FAQs

General

  1. How do I prepare the nanoparticle formulation?
    Preparation is simple and easy, requiring only a pipette. You can download specific protocols from the Products – DIVERSA (diversatechnologies.com) on our website. We are the exclusive provider offering a diverse range of nanoparticle reagents that can be easily prepared without the need for specialized equipment. Videos are also available at our website and at our youtube channel https://www.youtube.com/@diversatechnologies9233.
  2. Are DIVERSA formulations compatible with nucleic acid transfection or viral infection?
    Yes, DIVERSA Delivery Nanoparticles are compatible with transfection as the delivery is not damaging the cells. We also offer specific nanoparticlesfor gene and protein delivery, a real alternative to reagents that are not translatable to in vivo settings and damage the cells, and to viral vectors.
  3. Do DIVERSA formulations contain cholesterol?
    No, DIVERSA Nanoparticles do not contain cholesterol, which is an advantage if you are aiming to deliver your therapeutic molecule for diseases affected by cholesterol imbalances. For example, liposomes typically contain cholesterol and cannot be used in those cases.
  4. 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).
  5. How and where is the delivery of compound carried out?
    The formulation 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.
  6. Can I direct the nanoparticles specifically to the target cells?
    Yes, the nanoparticles forming each DIVERSA Delivery Nanoparticles reagent can be surface decorated with ligands or biomolecules such as antibodies, aptamers, etc. to direct our formulation to the target site. For this customized product contact DIVERSA.
  7. How do I select the best nanoparticle reagent? DIVERSA’s nanoparticles are classified for type of molecule, small molecules, proteins, peptides and nucleic acids, making it simple to decide.
  8. What is the maximum concentration of small drug, peptide, and protein that I can encapsulate/associate?
    This depends 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.
  9. 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), and using ultrafiltration devices as Amicon®.
  10. What is the exact membrane type of the Amicon® filters used in the protein/peptide/mRNA reagents?
    Considering the manufacturer information, the Amicon® filters are composed by a regenerated cellulose membrane where the pore size depends on the molecular weight of the biomolecule.
  11. 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 990 µL of milliQ water.
  12. Can I filter the formulation?
    Yes, if necessary, DIVERSA formulations can be filtered using 0.22 µm filters of PES membrane.
  13. Can I use buffers other than MilliQ water?
    Yes, please check the formulation protocol.
  14. What is the pH range that the DIVERSA Nanoparticles can withstand?
    DIVERSA Nanoparticles maintain their properties irrespective of the pH (as tested in a wide range from pH between 3 and 10).
  15. What if I do not find specific nanoparticle reagents and protocols for the type of active/biomolecule of my interest?
    If you can’t find specific nanoparticle reagents and protocols for your desired active/biomolecule, please contact us or reach out to our support@diversatechnologies.com for guidance.
  16. 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. The formulations can be adapted to specific client’s requirements upon consideration of the dose, route of administration and targeted cells and organs.
  17. Is it possible to target DIVERSA formulation to specific cell subpopulations/organs?
    DIVERSA formulation can be surface-decorated with ligands or biomolecules such as antibodies, aptamers, proteins, etc. for specific targeting. Other formulation approaches can also be explored to improve access to a specific cell subpopulation/organ. Please, contact DIVERSA for advice and customized solution.
  18. Which administration routes have you tried in vivo?
    The formulations can be tailored for different administration routes. They have been tested for s.c. (subcutaneous), i.v. (tail vein injection and retroorbital), i.a. (intra-articular), i.p. (intraperitoneal) and i.m. (intramuscular) administration.
  19. What is the estimated stability time of the formulation after its formation?
    The blank formulation (without molecule) is stable for 60 days at 4 ºC. Please check the specific protocols for each reference to understand specific stabilities.
  20. 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.
  21. Can you produce the formulations at scale in GMP conditions?
    Yes. We are collaborating with CDMOs with GMP capabilities for scaling-up for clinical development.

 

