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Intracellular delivery of biomolecules mediated by a surface with pores

A cell and pore distribution technology, applied in animal cells, stress-stimulated microbial growth methods, biochemical equipment and methods, etc., can solve problems such as low throughput, modification or damage, and difficulty in implementation

Active Publication Date: 2018-06-08
SQZ BIOTECH CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods suffer from a number of complications, including nonspecific molecule delivery, modification or damage to payload molecules, high cell death, use of materials that may not generally be recognized as safe (GRAS) materials, low throughput, and / or difficult implement
Furthermore, these intracellular delivery methods are ineffective at delivering molecules to sensitive cell types such as primary immune cells and stem cells
Therefore, there is an unmet need for intracellular delivery technologies that are highly effective in delivering a range of molecules to a variety of cell types

Method used

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  • Intracellular delivery of biomolecules mediated by a surface with pores
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  • Intracellular delivery of biomolecules mediated by a surface with pores

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0140] 1. A method for delivering a compound into a cell, the method comprising passing a suspension of cells across a surface containing pores, wherein the pores deform the cell causing disturbance of the cell so that the compound enters the cell , wherein the cell suspension is contacted with the compound.

[0141] 2. The method of embodiment 1, wherein the surface is a membrane.

[0142] 3. The method of embodiment 1, wherein the surface is a filter.

[0143] 4. The method of any one of embodiments 1-3, wherein the surface is a tortuous path surface.

[0144] 5. The method of any one of embodiments 1-4, wherein the surface comprises a material selected from one of the following: polycarbonate, polymer, silicon, glass, metal, nitrocellulose, cellulose acetate, graphite , nylon, polyester, polyethersulfone, PTFE and ceramics.

[0145] 6. The method of any one of embodiments 1-5, wherein the inlet of the hole is wider than, narrower than, or as wide as the hole.

[0146] 7. ...

Embodiment 1

[0479] Example 1: Delivery of dextran particles to HeLa cells

[0480] introduction

[0481] To evaluate filter-mediated delivery of molecules into cells, HeLa cells mixed with fluorescent dextran particles were passed through filters containing pores of defined size, and intracellular particle delivery was evaluated by FACS analysis.

[0482] Materials and methods

[0483] Polycarbonate membrane filter obtained from STERLITECH TM (PCT8013100). These polycarbonate filters are produced by exposing carbonate membranes to charged particles in nuclear reactors to produce pores that are relatively uniform in size but have a random distribution. The resulting pores range in diameter from 0.010 μm to 35 μm. The filter pores are approximately 10 μm thick. Exemplary images of polycarbonate filters and filter pores are shown in Figure 1A and B. Filters with pore sizes of 8 μm, 10 μm, 12 μm, and 14 μm were used in the studies described herein. Additional materials used in the fil...

Embodiment 2

[0495] Example 2: Delivery of dextran particles to human T cells through 5 μm sized filter pores.

[0496] introduction

[0497] To evaluate filter-mediated delivery of molecules into primary immune cells, primary human T cells mixed with fluorescent dextran particles were passed through 5 μm sized filter pores and intracellular particle delivery was determined by FACS analysis. Comparison of filter-mediated delivery of dextran particles to primary human T cells under manual syringe pressure or constant pressure.

[0498] Materials and methods

[0499] Peripheral blood mononuclear cells (PBMC) were first isolated from fresh human blood by Ficoll separation. T cells were then separated from PBMCs by negative selection using magnetic columns. Spin T cells 4x 10 6 cells / mL were suspended in optiMEM medium. Pipette 100 μl of cells to use as a negative control. Suspend the polycarbonate membrane filter in PBS using tweezers to wet the membrane filter. Remove the cover from t...

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Abstract

The present disclosure pertains to methods and devices for delivering a compound into a cell, including passing a cell suspension through a surface containing pores, wherein the pores deform the cellthereby causing a perturbation of the cell such that the compound enters the cell, wherein the cell suspension is contacted with the compound.

Description

[0001] Cross references to related applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 214,820, filed September 4, 2015, and U.S. Provisional Application No. 62 / 331,363, filed May 3, 2016, which are hereby incorporated by reference in their entirety and enter. field of invention [0003] The present disclosure generally relates to methods for delivering a compound into cells by passing a suspension of cells through a surface containing pores. Background of the invention [0004] Intracellular delivery is a central step in the research and development of novel therapeutics. Existing technologies aimed at intracellular delivery of molecules rely on electric fields, nanoparticles or pore-forming chemicals. However, these methods suffer from a number of complications, including nonspecific molecule delivery, modification or damage to payload molecules, high cell death, use of materials that may not generally be recognized as safe (GRAS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M35/04C12N5/07C12N5/071C12N5/0735C12N5/074C12N5/0775C12N5/078C12N5/0781C12N5/0783C12N5/0784C12N5/0786C12N5/0787C12N5/0789C12N5/079C12N5/0793C12N5/09C12N15/11C12N15/113
CPCC12M35/04C12N15/90C12N5/06
Inventor J·B·吉尔伯特K·弗鲁H·伯恩斯坦A·R·沙雷
Owner SQZ BIOTECH CO