LNP for siRNA delivery

In 293T, HepG2, A549, and HeLa cell lines, NP-3 (0.05–1.6 mg/mL; 24 hours) does not lessen cell cytotoxicity; however, DPPC and DMG–PEG loaded nanoparticles do. Furthermore, a maximal transfection efficiency of approximately 76% eGFP positive cells is obtained upon transfection of DPPC/DMG-PEG/(lPEI/DNA) nanoparticles (NP-3) into 293 cells[1].

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Synthesis and Characterization of DPPC/DMG-PEG/(lPEI/DNA) Nanoparticles[1]
The plasmid DNA was diluted in 25 mM HEPES buffer to a final concentration of 200 μg/mL, and the pH was adjusted to 6.5. The lPEI was dissolved in water at a predetermined concentration to obtain lPEI/DNA complexes with final nitrogen to phosphate (N/P) ratios ranging from 6 to 16. DPPC and Chol were weighed and dissolved in ethanol to concentrations of 3 and 1.3 mg/mL, respectively, to form DPPC/Chol solution. DPPC, Chol, and DMG-PEG were weighed and dissolved in ethanol solution at 3, 1.3, and 1 mg/mL, respectively, to form DPPC/DMG-PEG/Chol solution. The preparation processes were performed on a two-step FNC setup (Figure 1). With a 2-inlet confined impinging jet (CIJ) device, lPEI solution and plasmid DNA solution were impinged at a flow rate of 25 mL/min to form lPEI/DNA nanoparticles. The obtained lPEI/DNA nanoparticles were introduced into a 3-inlet jet FNC device through inlets 2 and 3; and the DPPC/Chol or DPPC/DMG-PEG/Chol solution was introduced through inlet 1 at flow rates of 1, 5, 10, 15, 20, 25, or 30 mL/min (same flow rates for all three inlets) to achieve coating of the lPEI/DNA nanocomplex core with the lipids. The products were dialyzed against water overnight to remove the ethanol. To demonstrate the benefits of the FNC method, a traditional fabrication method involving evaporation rotation, hydration, and incubation overnight was also used to coat the lPEI/DNA nanocomplex with DPPC and DMG-PEG. The size distribution of uncoated lPEI/DNA nanoparticles (NP-1), DPPC/(lPEI/DNA)-coated nanoparticles (NP-2) and DPPC/DMG-PEG/(lPEI/DNA)-coated nanoparticles (NP-3) were determined by a Malvern Zetasizer NanoZS90 3 times. Nanoparticle samples were stained by phosphotungstic acid; and the morphology was then examined under a transmission electron microscope. In order to study the structure of selected nanoparticle samples, cryo-TEM was used. Briefly, carbon-coated copper grids were subjected to plasma treatment (N2 glow discharge) for 40 s before sample loading. The imaging grids were prepared using Vitrobot with a chamber that was maintained at 95% humidity. An aliquot of 6 μL of the concentrated sample solution was added dropwise to a treated grid and allowed to sit for 1 min. The grid was then blotted and plunged instantly into a liquid ethane reservoir precooled by liquid nitrogen to produce a thin vitreous ice film on the surface of the grid. Afterward, the grid was transferred to a cryo-holder and kept at liquid nitrogen temperature for no more than 24 h before imaging. The cryo-holder temperature was also maintained at liquid nitrogen temperature during imaging to prevent sublimation of vitrified water. Imaging was performed on an FEI Tecnai 12 Twin TEM operating at 100 kV. All images were taken by a 16-bit 2k × 2k FEI Eagle bottom mount camera or a MegaView III wide-angle camera[1].

To confirm the permeability of NP-3 (oral administration; 150 μg DNA per animal; single dose), luciferin substrate is intraperitoneally administered at 12, 24, and 36 hours after delivery. 12–24 hours after treatment, the NP-3 group still exhibits high levels of luciferase expression in the intestinal, lung, and liver regions. Additionally, from 12 to 24 hours after postoral injection, NP-3 shows a 1.5-fold increase in signal strength compared to the NP-1 or NP-2 group[2].

Cytotoxicity and Transfection Efficiency of DPPC/DMG-PEG/(lPEI/DNA) Nanoparticles in Vitro[1]
All cells used in this study, including 293T (human embryonic kidney cell), A549 (adenocarcinomic human alveolar basal epithelial cell), HepG2 (human hepatocellular cancer cell), and HeLa (human cervical cancer cell) cell lines were purchased from American Type Culture Collection. All cell lines were incubated in DMEM media supplemented with penicillin (100 units/mL), streptomycin (50 units/mL), and fetal bovine serum (10%) at 37 °C in a humidified incubator with a 5% CO2 atmosphere. To assess the toxicity of the nanoparticles, cells were seeded at a density of 2 × 105 cells/mL in a 96-well plate for 24 h and then treated with serial concentrations of nanoparticles for 72 h. After that, MTT dissolved in PBS was added to each well. After 4 h of incubation, the medium in each well was then removed and DMSO was added to each well instead. The absorbance of each well at 570 nm was then measured by a microplate reader. To evaluate the transfection efficiency of the nanoparticles, cells were seeded at a density of 1 × 106 cells/mL in a 24-well plate for 24 h. After that, cells were treated with DPPC/DMG-PEG/(lPEI/DNA) nanoparticles with different N/P ratios (equivalent to 1 μg plasmid DNA per well) and incubated for 24 h. Cells were observed under a fluorescence microscope then harvested and analyzed via flow cytometry to determine the transfection efficiency based on the population of eGFP-positive cells.