FluoGreen Delivery Nanoparticles

  1. What is the concentration of the fluorophore in DIV000-F1?
    The concentration of the fluorophore is 4 µg/mL in the final DIVERSA FluoGreen Delivery Nanoparticles.
  2. What if I need to increase the concentration of the FluoGreen in the DIVERSA FluoGreen Delivery Nanoparticles?
    You can increase the FluoGreen concentration in the DIVERSA Fluogreen Delivery Nanoparticles by concentrating the formulation. If necessary, DIVERSA Nanoparticle formulation can be concentrated using a SpeedVac or Rotavap under mild conditions (avoid exceeding 35 °C or overdrying the samples), and using ultrafiltration devices as Amicon®. Samples can be concentrated up to 4-fold their original volume, reaching a final volume of 250 µL. For higher concentrations, please contact our Customer Support Department.
  3. In which fluorescence channel would the fluorophore in DIV000-F1 be detected?
    The fluorophore in DIV000-F1 is detected in the green channel corresponding to excitation and emission wavelengths of FITC (fluorescein isothiocyanate).
  4. Can I use GFP-labelled molecules with DIVERSA FluoGreen Delivery Nanoparticles?
    If your molecule is GFP-labelled, it is not compatible with DIVERSA FluoGreen Delivery Nanoparticles due to our formulation is also labelled with a green fluorophore. However, contact DIVERSA to obtain a customized reagent labelled with other fluorochromes.
  5. Is the green fluorochrome the only available fluorochrome so far?
    Currently, we offer the green fluorochrome. However, you can contact DIVERSA for a customized formulation, where we can incorporate alternative fluorochromes such as Cy5, Cy7.5, and other fluorescent dyes.
  6. Is the fluorochrome pH sensitive?
    No, it is not. It can be used at a wide range of pH, from acid to neutral.
  7. How stable is the signal from the DIVERSA FluoGreen Delivery Nanoparticles?
    The green signal of the green fluorochrome is stable for up to 1 year at -20 ºC, according to the manufacturer. Prior to formulation, DIVERSA FluoGreen Delivery Nanoparticles, should be stored at these conditions to preserve the stability. Upon formulation, you can use it for up to 60 days if it is stored at 4 ºC and protected from light. Regarding stability upon addition to cell cultures, we have tracked it in live cells for up to 8 days.
  8. Does the fluorescence of the formulation come from molecules that have any biological effect?
    No, it does not. The fluorescence arises from molecules that are covalently linked to the lipids but have no intrinsic effect. The fluorescence does not interfere with the efficacy of the formulation or the activity of the associated drug/ biomolecule.
  9. Can I use DIVERSA FluoGreen Delivery Nanoparticles for in vivo studies?
    If the purpose is to extract organs/cells and perform analysis by flow cytometry or confocal microscopy, then the answer is yes. If the purpose is in vivo tracking using whole-body imaging systems, it is necessary to use other fluorophores like Cy7.5. DIVERSA can offer this customized reagent. Other available reagents are labeled formulations with Cy5. For specific recommendations and a customized and optimized prototype, contact DIVERSA.

Small Molecule Delivery Nanoparticles

  1. What should I do If my small molecule is not soluble in ethanol?
    If your drug is not soluble in ethanol, you can use any solvent listed in Table 1 of your DIVERSA Small Molecule Reagent protocol. You can also sonicate the vial containing the drug prior to addition to the DIVERSA Reagent vial.
  2. Does the DIVERSA Small Molecule Delivery Nanoparticles work with hydrophilic inorganic molecules?
    No, our DIVERSA Reagent works best with small hydrophobic and amphiphilic molecules. However, contact DIVERSA for a customized reagent for hydrophilic small molecules.
  3. What is the maximum concentration of drug that I can encapsulate/associate?
    This depends 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.
  4. How can I determine the amount of drug that can be encapsulated?
    In DIVERSA Small Molecule Delivery Nanoparticles there are optimization steps, please refer to it and check. If you have specific questions, please refer to our Customer Support Department. DIVERSA also offer a characterization service under request, customize nanoparticle reagents and co-development Services.
  5. Can I encapsulate a small molecule to specifically direct it to target cells?
    Yes, DIVERSA Small Molecule Delivery Nanoparticles can encapsulate small molecules and the nanoparticles can be surface decorated with ligands or biomolecules such as antibodies, proteins, aptamers, etc to direct them to target cells. For this customized product contact with DIVERSA.
  6. 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.

