Figure 7. Cytokine analysis of samples from mice injected with Invivofectamine 3.0 Reagent. Invivofectamine 3.0 Reagent complexed with Ambion in vivo siRNA targeting FVII was injected at a dosage of 0.25 mg/kg (with and without the addition of dexamethasone (Dex)). Following injection, blood samples were collected at 2, 6, 24, and 48 hr, and cytokine levels were measured using a multiplexed bead-based immunoassay kit. As a control, samples from individuals not treated with the reagent were subjected to the same cytokine panel assay. Each bar comprises the data from three replicates.
A breakthrough in vivo transfection reagent
As a breakthrough reagent for in vivo RNAi delivery, Invivofectamine 3.0 Reagent improves transfection performance, including up to 85% knockdown achieved using microgram levels of siRNA.
- Easy to use—siRNA complexes are ready to deliver in just a few steps
- Effective, targeted knockdown—up to 85% knockdown observed with targets tested
- Use less siRNA sample—up to 90% less siRNA than required with previous reagent for effective knockdown
- Sustained knockdown—prolonged knockdown observed following a single injection
- Low toxicity—complexes exhibit extremely low in vivo toxicity
Ordering information for Invivofectamine 3.0 Reagent
Lipid nanoparticles for in vivo delivery
Vivofectamine Delivery Solutions are highly advanced lipid nanoparticles (LNPs) that have been selected from a large, diverse lipid library and extensively screened for performance, safety and efficacy. Designed especially for selectively and efficiently delivering nucleic acid payloads to a variety of targets, these products and services are available in formats ranging from individual ionizable lipids to pre-mixed lipid compositions and as a formulation service.
Research article
Liver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis
Prepare RNAi-Invivofectamine complexes easily for siRNA in vivo delivery
Creating complexes of Invivofectamine 3.0 Reagent and RNAi duplexes for in vivo delivery is easy: simply mix, incubate (30 min), dilute, and inject (Figure 1).
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Figure 1. An injection-ready RNAi/Invivofectamine 3.0 Reagent complex can be prepared quickly and in just a few steps. Step 1 illustrates the combination of the complexation buffer and siRNA duplex. Step 2 shows the mixing of the Invivofectamine 3.0 Reagent and diluted siRNA. Step 3 shows the shaking of the complex. Step 4 illustrates incubation at 50°C for 30 minutes. Step 5 shows the addition of the now ready complex to the lateral caudal vein, on top of the dorsal vein.
Effective knockdown of target mRNA with Invivofectamine 3.0 Reagent
Complexes of Invivofectamine 3.0 Reagent and Ambion in vivo siRNA targeting Factor VII or Stealth RNAi PPIB have been successfully delivered by mouse tail vein injection to liver tissue, as evidenced by effective knockdown of Factor VII and PPIB at the mRNA level (Figure 2).
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Figure 2. Use of Invivofectamine 3.0 transfection reagent demonstrates targeted gene knockdown in the liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with siRNA targeting mRNA for Factor VII (FVII) or PPIB, injected at doses of 1 mg per kilogram mouse body weight (mg/kg), achieved as much as 85% knockdown of target mRNA levels (knockdown assessed via TaqMan assay).
In vivo lipofectamine reagent requires less siRNA to achieve effective knockdown
Complexes of Invivofectamine 3.0 Reagent and siRNA in a range of amounts were introduced via tail vein injection. FVII protein levels in the serum were measured using a chromogenic assay 24 hours after injection (Figure 3). The amount of knockdown is correlated with the amount of siRNA in the complex of Invivofectamine 3.0 Reagent and siRNA, which was introduced via tail vein injection. The ED50 of Invivofectamine 3.0 Reagent is 0.1 mg/kg, compared to previous levels of 1.0 mg/kg.
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Figure 3. Invivofectamine 3.0 Reagent and siRNA targeting FVII produce dose-response knockdown in liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with Ambion in vivo siRNA targeting FVII was injected at doses ranging from 0.02 to 2 mg/kg. Blood serum was isolated and assayed for FVII protein levels (Biophen® chromogenic assay).
