CellInject™ internalization into model cells

Fade of CellInject™ particle system, loaded with fluorescence dye on Saccharomyces cerevisiae cells.
This experiment was built on the hypothesis that if the particle does not release the incorporated active substance trough passive diffusion or leak, the only way to effectively deliver it is to undergo enzymatic degradation. As far as CellInject™ is not susceptible to lipases, such a digestion can only be possible intracellularly.
A. Saccharomyces cerevisiae cells treated with dye solubilizate. No dye internalization and no fluorescence is observed.

B. The dye is integrated within CellInject™ Technology. Massive fluorescence due to dye-RNA complexes.
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The dye solubilizate (section A) gives very low diffuse emission with no emission spots detected within the examined cells. The absence of emission is illustrative for inability of the dye to enter cells.
CellInject™ system (section B) induces strong and sustained fluorescence after 60th min. of treatment within cells until the end of the experiment (13 hours). The fluorescence occurs as a result of CellInject™ degradation by intracellular enzymes with release of free dye* which further forms fluorescent complexes with intracellular RNA.
*The dye has been synthesized for the purpose and possesses high lipophilicity and strong emission after binding to nucleic acids.

  • Such an in-cell specific release can be used in cases when higher concentration of the active substance is needed intracellularly.
  • Due to the lack of “wasting” of the active substance within the surrounding fluids (e.g. body fluids) its effective levels can be drastically lowered.

Evaluation of toxicitty / safety

Toxicity on Cells
The spontaneously immortalized human HaCaT keratinocyte cell line was used as a model.
CellInject™ samples show no observable effects on viability of HaCaT cells, MTT test. Interestingly, the increase in lipid concentration from 1% to 5% show even less toxicity on HaCaT cells. A possible explanation can be related with antioxidant and nutritive value of the lipid composition.
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Viability cell count (HaCaT) after 30 min incubation with different formulations.
  • CellInject™ samples show no observable effects on viability of HaCaT cells, MTT test.
Evaluation of nasal epithelium safety/toxicity
(20 albino rabbits, 30 days exposure)

As far as all the ingredients in CellInject™ are Pharmacopoeial (EP, USP) and/or GRASS listed by FDA, possible toxicity and safety concerns can only arise from the specific particle size and from the expected high intracellular drug concentrations. Nasal mucosa has been chosen as a potential place for application of the Cellinject™ and for its delicate structure.
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Light photomicrographs of cross-sections from biopsy samples of treatment groups.
  • The microscopic examination of rabbit nasal mucosa biopsates taken from 30 days treated rabbits showed no pathological changes in the nasal epithelial membrane and its adjacent layers.
Evaluation of eye safety/toxicity
The eye test was conducted to test the safety of the CellInject™ on eye tissues integrity.

Study details:
• Modified Draize test
• 9 New Zealand rabbits
• Group I – Single application
• Group II – 14-day-course of daily application
• 100 µL dose of CellInject™ is applied in right eye
• Evaluation at 24 after last administration with and without fluorescein
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Toxicity after single application
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Toxicity after 14-day-application
  • No toxicity or irritation was observed after single and 14-day-application.
  • No difference between the treated and untreated eyes of all animals.
  • Very low score in the Draize test (practically zero).

Efficacy of CellInject™

А. Evaluation of the effect of pre-treatment with CellInject™ loaded with Loratadine on histamine induced wheals.
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Evaluation of the effect of topical application of CellInject™ loaded with Loratadine vs Fenistil gel on histamine induced wheals on rabbits (n=6, mean+/-SD).
  • CellInject™ loaded with 0.1% Loratadine showed faster and stronger antihistamine effect compared to the marketed product Fenistil gel.
B. Effect of intradermal injection of placebo CellInject™ on histamine induced wheals.
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Studying the effect of topical application of placebo on the size and texture of the wheals, a further coadministration experiment was conducted with placebo CellInject™ and histamine intradermal injection.
Surprisingly, the wheals were smaller in the group treated with histamine solution/CellInject™. This comes to show that placebo CellInject™ in fact decreased the effect of injected histamine. It is likely that the composition of the Cellinject™ ingredients, namely, carnauba fatty acid esters and their derivatives, tocotrienols and tocopherols possess cumulative anti-inflammatory effect.
Evaluation of the effect of intradermal injection of placebo CellInject™ on histamine induced wheals on rabbits (mean, n=6, +/-SD).
  • Placebo CellInject™ decrease the effect of injected histamine.