Cell Type-Specific Avalanche™ Transfection Reagents were developed in three major steps:

1. Design and produce candidate ingredients and formulations based on modern transfection theories and combinatorial chemistry.

2. Empirical screening/confirmation

3. Repeat from step 1 to step 2 to get the final optimized formula for each specific cell type or cell line.

Step 1: New ingredient development

• The R&D department is composed of a group of scientists who specialize in cell biology, molecular biology, lipid chemistry, biophysics, cell dynamics, cell metabolisms, synthetic chemistry, as well as combinatorial chemistry.

The technical specifications of all of the major commercial transfection reagents, their ingredients, and their pros and cons are understood: o Different types of cationic lipids (different number and length of hydrophobic chains, modification status of the hydrophobic chain, such as cholesterol, fluorinated, ester bond, and biodegradability, etc.);

• Cationic polymer (different backbones, number of nitrogen content, different length, branch/linear situation, etc.);
• Peptides (cationic peptides, receptor ligand sequences, etc);
• Dendrimers (activated and non-activated)
• Calcium phosphate and other cationic molecules

•Based on the above information and knowledge, and by using combinatorial chemistry and synthetic chemistry, thousands of new ingredients were developed that are believed to have the potential to possess higher transfection efficiency and lower toxicity as compared to the ingredients used in other commercial products.

Step 2: Empirical screening/confirmation

• Using high-throughput screening technology, those thousands of new ingredients were evaluated, as well as compounds in some pre-existed compound libraries, and chose the ones that have higher transfection efficiency and lower toxicity in several commonly used cell lines. Step 3: Make new hybrid ingredients and formulations, and evaluate again.
• Many of the above ingredients work in totally different mechanisms during the process of transfection. Like many ingredients in some commercial transfection reagents, each of the above ingredients has its advantages and disadvantages. The scientists were convinced that some of them might synergistically work together to promote transfection. Because of this, comprehensive strategies to chemically link different ingredients together were developed, thus creating a series of new hybrid ingredients that might keep the advantages of each of the constituent ingredients and hide the disadvantages, resulting in better performance than any of the individual constituent ingredients. The hybrid ingredients were evaluated again using our high-throughput screening technology, and the more effective hybrid ingredients were identified.
• Once the effective ingredients were identified, and the hybrid ingredients were ready, the ideal formulations to facilitate the transfection process were created. Similar to the chemical linking of different ingredients, mixing different ingredients of different mechanisms together in different amounts and ratios is also an effective way to achieve the synergistic effect of different ingredients on transfection. Approximately 500 candidate formulations were made from the above candidate ingredients and hybrid ingredients.
•  Next, the candidate formulations were screened on several representative cell types, including, but not limited to endothelial, fibroblast, and epithelial cells, and 172 formulations that have better transfection efficiencies on all or part of the representative cell types were then identified.
• High throughput test screening of those 172 final candidate formulas was performed on each major type of primary cells and cell lines in comparison with 4 of the most popular commercial transfection reagents, in order to identify the best one for each specific cell type or cell line (data graphs from this screening are shown in the data section of each respective cell type/cell line specific transfection reagent). The one that showed the optimal balance of potency & low cytotoxicity among those candidate formulas after flow cytometry analysis on the percentage of 7AAD positive cells was later named as the respective cell type Avalanche® Transfection Reagent.
• In the final step, the protocols were optimized for each cell type specific transfection reagents, including the amount of each formula and the ratio of the formula/nucleic acid, for each respective type of cells in order to maximize efficiency/viability, as well as for conveniences.
• Now, with the combined efforts of the scientists at EZ Biosystems, a comprehensive series of Cell type/Cell line Specific Avalanche® Transfection Reagents for more than 150 cell lines and primary cells is available. The number is still growing. The goal is to ensure that scientists of life sciences always have access to the right transfection reagents with the highest possible transfection efficiency and the lowest toxicity for any of their primary cells or cell lines.