Protein Delivery Nanoparticles

  1. What is the role of the modification of the protein?
    The protein modification facilitates its association to DIVERSA formulation by following easy and mild procedures that do not compromise its structure and activity. DIVERSA Reagent does not include cationic components.
  2. How can I get the modified proteins?
    All you need for the protein modification step is provided in the DIVERSA Protein Delivery Nanoparticles. You do not need specific equipment other that a micropipette and a centrifuge.
  3. Can the protein modification alter its biological function?
    The proposed modifications do not alter the native protein biological function. If you have any doubts, please contact DIVERSA for advice depending on your specific molecule. DIVERSA can provide alternative procedures.
  4. Are there any additional modifications that can be suitable to my protein?
    In case your molecule requires a different modification, please contact DIVERSA for advice. DIVERSA TECHNOLOGIES have expertise in the association of proteins following alternative approaches, although the modification that DIVERSA proposes has competitive advantages in terms of loading, stability, and functionality.
  5. What if I need to work with higher protein concentration than the ones provided in Table 2 of DIVERSA Protein Delivery Nanoparticles protocol?
    You can concentrate the formulation by using a SpeedVac or Rotavap in mild conditions (avoid surpassing 35 °C or drying out the samples), and using ultrafiltration devices as Amicon®. Samples can be concentrated up to 4- to 6-fold its original volume (i.e., to a final volume 250 µL). Alternatively, contact DIVERSA for advice depending on your specific protein.
  6. Can DIVERSA reagents be used to increase the intracellular concentration of a specific protein replacing gene transfection experiments?
    DIVERSA reagents can indeed led to an increased in the intracellular content of the associated protein in much simple and easy way in relation to conventional transfection protocols. The reagents can be added when the cell culture media is replaced, or at specific time points. Please contact DIVERSA’s team for advice.
  7. Can the DIVERSA Reagent divided into aliquots to associate more than one protein?
    Yes, the DIVERSA Reagent can be divided into aliquots to conjugate more than one protein, as per protocol. However, you will need to buy additional Amicon® filters.
  8. How can I verify if the protein is associated with the DIVERSA formulation?
    The efficiency of the protein conjugation to the DIVERSA formulation is high, and the process has been validated with a wide range of proteins. If you still want to have an additional evidence, you perform electrophoresis in polyacrylamide gels (SDS-PAGE), where the percentage of the gel depends on the molecular weight of the protein, and detect if some free protein is migrating.
  9. Can the final DIVERSA Protein Delivery Nanoparticles be purified to remove the excess of not-conjugated protein?
    This is not a necessary step due to the conjugation of the protein to the DIVERSA formulation is very high. However, it you are interested, it is possible to remove the not-conjugated protein using filter systems, where the filter characteristics depend on the molecular weight of the protein since the cut-off must allow the passage of the not-conjugated protein. The purification material is not provided for DIVERSA Protein Delivery Nanoparticles.
  10. Is it better to transfect my protein or deliver it with DIVERSA Reagents?
    DIVERSA Protein Delivery Nanoparticles ensures that the protein can be internalized into the cells in 2 hours, following a very simple protocol.

 

Peptide Delivery Nanoparticles

  1. Is possible to associate cationic peptides in DIVERSA Peptide Delivery Reagent?
    No, the DIVERSA Peptide Delivery Nanoparticles is to associate anionic peptides through covalent interaction with cationic compounds present in the formulation. However, DIVERSA offers a customized reagent to encapsulate cationic peptides.

mRNA Delivery Nanoparticles

  1. Is the mRNA encapsulated in the DIVERSA formulation?
    Yes, the mRNA molecules are internalized into the core of the DIVERSA formulation.
  2. Can I use mRNA encoding any protein?
    Yes, DIVERSA mRNA Delivery Nanoparticles can be loaded with any mRNA encoding for your protein of interest. We recommend using the mRNA at 1 mg/mL.
  3. What is the maximum amount of mRNA to encapsulate?

     

    DIVERSA mRNA Delivery Nanoparticles can encapsulate 5 µg of mRNA per formulation. However, higher amounts of mRNA can be encapsulated, but the lipid proportion must be adapted. For customized prototypes, contact DIVERSA.

     

  4. Can I use another type of polynucleotides instead of mRNA?
    Yes, DIVERSA mRNA Delivery Nanoparticles can be loaded with any polynucleotide such as pDNA, siRNA, aptamers. For specific customized nanoparticle reagents, contact DIVERSA.
  5. Can DIVERSA formulation encapsulate more than one nucleic acid?
    Yes, DIVERSA Nanoparticles can encapsulate together more than one nucleic acid. Consider that the mRNA nanoparticle reagent is design for 5 µg of mRNA (or mixture) per formulation. For higher amounts, please ask for customized prototypes DIVERSA.
  6. Can the surface of the DIVERSA Nanoparticles be decorated with ligands to direct the mRNA specifically to the target cells?
    Yes, DIVERSA Nanoparticles can be decorated with ligands such as aptamers, to direct the mRNA to the target cells. For this customized product, contact DIVERSA.
  7. Can I use mRNA at different concentration?
    It is recommended to use the mRNA at 1 mg/mL for maximum reproducibility. If the concentration of your mRNA is lower, you could concentrate it using an Amicon or by SpeedVac.
  8. What should I do if I cannot concentrate my mRNA at 1 mg/mL?
    If you cannot concentrate your mRNA at 1 mg/mL , add the volume of your mRNA corresponding to encapsulate 5 µg into the total volume of 900 µL of Citrate Buffer.
  9. What is the concentration of the DIVERSA mRNA Delivery Nanoparticles?
    The concentration of DIVERSA mRNA Delivery Nanoparticles depends on the final volume obtained after the buffer exchange step, over 200-250 µL.
  10. For in vivo studies, what is the volume of DIVERSA mRNA Delivery Nanoparticles that can be administered?

     

    The volume of DIVERSA formulation that can be administered is 100 uL of formulation per mouse. However, this volume can be modifiable with respect to the amount of mRNA you want to administer. In addition, formulations may be placed in injections buffers to suitable in vivo administration (e.g. 0.9% physiological saline, 10% w/v glucose solution, sodium DL-lactate solution).