Invloed van koolstofnanobuisjes en zijn derivaten op tumorcellen in vitro en biochemische parameters, cellulaire bloedsamenstelling in vivo

Resultaten en discussie

Scanning-elektronenmicroscopie van MWCNT's

Volgens protocol [38] was de gemiddelde diameter van CNT's 10-20 nm en was het specifieke oppervlak bepaald door argondesorptie 200-400 m ² /G. Bulkdichtheid binnen 20–40 g/dm ³ . In het bijzonder worden de agglomeraten in de vorm van verstrengelde buizen met afmetingen van 20-500 μm verkregen tijdens een industriële CNT-productie waarbij de CVD-methode wordt toegepast (Fig. 3).

SEM-afbeeldingen van CNT's, 1) schaal 0,5 m; 2) schaal 5 m

Zetapotentieel van suspensies van nanobuisjes

CNTox en CNT duiden op een significante aggregatiestabiliteit, die direct afhangt van de waarde van de zeta-potentiaal. CNT-DOX heeft een kleinere zeta-potentiaal dan CNTox en CNT, en een hoge reproduceerbaarheid van de meting, die de homogeniteit van de deeltjes aangeeft (tabel 1). Een kleine waarde van de zeta-potentiaal van CNT-FITC kan duiden op significante deeltjesgroottes of hun kleine concentraties, zoals blijkt uit de hoge ruisverhouding bij meting.

FTIR-spectroscopie van CNT's, CNT-Dox en CNT-FITC

De bindingen tussen CNTox en doxorubicine (DOX) en fluoresceïne (FITC) werden geschat op basis van infrarood spectrale gegevens zoals aangetoond in Fig. 4.

Infraroodspectra van CNTox (CNT-O), CNT-DOX (CNT-D), CNT-FITC (CNT-F)

De aanwezigheid van een sterke band op υ = 3311 cm ⁻¹ (CNT-O) en υ = 3451 cm ⁻¹ (CNT-D) toegeschreven aan de CON-H koppelingsfluctuaties, evenals de aanwezigheid van een sterke band bij υ = 1634 cm ⁻¹ (CNT-O) en υ = 1631 cm ⁻¹ (CNT-D) toegeschreven aan de O-CNH-bindingstrillingen, wat duidelijk het type amidebinding tussen CNT en Dox aangeeft. IR toonde ook de absorptie van C-H-bindingen aan, die zich op 2969-2834 cm ⁻¹ bevindt en een brede band van 1460–1407 cm ⁻¹ uit het C-O-H-fragment. We kunnen dus concluderen dat als gevolg van chemische reacties CNT's werden gefunctionaliseerd met een antitumormiddel (doxorubicine) en een fluorescerend label (FITC).

Cytotoxiciteit van CNT's in verschillende stadia van functionalisering op monolaag- en sferoïden-celgroeimodellen

De volgende stap was het bepalen van de cytotoxiciteit van geoxideerde CNT's (CNTox), CNT's gefunctionaliseerd met doxorubicine (CNT-Dox) en fluorescerend label (CNT-FITC). De resultaten van de MTT-test op monolaagcultuur van HT29 worden getoond in Fig. 5.

Levensvatbaarheid van tumorcellen HT29 in monolaagcultuur na incubatie gedurende 48 uur met CNT's en zijn derivaten (CNT-Dox en CNT-FITC). Statistische significantie:*р ≤ 0.05 of **р ≤ 0.01

