Method for tumour growth inhibition induction in experiment

FIELD: medicine.

SUBSTANCE: invention refers to medicine and may be used for tumour growth inhibition induction in experiment. For this purpose, the Pliss's lymphosarcoma cells 1 ml in the concentration of 20x106/ml proincubated for 30 min at 37°C with 100 mcl with a one-wall carbon nanotube suspension of the length of 100-200 nm functionalised by MH2-groups in the concentration of 10 mcg/ml. The tumour growth inhibition is specified as 97% for 20 days and prolonged life length is stated to increase in 1.68 times as compared to reference tumour carrier animals.

EFFECT: invention induces the tumour growth inhibition and prolongs life length of experimental animals ensured by the combined incubation of tumour cells and one-wall carbon nanotubes.

3 tbl

 

The invention relates to medicine, namely to experimental research in Oncology, and can be used to assess antitumor activity werdnig nanotubes (NT).

Carbon is the basis of biological macromolecules, which prompted researchers to develop new biocompatible materials carbon nature (fullerenes, graphenes, carbon nanotubes). Functionalization of their different chemical groups allows them to react in vivo and show various kinds of biological activity depending on their properties.

Known cytotoxic effect of nanosized particles of different nature (patent RU №2392668, publ. 20.06.2010,, bull. No. 17), in particular, single-walled carbon HT on the tumor cell culture (Amoopi, P.Drockery, U.Greiser, M.Murphy, V.Barren. Carbon nanotubes and mesenchimal stem cells: biocompatibility, proliferation and differentiation // nano-letters, 2008. V.8. No. 8. 2137-2143). Other authors report the absence of such action (III, Rahmaninov, Asselineau, Reapedgerne, Oaapn. Research proliferative activity and cell viability of fibroblasts and glioblastoma on different types of carbon nanotubes // bull. the experimental. Biol. and the honey. 2012. T. No. 2. S-231). A well-known effect of the destruction of tumor cells under combined action of carbon nanoblock, covered with gold, and various physical factors (.Loo,A.Lowery, N.Halas, J.West, R.Drezek. Immunotargeted nanoshells for integrated cancer imaging and therapy // nano-letters, 2005. V.5. No. 4, 709-710). There is information about selective absorption of different nanosized particles of tumor cells (Acheh, G.Khaled, J.Fang, H.Maeda Exploiting the enhanced permeability and retention effect for tumor targeting // Drug Discov. Today, 2006. No. 11, 812-818). A definite answer, whether single-walled carbon HT cytotoxic or antiproliferative action, the literature provides no; perhaps it depends on the length, functionalization, and other characteristics of the nanotubes. Is the development and experimental study of the effect of conjugates of HT drugs for targeted delivery to the tumor (Jinjun Shi, A.R.Votruba, O.C.Farokhzad, R.Langer. Nanotechnology in drug delivery and tissue engineering: from discovery to application // nano-letters, 2010. 10. 3223-3230).

In the above works do not consider the possibility of inhibition of tumor growth in vivo under the action of short otnoshenij carbon HT, functionalized groups, allowing them to react with protein molecules (NH2and COOH).

As a prototype we have used the work (Ashwin A. Bhride, Vyomesh Patel, Julie Gavard, et al. Targeted killing of cancer cells in vivo and in virto with EGF-directed carbon nanotube-based drug delivery // ACS NANO. 2009. V.3. No. 2. 307-316), which examined the effect of single-walled carbon HT length 50-300 (110±50) nm and a diameter of about 10 nm, functionalized cisplatin and EGF on tumor growth in vivo. The authors floor is constrained 10-day growth inhibition of transplantable tumors HN12, overexpressing EGF, after preprocessing of functionalized nanotubes and intravenous inoculation of Nude mice. However, this method requires complex design nanoconjugates and has a specific focus against EGF-expressing tumors. In addition, the complexity of the model is to use tumor, perelivayuschiesya on Nude mice, which, if you keep in mind the further stages of the study of the effects of NT in a clinical setting, they do not match.

The aim of the invention is the induction of inhibition of growth of transplantable tumors in the experiment using functionalized NH2groups of carbon nanotubes.

This objective is achieved in that white outbred rats weighing 200-250 g contained in standard vivarium conditions, under the skin of the back perejivaut 1 ml suspension of tumor cells lymphosarcoma (LSA) Plissa containing 20×106cells in 1 ml, incubated at 37°C for 30 min with 100 µl of the suspension of NT NH2or NT COOH, 10 µg/ml in 4% proxanol is chosen, preventing their aggregation, thus, for each tumor cell has 5×10-5ng HT. Control rats perform a similar perepevku LCA, incubated at 37°C for 30 min with 100 ál of 4% aqueous solution of proxanol is chosen-268, - nonionic surfactants, is what the student component of the commercial product of perftoran. Assess the dynamics of tumor growth and life expectancy of animals-carriers of tumour. In the animals studied groups show statistically significant differences on these indicators: perebivka tumors, preincubating with HT NH2contributes to the inhibition of its growth and increase in life expectancy of animals-carriers of tumour compared with control animals and rats, which transplanted tumor cells, proinsurance with HT COOH.

Thus, objective, namely the induction of inhibition of growth of transplantable tumors (LCA plisse) using carbon HT, functionalized NH2groups, seems to be reached.

The invention "Method of induction inhibition of tumor growth in the experiment is new, because it is unknown in medicine, in particular in Oncology, experimental studies to evaluate the antitumor activity of carbon NT.

