- How do I concentrate the formulation?
If necessary, 1 mL of DIVERSA formulations can be concentrated by using a SpeedVac or Rotavap in mild conditions (avoid surpassing 35 °C or drying out the samples). Samples can be concentrated up to 4-fold its original volume (i.e., to a final volume 250 µL).
- Can I filter the formulation?
Yes, if necessary, DIVERSA formulations can be filtered using 0.22 µm filters.
- Can I use buffers other than milliQ water?
Yes, please check Table 1 of your DIVERSA REAGENT protocol for other recommended buffers.
- How can I measure the size of the final formulation?
Diameter size can be measured by Dynamic Light Scattering (DLS) analysis adding to the cuvette 10 µL of DIVERSA formulation with 90 µL of milliQ water.
- What is the concentration of the fluorophore in the DIVERSA FluoGreen reagents?
The concentration of the fluorophore is 4 µg/mL in the final DIVERSA FluoGreen reagents.
- Is the fluorochrome pH sensitive?
No, it is not. It can be used at a wide range of pH.
- Do you only offer the DIVERSA reagents labelled with a green fluorochrome?
At the moment, yes. Alternatively, you can contact DIVERSA for a customized formulation. We can add alternative fluorochromes as for example Cy5, Cy7, and other fluorescent substances.
- How stable is the signal from the FluoGreen reagents?
The green signal of the green fluorochrome is stable for up to 1 year at -20 ºC according to the manufacturer. We have tracked it in live cells for up to 8 days.
- Does the fluorescence of the formulation come from molecules that have some biological application?
No, they are molecules that are covalently linked to the lipids and emit fluorescence, they do not have intrinsic activity.
- What should I do if my drug is not soluble in EtOH?
You can use any solvent listed in Table 1 of your DIVERSA REAGENT protocol. You can also sonicate the vial containing the drug prior to addition to the DIVERSA REAGENT vial.
- What is the role of the modification of the protein?
The modification of the proteins facilitates their association to DIVERSA REAGENT formulation by following easy and mild procedures that do not compromise its structure and activity. DIV031 does not include cationic components.
- Can the protein modification alter its biological function?
The proposed modifications do not alter the native protein biological function. If in doubt, please contact DIVERSA for advice.
- Are there additional modifications that can be suitable to my protein?
In case your molecule requires a different modification, please contact DIVERSA for advice.
- How can I get the modified proteins?
All that you need for the protein modification step is provided in the DIVERSA REAGENT.
- What if I need to work with higher peptide/protein concentrations than the ones provided in Table 2 of my DIVERSA REAGENT protocol?
You can concentrate the formulation (see question 1 in General), or alternatively, contact DIVERSA for advice depending on your specific peptide/protein.
- Can I use DIVERSA formulations for in vivo studies?
Yes, DIVERSA formulations can be used in vivo. For specific recommendations and a customized and optimized formulation, contact DIVERSA.
- What is the maximum concentration of small drug, peptide and protein that I can encapsulate/associate?
This will depend on the specific properties of each molecule. For small drugs, it can range from 1% to 15% (55 µg – 0,83 mg) with respect to the total amount of lipids (5,5 mg) per vial. We recommend starting with 1%, and then increasing the concentration gradually. For proteins and peptides, it depends on the selected DIVERSA REAGENT and on the specific molecule, but it is possible to load at least 150 ug of protein/peptide per vial.
- I would like to do experiments with fluorescent tracking, but I still do not know how my small drug will associate with the DIVERSA reagent.
We recommend that you first use DIV010 to optimize the encapsulation method and afterwards use that concentration of small drug in DIV010F1.
- Which administration routes have you tried in vivo? We have tested in healthy mice to get an insight, but detailed biodistribution should be done with relevant models and with encapsulated drugs / molecules as this affects the biodistribution. We have tried s.c. (subcutaneous) administration using one or more imaging modalities as we have optimized labeling (PET, SPECT and fluorescent tracking), i.v. (tail vein injection and retroorbital), i.a. (intra-articular), and i.p. (intraperitoneal).
- What is the estimated stability time of the nanoemulsion after its formation?
Once formulated and with the drug/biomolecule loaded/associated, we recommend using the formulation within 24-48 h, according to the provided protocol. The blank nanoemulsion (without molecule) is stable for 60 days at 4 ºC.
- Is it appropriate to evaluate parameters such as pH and viscosity? If yes, what are the appropriate techniques to carry out these assessments?
It is not necessary for first in vitro testing. There is not a big increase in viscosity and the pH is suitable for in vitro testing. It is also possible to use buffers to formulate, as indicated in the DIVERSA REAGENT protocol.
- In order to quantify the percentage of encapsulated compound by HPLC, in your opinion, is it enough to carry out a simple filtration of the nanoemulsion with a 0.45 µm membrane to obtain the percentage of free compound and then calculate the percentage of encapsulation through the subtraction of the total and free concentrations, in relation to the total concentration? Or do you advise another separation method to quantify by HPLC the percentage of free/encapsulated compound?
Filtration through 0,45 µm or 0,22 µm will work to eliminate free precipitated compound. The filtered fraction that has passed through the filter should be analyzed.
- How does the cargo enter the cell?
Based on our experiments so far, we can say that there is a mix of active entrance (endocytosis) and passive (fusion).
- Are the formulations homogeneous in size?
Yes, they are always around 200nm in diameter. You can measure their size after encapsulation/association of your molecule following the procedure in question 4 in General.
- How and where is the delivery of compound carried out?
The nanoemulsiones get internalized by fusion of the membranes and endocytosis and deliver the drug intracellularly, partly by diffusion and partly because they are degraded and metabolized.
- Can you produce the formulations at scale in GMP conditions?
Yes. We are collaborating with two CDMOs with GMP capabilities to produce our DIVERSA REAGENT formulations. We can now produce up to 1L.