Als resultaat werd aangetoond dat CNTox, CNT-Dox en CNT-FITC een matig cytotoxisch effect hebben bij concentraties van 12,5 μg/ml (81%). Het verhogen van de CNTox-concentratie van 25 naar 50 en 100 μg / ml leidde tot een dosisafhankelijke afname van de levensvatbaarheid van tumorcellen tot 71,8-69,6-62,5% dienovereenkomstig in vergelijking met controle. Bij een concentratie van CNTox tot 200 μg/ml nam de levensvatbaarheid van HT29 af tot 39,2%. Op dat moment vertoonde CNT-Dox bij lage concentraties (12,5-50 g/ml) geen statistisch significante cytotoxiciteit, vergeleken met CNTox. Bij hoge concentraties (200 g/ml) had CTN-Dox zelfs minder cytotoxiciteit dan CNTox (50%). Tegelijkertijd hadden CNT's, gefunctionaliseerd met fluoresceïne, een relatief hoger cytotoxisch effect op tumorcellen. Na incubatie met CNT-FITC in een concentratie van 25 g/ml, verminderde de overleving van HT29-cellen tot 55% en bij 100-200 μg/ml tot respectievelijk 23 en 7%. In dat geval speelden CNT's dus de rol van nogal inert celsubstraat. Meer dan dat, CNT's geïmmobiliseerd Dox en verminderde Dox cytotoxiciteit. In eerdere studies [33] hebben we aangetoond dat primaire nanobuisjes hydrofobe eigenschappen hebben, een matig cytotoxisch effect hebben en de vorming van een groot aantal cellulaire aggregaten stimuleren. Volgens de literatuur [39] verandert de oppervlaktelading van CNT's tijdens de oxidatie. CNT's worden hydrofiel en het leidt tot de vorming van kleinere aggregaten en verhoogt het cytotoxische effect van CNT's. De gegevens die we in eerdere studie ontvangen, bevestigen deze tendens. CNTox verminderde de overleving van tumorcellen met ten minste 60% in vergelijking met controle, CNT-Dox met 45% en CNT-FITC met 97% (bij 200 μg/ml). Verklaring van de verkregen gegevens kan zijn in rapporten van de andere auteurs dat functionele liganden in de meeste gevallen het oppervlakte-zeta-potentieel van CNT's veranderen, de oplosbaarheid van CNT's stimuleren en de celpenetratie van CNT's verhogen [40]. Het vermogen van CNT's om aggregaten te vormen na oxidatie en functionalisering neemt af, terwijl de permeabiliteit door het celmembraan toeneemt en de reactiviteit van cellulaire organellen toeneemt. Meer dan dat, CNT-Dox heeft bij hoge concentraties een lager cytotoxisch effect dan CNTox. Tegelijkertijd kan volgens de verkregen gegevens worden aangenomen dat de peptidebinding doxorubicine op het oppervlak van de CNT houdt. Peptidebinding tussen CNT's en Dox is voldoende stabiel in celkweekomstandigheden. Het breekt niet af en neemt doxorubicine in inactieve vorm in. Tegelijkertijd wordt fluoresceïne-natriummoleculair (C₂₀ H₁₂ O₅ ), een ligand ter grootte van 332.311 g/mol, dissocieert vrij gemakkelijk van het oppervlak van de CNT's en komt de cellen binnen. In de resultaten zijn er onomkeerbare schendingen van DNA-structuren, kernen en mitochondriën [41].

Om de cytotoxiciteit van CNT's en zijn derivaten en de impact op de adhesieve eigenschappen van tumorcellen te verifiëren, werd het gebied van de HT29-cel 2D-kolonies geanalyseerd. De resultaten worden getoond in Fig. 6. Er werd aangetoond dat CNT's en hun derivaten een invloed hebben op het adhesief vermogen van tumorcellen en de vorming van tumorcelkolonies in monolaagcultuur. Incubatie van tumorcellen met CNTox (monster # 1) in concentraties van 12,5, 50,0 en 200 μg/ml leidde tot een dosisafhankelijke afname van 2D-celkolonies met 55,2%, 57,8% en 78,3% dienovereenkomstig, vergeleken met controle. Tegelijkertijd veroorzaakte het monster van CNT-Dox (monster # 3) in dezelfde concentraties niet zo'n effect. Het gebied van 2D-kolonies nam af met respectievelijk 34,8%, 61,6% en 82,4%. De CNT-FITC (monster #5) vertoonde de grootste invloed op tumorcellen in monolaagcultuur. Het gebied van 2D-kolonies na incubatie met CNT-FITC nam af met respectievelijk 59,8%, 85,2% en 89,8%. Opgemerkt moet worden dat de verkregen resultaten dezelfde tendens hebben als de MTT-assay. Maar de resultaten van de MTT-analyse toonden niet zo'n significant cytotoxisch effect. De resulterende discrepanties kunnen het gevolg zijn van niet-cytotoxische effecten van CNT's op de celkolonie en, gedeeltelijk, van een anti-adhesieve invloed. Eerder hebben we aangetoond dat CNT's minder cytotoxisch vermogen hebben dan anti-adhesie. Volgens onze gegevens stimuleren CNT's de migratie van tumorcellen naar de suspensiefractie en de vorming van meercellige tumorsferoïden (MTS). De gevoeligheid van cellen voor antitumormiddelen in monolaag- en sferoïde culturen is anders. Daarom hebben we in de volgende stap de vorming van MTS door tumorcellen HT29 in aanwezigheid van CNTox, CNT-Dox en CNT-FITC onderzocht. De concentraties van CNT's waren hetzelfde als in eerdere experimenten. In figuur 7 werd aangetoond dat CNTox (monster # 1) de vorming van MTS kan stimuleren. Verhoging van de CNTox-concentratie gaat gepaard met een dosisafhankelijke toename van het mediane volume van MTS. Bij concentratie CNTox van 12,5, 50, 200 g/ml, neemt het mediane volume van MTS toe van 1,79 tot 2,18 en 10,98 mm ³ ; het is bijna tien keer. Derivaten van CNT's veroorzaken geen dergelijk effect op tumorcellen. CNT-Dox (monster # 3) stimuleert de vorming van MTS in 2,83 keer. Tegelijkertijd leidt incubatie van 3D-tumorcelcultuur met CNT-FITC (monster #5) tot een 2,4-voudige afname van het MTS-volume.