The novelty of the invention lies in the fact that rats experimental groups subcutaneously in the region of the back perejivaut 1 ml suspension of tumor cells LCA Plissa containing 20×106cells in 1 ml, incubated at 37°C for 30 min with 100 µl of the suspension of NT, functionalized NH2or COOH, HT concentration is 10 μg/ml; determine the impact of the introduction of HT on growth LCA Plissa, the duration of the ity of life of rats and reveal in the control group, where the transplantable material incubated with proxanol is chosen, there is a gradual increase in tumors with subsequent necrotization, decay and death of animals-carriers of tumour after 22 days after inoculation; in the experimental group, where perepevku conducted after inactivated with HT COOH, differences from control were observed, while in the experimental group, where perepevku conducted after inactivated with HT NH2comes inhibition of tumor growth, the death of the animal occurs later (after 38 days after inoculation).

Thus, in the experiment it is shown that under the action of NT NH2is inhibition of the release of transplantable tumors, and NT COOH such activity do not show.

The invention is industrially applicable as it can be used in biomedical research while conducting experiments in the field of Oncology.

The method of induction inhibition of tumor growth in the experiment is as follows: HT, functionalityand NH2or COOH-groups before the experiment were bred 4% proxanol is chosen up to a concentration of 1 μg/ml, and to prevent aggregation was treated with ultrasound for 1 min, a frequency of 44 kHz, the amplitude of 30-50 μm; rats-males (weight 200-250 g) experimental groups subcutaneously in the back area transplanted 1 ml suspension of tumor cells LCA Square the SSA, containing 20×106cells in 1 ml, incubated at 37°C for 30 min with 100 µl of the suspension HT; control animals were injected transplantable material after incubation with proxanol is chosen; HT concentration is 10 μg/ml, i.e. for each tumor cell has 5×10-5ng HT. Assess the dynamics of growth LCA Plissa and reveal that in control rats and in animals in which the tumor transplanted after inactivated with HT COOH, there is a gradual increase in tumors with subsequent necrotization, decay and death of animals-carriers of tumour through 22-23 days after inoculation, and animals, which transplantable material was administered after inactivated with HT NH2is inhibition of tumor growth, the death of the animal occurs at a later date (after 38 days after inoculation). As can be seen from the data presented in table 1, the braking index (it) tumor growth in animals of the experimental group when perebivke LCA Plissa, incubated with HT NN2is 88,9% after 10 days, which is 97.6% 13 days and 97% after 20 days, and when perebivke LCA Plissa, incubated with HT COOH, inhibition is observed only after 10 days after inoculation, whereas it significantly less (44%), and then the tumor grows as well as in the control group.

Dynamics of tumor growth in rats after inoculation of tumor cells, incubated with HT NH2within 30 min 37°C
Table 1
Groups of ratsDays after inoculation, tumor volume (cm3)
10th13th20th27th33
Control (proxanol is chosen)0,09±0,019822,97±2,849,55±4,95
Experienced (NT NH2)0,01±0,0022*0,55±0,26*1,42±0,62*of 4.44±2,0*22,3±3,38
% inhibition =(V K-V/V)×100%88,997,697--
HT a=V/V941,834,0--
Experienced (NT COOH)0,05±0,009 19,23±2,354,83±5,5--
% inhibition =(V K-V/V)×100%4416-10--
It a=V/V1,81,190,9--
Note. * - statistically significant differences from control (P<0,05)

As can be seen from the data presented in table 2, the tumor is slower than in the control, is formed in rats of the experimental group after inoculation of the cells, incubated with HT NH2while the death of rats of this group occurs at a later date. In the control group, the tumor has formed all 5 rats 10 days after inoculation, their downfall started on the 20th and ended on the 27th day. In the experimental group after inoculation of tumor cells, incubated with HT NH2after 10 days the swelling was gone in 3 rats from 5, then another 1, and 1 - never came out; the death of 4 rats came from 33 to 40 days, 1 animal lived without tumor 6 months and were killed for another reason. After inoculation of tumor cells, incubated with HT COOH, LCA Plissa develops what I similar to control.

Table 2
The impact of the introduction of tumor cells, incubated with HT NH2for 30 min at 37°C, on the death of rats-carriers of tumour
Groups of ratsThe number of rats with tumors/number of live rats
10th13th20th27th3340
Control (proxanol is chosen)5/55/54/4000
Experienced (NT NH2)3/54/54/54/54/50/1
Experienced (NT COOH)5/55/54/42/200

As can be seen from the data presented in table 3, long is th life carriers of tumour maximum in the experimental group after inoculation of the cells, incubated with HT NH2and statistically significantly higher than that in control group than in animals after inoculation of tumor cells, incubated with HT COOH.

Table 3
The life span of rats after inoculation LCA Plissa, incubated with tumor cells 30 min 37°C
Groups of ratsLifetime (days)
Control (proxanol is chosen)22,6±1,54
Experienced (NT NH2)38±1,0*
Experienced (NT COOH)23,3±1,85
Note. * - statistically significant differences from control (P<0,05)

Thus, preincubate tumor cells with HT ML causes growth inhibition LCA Plissa 97% for 20 days and the increase in life expectancy of animals-carriers of tumour 1.68 times; HT COOH show no such effect.

Technical and economic efficiency "Method of induction inhibition of tumor growth in the experiment is that preincubation NT NH2with transplantable tumor cells you which indicates the inhibition of its growth and the increase in life expectancy of animals-carriers of tumour.

The method of induction inhibition of tumor growth in the experiment, including perepevku tumor cells after incubation in vitro with single-walled carbon nanotubes, characterized in that the experimental rats subcutaneously previvous 1 ml of tumor cells LCA Plissa at a concentration of 20·106/ml, preincubating for 30 min at 37°C With 100 µl of suspended single-walled carbon nanotubes with a length of 100-200 nm, functionalized NH2groups, in a concentration of 10 μg/ml, establish the inhibition of tumor growth by 97% within 20 days and the increase in life expectancy of animals 1.68 times as compared with the control animals-carriers of tumour.



 

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