MICROFLUIDIC DEVICE FOR FORMING MONODISPERSE LIPOPLEXES

• US 20070264320A1
• Filed: 05/08/2007
• Published: 11/15/2007
• Est. Priority Date: 05/09/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Lee, Abraham P., Hsieh, Albert Tsung-Hsi
• Granted Patent: US 7,811,603 B2
• Time in Patent Office: Days
• US Class Current: 424/450
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0436 acoustic forces, e.g. surface acoustic waves [SAW]
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 3/0241 Drop counters; Drop formers making arrays for combinatorial libraries B01J19/0046; automation of dispensing for analysis G01N35/10
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08

Abstract

A method for forming monodisperse lipoplex assemblies includes providing a microfluidic device having a main microfluidic channel coupled to first and second reactant inlet channels. The first inlet channel is used to deliver a cationic lipid to the main channel while the second channel is used to deliver a nucleic acid. A droplet generation zone is provided in the main channel at the intersection of first and second carrier channels that contain a hydrophobic fluid. The cationic lipid, nucleic acid, and the hydrophobic fluid are then pumped through the device. In the droplet generation zone, shear force from the hydrophobic fluid pinches off droplets. The cationic lipid and nucleic acid are mixed in the generated droplets as the droplets move within a mixing region. A plurality of splitting channels may be coupled to the outlet of the device to produce smaller, monodisperse droplets having picoliter volumes.

Claims

1. A method of forming monodisperse lipoplexes comprising:

providing a microfluidic device having a main channel including a droplet generation zone, the droplet generation zone comprising an intersection between the main channel and first and second carrier channels configured to receive a hydrophobic fluid, the microfluidic device further comprising first and second inlet channels coupled to the main channel at a region upstream from the droplet generation zone;

providing a first reactant in fluid communication with the first inlet channel, the first reactant comprising a cationic lipid;

providing a second reactant in fluid communication with the second inlet channel, the second reactant comprising a nucleic acid;

providing a hydrophobic fluid in fluid communication with the first and second carrier channels; and

flowing the first and second reactants and the hydrophobic fluid into the droplet generation zone so as to generate a plurality of lipoplex droplets.

2. The method of claim 1, further comprising mixing the lipoplex droplets by passing the same through a mixing region downstream of the droplet generation zone.

3. The method of claim 1, wherein the nucleic acid comprises DNA, RNA, or shRNA.

4. The method of claim 1, wherein the hydrophobic fluid comprises oil.

5. The method of claim 1, further comprising splitting the generated lipoplex droplets into a plurality of smaller sized lipoplex droplets.

6. The method of claim 5, wherein the splitting is accomplished using a plurality of branch channels.

7. The method of claim 5, wherein the smaller sized lipoplex droplets have volumes less than about 50 picoliters.

8. The method of claim 6, further comprising delivering the smaller sized lipoplex droplets into a common incubation chamber coupled to each branch channel.

9. The method of claim 6, further comprising splitting the already-split lipoplex droplets into a plurality of smaller sized lipoplex droplets.

10. The method of claim 9, wherein the splitting is accomplished using a plurality of secondary branch channels.

11. A microfluidic device for forming monodisperse lipoplex assemblies comprising:

a substrate including a main microfluidic channel coupled to first and second reactant inlet channels, the main microfluidic channel including droplet generation region, the droplet generation zone comprising an intersection between the main microfluidic channel and first and second carrier channels configured to receive a hydrophobic fluid;

a mixing region disposed downstream of the droplet generation zone; and

a plurality of branch channels coupled to an output of the mixing region.

12. The device of claim 11, wherein substrate comprises PDMS covered with glass.

13. The device of claim 11, further comprising a plurality of pumps operatively coupled to the first and second reactant inlet channels and the first and second carrier channels.

14. The device of claim 11, further comprising an incubation region formed in the substrate, the incubation region being coupled to the output of the plurality of branch channels.

15. The device of claim 11, wherein the plurality of branch channels are configured to split into a plurality of smaller branch channels.

16. The device of claim 11, wherein one of the first and second reactant inlet channels are coupled to a source of nucleic acid and the other of the first and second reactant inlet channels is coupled to a cationic lipid.

17. The device of claim 11, wherein the mixing region comprises a tortuous microfluidic channel.

18. The device of claim 11, wherein the branch channels have widths less than about 50 micrometers.

19. A system for generating monodisperse lipoplexes comprising:

a microfluidic device including a main microfluidic channel coupled to first and second reactant inlet channels, the first reactant inlet channel being operatively connected to a source of cationic lipid, the second reactant inlet channel being operatively connected to a source of nucleic acid, the main microfluidic channel including droplet generation region comprising an intersection between the main microfluidic channel and first and second carrier channels configured to receive a hydrophobic fluid;

a mixing region disposed downstream of the droplet generation zone;

pumping means configured for flowing the cationic lipid and nucleic acid into the first and second reactant inlets;

secondary pumping means configured for flowing the hydrophobic fluid into the first and second carrier channels; and

a plurality of branch channels coupled to an output of the mixing region.

20. The system of claim 19, further comprising a chamber operatively coupled to the plurality of branch channels.