Sustained knockdown after a single injection of Invivofectamine 3.0 Reagent/siRNA complex
A single injection of an Invivofectamine 3.0 Reagent/siRNA complex results in significant knockdown at day 1 and for up to 3 weeks (Figure 4). Higher amounts of siRNA in the injected complexes resulted in longer-lasting knockdown over the range tested.
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Figure 4. Study of dosage effect using three concentrations of siRNA targeting Factor VII (FVII). siRNA molecules were complexed with Invivofectamine 3.0 Reagent and injected at doses of 1, 0.5, and 0.25 mg/kg. Serum was collected on days 2, 5, 9, 16, 23, and 30, and serum was analyzed for FVII protein knockdown by a chromogenic assay.
Gene expression knockdown in vivo delivery using Invivofectamine 3.0 Reagent results in expected phenotypic changes
Apolipoprotein B (ApoB) is the primary apolipoprotein of low-density lipoprotein (LDL), which carries cholesterol to tissues. Studies monitoring reduced expression of ApoB have shown decreased levels of cholesterol and triglyceride. With high-efficiency knockdown, Invivofectamine 3.0 transfection reagent complexed with siRNA targeting ApoB demonstrates specific reduction in levels of both cholesterol and triglyceride (Figure 5). The specificity and effectiveness of Invivofectamine 3.0 Reagent helps give researchers confidence that the observed phenotypes are attributed to specific target knockdown and not to nonspecific toxicity.
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Figure 5. Invivofectamine 3.0 Reagent and siRNA targeting ApoB achieve knockdown in liver after a single intravenous injection. Invivofectamine 3.0 Reagent complexed with ApoB siRNA was injected at doses of 1.5, 0.5, and 0.25 mg/kg. Delivery of the complex containing the dosage of 1.5 mg/kg siRNA resulted in >80% knockdown in mRNA and ~70% knockdown in protein levels. Blood serum was isolated and assayed for cholesterol and LDL content, and the results show that the knockdown of ApoB protein resulted in a significant reduction in cholesterol and LDL.
Low in vivo toxicity of Invivofectamine 3.0 Reagent
Blood chemistry analysis and cytokine measurements were taken at multiple points of time, following the injection of the reagent, in order to evaluate in vivo toxicity of the Invivofectamine 3.0 Reagent. (Figures 6 and 7). The results show that the levels of the assayed biomarkers in individuals transfected using Invivofectamine 3.0 Reagent were not significantly different from those that did not receive the reagent.
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Figure 6. Blood chemistry analysis of samples from mice injected with Invivofectamine 3.0 Reagent. Invivofectamine 3.0 Reagent complexed with Ambion in vivo siRNA targeting FVII was injected into mice at doses of 1 mg/kg [1], 3 mg/kg [3], or untreated [U]. Blood samples were collected at 2, 24, and 48 hr and were evaluated using clinical chemistry assays for several biomarkers (Antech): glucose (GLU, in mg/dL), alkaline phosphatase (ALP, in U/L), alanine aminotransferase (ALT, in U/L), aspartate aminotransferase (AST, in U/L), total bilirubin (TBIL, in mg/dL), cholesterol (CHOL, in mg/dL), and triglycerides (TRIG, in mg/dL). Each bar comprises the data from four replicates.
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Stability of Invivofectamine 3.0 Reagent after thawing
The recommended long-term storage temperature for Invivofectamine 3.0 Reagent is –20°C, but Invivofectamine 3.0 Reagent can be stored at 4°C for at least 14 days after thawing (Figure 8), allowing you to extend your experiment over several days without needing to refreeze the reagent. If desired, however, the reagent can be frozen and thawed up to 4 times without loss of performance (data not shown).
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Figure 8. Invivofectamine 3.0 Reagent stored at –20°C can be thawed and kept at 4°C for up to 14 days. Invivofectamine 3.0 Reagent was thawed and complexed with FVII siRNA and injected intravenously at 0.125 and 0.5 mg/kg doses. Aliquots of the same thawed reagent were used to create complexes for subsequent injections at 6 and 14 days after the first injection. After each siRNA injection, blood was collected and FVII protein levels evaluated (Biophen® chromogenic assay).
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