demonstrated to be efficient in survivin siRNA delivery in vivo study. Survivin down-regulation could inhibit tumor growth in HeLa xenograft models, indicating that survivin suppression by siRNA may contribute to tumor inhibition through both the proliferation inhibition and apoptosis promotion effects. Furthermore, our findings reveal the preliminary pharmacokinetic characteristics for anti-survivin siRNA nanoliposomes and serve as a fundamental role for further studies on its applicability for cancer therapy in vivo.
14
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Manning BD. Challenges and opportunities in defining the essential cancer kinome. Science signaling. 2009;2(63):pe15-pe15.
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Li Q, Lozano G. Molecular pathways: targeting Mdm2 and Mdm4 in cancer therapy. Clinical Cancer Research. 2013;19(1):34-41.
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Ozpolat B, Sood AK, Lopez-Berestein G. Nanomedicine based approaches for the delivery of siRNA in cancer. J Intern Med. Jan 2010;267(1):44-53.
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Zhou J, Shum KT, Burnett JC, Rossi JJ. Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges. Pharmaceuticals (Basel). 2013;6(1):85-107.
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Wang Y, Xu Z, Guo S, et al. Intravenous delivery of siRNA targeting CD47 effectively inhibits melanoma tumor growth and lung metastasis. Mol Ther. Oct 2013;21(10):1919-1929.
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Table legend: Table 1 Inhibitory effect of survivin siRNA nanoliposome on the growth of tumor in xenograft nude mice with human cervical cancer HeLa cells. Table 2 Average concentration of survivin siRNA nanoliposome in plasma after Intravenous administration (pg/ml) at different time points in three groups. Table 3 Pharmacokinetic parameters for survivin siRNA nanoliposome of each group after single dose.
Figure Legend: Fig.1 The antitumor effect of survivin-targeted small interfering RNA nanoliposomes on the volume of tumor by different injection methods in HeLa xenografts models. Compared to the NC-IV group (intravenous injection), the SU-IV group (intravenous injection) in 24 days and 28 days P<0.05, the DOX group from the seventh day P<0.05; Fig.2 Tumor samples were harvested for histological analysis (HE staining), and immunohistochemical analysis of survivin expression following survivin-targeted small interfering RNA nanoliposomes treatment.
19
Fig.3 Reverse transcription-quantitative polymerase chain reaction analysis of survivin expression following survivin-targeted small interfering RNA nanoliposomes treatment. Fig.4 Biodistribution of Cy5-labeled survivin-targeted small interfering RNA nanoliposomes formulated in LNPs (LNP-1). The biodistribution of survivin-targeted small interfering RNA nanoliposomes in the tumor and other important organs (such as hearts, livers, and lungs) using invivofectamine and LNP-1. Fig.5 Calibration curve of survivin siRNA nanoliposomes in plasma. Fig.6 Concentration-time curve of three groups (low, medium and high dose of survivin-targeted small interfering RNA nanoliposomes injection group) after single intravenous injection in different time points.
Table 1 Inhibitory effect of survivin siRNA nanoliposome on the growth of human cervical cancer HeLa xenograft nude mice Number of Mice Group
Body Weight (g)
Pre-treat
Post-trea
pre-treatm
post-treat
ment
tment
ent
ment
Inhibi Tumor
tion
Weight (g)
Rate (%)
NC-IV
6
6
22.2±0.75
22.6±2.40
0.83±0.12
/
DOX
6
5
22.1±1.83
21.6±1.24
0.40±0.46
51.81
SU-IT
6
6
22.3±1.29
23.6±0.58
1.08±0.36
N/A
SU-IV
6
6
21.2±1.40
21.7±2.38
0.58±0.27
30.12
20
Table 2 Average concentration of survivin siRNA nanoliposome in plasma after intravenous administration (pg/ml) at different time points in three groups. Time
Group A
Group B
Group C
(low dose:1 mg/kg)
(moderate dose:3 mg/kg)
(high dose: 6mg/kg)
5 minutes
336601.5
5674548.81
4704490.01
15 minutes
1042538.00
6837099.54
14631333.15
30 minutes*
56201.62*
645714.88*
568540.18*
45 minutes
821309.3
3920663.95
10313715.46
1 hours
18392.55
81355.16
108388.11
1.5 hours
98452.32
380561.31
1014007.80
2 hours
18694.05
465844.66
389596.55
3 hours
3674.25
4765.05
86880.87
4 hours
999.59
7259.11
64239.90
6 hours
2923.55
23813.45
7439.86
7 hours
17814.76
164815.22
359524.76
24 hours
22.04
44.18
258.36
30 hours
3.05
1.12
11.04
48 hours
16.00
5.51
12.96
Table 3 Pharmacokinetic parameters for survivin siRNA nanoliposome of each group after single dose Group A
Group B
Group C
(low dose:1 mg/kg)
(moderate dose: 3 mg/kg)
(high dose: 6 mg/kg)
t1/2 (hour)
3.60
2.64
2.80
Tmax (hour)
0.25
0.25
0.25
Cmax (pg/ml)
1042537.60
6837099.54
14631333.15
Parameters
21
AUC (h∙pg/ml)
952190.88
6800687.79
13803680.96
Vz (ml/kg)
5454.76
1682.54
1757.33
CL (ml/h/kg)
1050.12
441.13
434.67
MRT (hour)
1.70
1.97
2.10
t1/2, half-life time; Tmax, time when maximum concentration arrived; Cmax, maximum concentration; AUC, area under curve; Vz, apparent volume of distribution; CL, clearance; MRT, mean residence time.
Fig. 1
22
Fig. 2
23
Fig. 3
Fig. 4
24
Fig. 5
Fig. 6
25