Novel immunotherapy approach for melanoma based chitosan derivatives

Definition
Melanoma is a type of skin cancer that develops from the pigment-producing cells known as melanocytes.
Melanomas typically occur in the skin but may rarely occur in the mouth, intestines or eye.
Typically, people are cured if metastatic spread has not occurred. For those in whom melanoma has spread, immunotherapy, biologic therapy, radiation therapy or chemotherapy may improve survival.
Five-year survival rates in US 99% (localized), 25% (disseminated). Therefore, melanoma is one of the most dangerous and insidious type of cancer.

Figure 1. Variation in melanoma incidence

Figure 2. Clark’s melanoma invasion classification

Revelance
Figure 3.
Melanoma metastases

Revelance
Figure 4.
Melanoma metastases

Chitosan properties
Chitosan /ˈkaɪtəsæn/ is a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).
It is made by treating the chitin shells of shrimp and other crustaceans with an alkaline substance, such as sodium hydroxide.
Figure 5. Chemical formala of chitosan

Peptides
Chitosan nanoparticles
Figure 6. Chitosan
nanoparticles

Chitosan nanoparticles
Figure 7. Chitosan
nanoparticles

Chitosan properties
Therefore, this substance can be used to create a series of drugs.
We can use for the treatment of melanoma:
Chitosan based vaccines
Modified delayed release preparations of cytokines
Various targeted drug carriers

Purpose of the study
Creation of a complex of drugs for immunotherapy of melanoma

Research objectives
1. Development methods of synthesis, purification and standardization of highly purified chitosan. (chemical part)
2. Getting set of drugs for testing.
3. Estimate the toxicity of drugs at in vitro experiments (MTT test) and in vivo.
4. Determine the allergic activity of chitosan derivates at in vitro experiments and in vivo. (allergic part)
5. Estimate the immunostimulating properties of highly purified chitosan derivatives. (immunology part)
6. Explore the benefit of using nanoparticles and covalently bonded chitosan derivatives with interleukins;
chitosan-based vaccine structures and targeted platforms to increase the therapeutic efficacy of therapy melanoma
at in vivo experiment.
Горшенин Д.С., Жернов Ю.В., Кривцов Г.Г., Хаитов М.Р. Применение хитозана и его производных в иммунотерапии злокачественных новообразований. Иммунология. 2020, 41 (5): 00–00. DOI: 10.33029/ 0206-4952-2020-41-5-00-00

Chemical part of experiments
Methods for drug standardization:
1. Mass spectrometry (MS)
2. High-performance liquid chromatography (HPLC)
3. Infrared spectroscopy (IR spectroscopy)
4. Viscometery
5. Dynamic light scattering (DLS)
6. Electrophoresis
The main parameters of chitosan, which determine the immuno- and allergic activity:
1. Molecular weight
2. Weight average distribution
3. Degree of deacetylation
4. Solubility at physiological pH
5. Degree of contamination with protein
andpeptide fractions
6. Degree of endotoxin contamination and other TLR agonists

Mass spectrometry (MS)
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are typically presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio.
Methods M.-s. are used in various fields: in the nuclear industry, medicine, biology, metallurgy, geology, space. research, when conducting elemental analysis of high-purity substances and determining the absolute amount of substances by isotopic dilution, when controlling the quality of food and water, etc.
These methods are used to analyze complex mixtures and determine the structure of molecules.
Figure 8. Photo of Mass spectrometr

Allergic part
Methods for estimate allergic potential:
Histamine release in heparinized whole blood
Basophil activation test
Lymphocyte activation test
Experiments in vivo

Basophil activation test
Figure 9.
Scheme of
BAT

Immunology part
Methods for estimate immunological potential:
Determine the activity of endocytosis and pinocytosis by fluorescently labeled chitosan.
Fix the main parameters of chitosan that determine the immunoactivity (include endotoxin level).
Test dendritic cell activation.
Test T - cell activation.
In vivo experiments.

Last part of research
In vivo (mouse models) experiments of chitosan based complex for activity to melanoma
Figure 9. Scheme of in vivo experiments

NRC Institute of Immunology FMBA of Russia
Federal State Budgetary Scientific Institution
«I. Mechnikov Research Institute of Vaccines and Sera»