In Vivo Fluorescence Imaging of Absorption Study[1]
In order to verify the mucus permeability of DPPC/DMG-PEG/(lPEI/DNA) nanoparticles, in vivo absorption studies in the GI tract were performed as previously described. Briefly, Balb/c mice were fasted for 8 h and then NP-1, NP-2, or NP-3 (plasmid DNA was labeled with SYTOX Orange) were administered via oral gavage. After 2 h, all mice were euthanized and their small intestines were collected and frozen in an OCT medium. The intestine tissues were sliced, stained, and observed under a confocal microscope (Leica SP8).

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Transfection Efficiency of DPPC/DMG-PEG/(lPEI/DNA) Nanoparticles in Vivo[1]
To access the general gene expression profile in vivo preliminarily, we then used a reporter luciferase plasmid to monitor the transfection efficiency. Balb/c mice were randomly divided into three groups for the three nanoparticle groups (NP-1, NP-2, and NP-3). All nanoparticles carried plasmid DNA encoding luciferase. At 8 h before administration, all mice were fed only with water. The nanoparticles were administered via oral gavage to each group at a dose equivalent to 150 μg DNA per mouse. Gene expression in mice was analyzed by whole-body imaging on an IVIS spectrum imaging system at 12, 24, and 36 h postadministration after intraperitoneal (i.p.) injection of luciferin substrate. At 36 h, mice were euthanized to quantify transgene expression levels using tissue homogenates, according to our previous protocol. This process was repeated with a separate set of Balb/c mice, randomly divided into three groups and administered with NP-1, NP-2, and NP-3, this time with plasmid DNA encoding GLP-1. Mice were euthanized at 12 h and 24 postinjection. Organs including the liver, lungs, and small intestine were collected for the GLP-1 expression analysis.

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Therapeutic Efficacy of DPPC/DMG-PEG/(lPEI/DNA) Nanoparticles in Vivo[1]
BKS-Leprem2Cd479/Nju mice, also known as db/db mice, were used as a type II diabetes model. To test the hypoglycemic effect in vivo, db/db mice were randomly divided into a control group (PBS) and three other groups receiving NP-1, NP-2, or NP-3 with GLP-1 plasmid DNA by oral gavage. Before the experiment, the mice were fed only with water for 8 h. Oral gavage of PBS solution for the control group or nanoparticles with a dose equivalent of 150 μg GLP-1 plasmid DNA per mouse for the experimental group was conducted every other day. A total of three doses were given over the course of 6 days. Mice were given free food access for 6 h at 12 h post nanoparticle dosing. The blood glucose level (BGL) was monitored every 2 h in the daytime and every 6 h at nighttime by a blood glucose meter. After 6 day monitoring, all mice were euthanized, with blood samples and organs (including heart, liver, spleen, lungs, kidneys, stomach, small intestines, and pancreas) collected.

[1]. Surface Coating Approach to Overcome Mucosal Entrapment of DNA Nanoparticles for Oral Gene Delivery of Glucagon-like Peptide 1.ACS Appl Mater Interfaces. 2019 Aug 21;11(33):29593-29603.

Oral delivery of nucleic acid therapy is a promising strategy in treating various diseases because of its higher patient compliance and therapeutic efficiency compared to parenteral routes of administration. However, its success has been limited by the low transfection efficiency resulting from nucleic acid entrapment in the mucus layer and epithelial barrier of the gastrointestinal (GI) tract. Herein, we describe an approach to overcome this phenomenon and improve oral DNA delivery in the context of treating type II diabetes (T2D). Linear PEI (lPEI) was used as a carrier to form complexes with plasmid DNA encoding glucagon-like peptide 1 (GLP-1), a common target in T2D treatments. These nanoparticles were then coated with a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol)-2000 (DMG-PEG) to render the nanoparticle surface hydrophilic and electrostatically neutral. The surface-modified lPEI/DNA nanoparticles showed higher diffusivity and transport in the mucus layer of the GI tract and mediated high levels of transfection efficiency in vitro and in vivo. Moreover, these modified nanoparticles demonstrated high levels of GLP-1 expression for more than 24 h in the liver, lungs, and intestine in a T2D murine model after a single dose, as well as controlled blood glucose levels within a normal range for at least 18 h with repeatable therapeutic effects upon multiple dosages. Taken together, this work demonstrates the feasibility of an oral plasmid DNA delivery approach in the treatment of T2D through a facile surface modification to improve the mucus permeability and delivery efficiency of the nanoparticles.[1]

C34H66O6

570.884252071381

570.485

160743-62-4

10257450

Typically exists as white to light yellow solids at room temperature

12.3

0

6

34

40

539

0

HYXWVUFYXOOASK-UHFFFAOYSA-N

InChI=1S/C34H66O6/c1-4-6-8-10-12-14-16-18-20-22-24-26-33(35)39-31-32(30-38-29-28-37-3)40-34(36)27-25-23-21-19-17-15-13-11-9-7-5-2/h32H,4-31H2,1-3H3

[3-(2-methoxyethoxy)-2-tetradecanoyloxypropyl] tetradecanoate

160743-62-4; mPEG-Dimyristoyl glycerol; DMG-PEG 2000; DMG-PEG PEG 2000; mPEG-DMG; DMG-PEG PEG5000; DMG-PEG PEG10000; DMG-PEG PEG30000;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 125 mg/mL (49.49 mM)
Ethanol: 100 mg/mL (39.59 mM)
H2O: 16.67 mg/mL (6.60 mM)

Solubility in Formulation 1: ≥ 10 mg/mL (3.96 mM) (saturation unknown) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (0.99 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (0.99 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (0.99 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (0.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.08 mg/mL (0.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 7: ≥ 2.08 mg/mL (0.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)