Het gebied van kolonies van HT29-tumorcellen in monolaagcultuur die werd geïncubeerd met CNTox (# 1), CNT-Dox (# 3), CNT-FITC (# 5). Statistische significantie:*р ≤ 0.05 of **р ≤ 0.01

De mediaan van het volume van meercellige tumorsferoïden. MTS werden gegenereerd in aanwezigheid van CNTox (monster #1), CNT-Dox (monster #3), CNT-FITC (monster #5). Statistische significantie:*р ≤ 0.05 of **р ≤ 0.01

Het is opmerkelijk dat CNT-Dox in de meeste experimenten niet zulke cytotoxische effecten op tumorcellen heeft, zowel in 2D als in 3D-cultuur, die kunnen worden vergeleken met het effect van een enkele doxorubicine. The mechanism of cytotoxic action of doxorubicin is based on penetration into the cell nucleus and intercalation between nucleotide pairs, violation of replication and DNA repair, protein synthesis and, as a result, cell death. The cause of the reduction of cytotoxic effect of the CNT-Dox may be the binding of the doxorubicin with CNT surface through peptide bonds. It prevents Dox dissociation from CNT surface and Dox penetration into the cell and cellular organelles. This fact may let to deliver the compound to certain tissues without causing a negative effect on “non-target” objects.

In this case, the next step of the study was controlled release of doxorubicin. For peptide bonds breaking, we used the commonly known peptidase trypsin as the release agent. The effect of trypsin on the Dox release from the surface of the CNT was investigated by spectral analysis. Since the free doxorubicin has a fluorescence peak at 495 nm, bounded doxorubicin has no such ability. It was analyzed how the increasing of trypsin concentration affected on concentration of free doxorubicin. The results are shown in our previous work [42]. Shortly, it was demonstrated that increasing the concentration of 0.05% trypsin from 11 to 20% in culture medium contributed to an increasing the concentration of free doxorubicin from 3.13 to 6.55 μg/ml. A further increasing the concentration of trypsin to 60% did not lead to increasing in free doxorubicin. However, the concentration of trypsin by about 66% stimulated release again and led to increasing the concentration of doxorubicin in two times, to 11.38 μg/ml. Thus, it was concluded that 0.05% trypsin has several effective concentrations. Under these conditions, peptide bonds between doxorubicin and CNT were broken and doxorubicin was released. Therefore, to analyze how CNT-Dox influence the tumor cells after Dox release, we incubated tumor cells in the w/o fetal bovine serum (FBS) nutrient medium with trypsin and in the presence of CNT-Dox. The survival of tumor cells was determined using the MTT test. The ratio of the nutrient medium, trypsin, concentration of CNT-Dox, and doxorubicin are given in Table 2. The results of incubation HT29 during 48 h are demonstrated on Fig. 8. Cell viability in the presence of trypsin—blue columns, alone doxorubicin—orange columns, and CNT-Dox with trypsin—pink columns. In results, it has been found that the simultaneous use of trypsin and CNT-Dox significantly increased the cytotoxic effect of CNT and doxorubicin compared to the separately use of these substances. So doxorubicin alone at concentrations from 0.05 to 1.0 μg/ml causes a dose-dependent decreasing the percentage of living HT29 cells from 7.18 to 45.7% relative to control (Fig. 8, orange columns). Incubation of CNT-Dox at concentrations of 20.0 to 1.25 μg/ml with trypsin at concentration from 0 t0 70% led to a dose-dependent decreasing in the percentage of living cells from 80.9 and 99.8% respectively (Fig. 8, pink columns). At the same time, incubation with trypsin alone leads to decreasing percentage of living cells only by 34.0–42.0% (Fig. 8, blue columns). Thus, we can conclude that we observe the synergistic effect of CNT-DOX and trypsin. Each individual component has a small cytotoxic effect on cells, and together the cytotoxic potential of substances increases several times.

Percentage of alive cells HT29 after 48 h of incubation in the presence of trypsin, doxorubicin, CNT-Dox. Statistical significance:*р  ≤ 0.05 or **р  ≤ 0.01

As functional groups (Dox) were attached to CNTs surface by peptide bonds as, in vivo Dox release will be realized in organs of the gastrointestinal tract with an increased content of proteases, peptidases. In organism, proteases are used for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food [43]. Other proteases are present in leukocytes (elastase, cathepsin G) and play several different roles in metabolic control. This is one of the fastest “switching on” and “switching off” regulatory mechanisms in the physiology of an organism. By complex cooperative action, the proteases may proceed as cascade reactions, which result in rapid and efficient amplification of an organism’s response to a physiological signal. Therefore, the authors suggest that CNT-Dox construction will be the most effective in the case of stomach, pancreas, liver, and small intestine cancer localization in case of parenteral administration of drug.

Influence of CNTs and Their Derivatives on Cell Blood Composition, Hepatic Enzyme System, and Proteins Turnover In Vivo

To determine the impact of the CNTox and CNT-Dox on the protein metabolism and the state of the hepatic enzyme system, the level of albumin (Al), total protein (Tp), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) was determined. Data are given in Fig. 9a, b. As a result, it was noted that CNT-Dox and Dox have influence on the hepatic enzymes activity, namely on the AST and ALP. AST level increased in mice serum from 1 group for 21.6%, 2 group for 93.9%, and 3 group for 126.4% compared with control. At the same time, the activity of ALP increased in mice serum from 1 group for 23.5%, 2 group for 119.1%, and 3 group for 147.8%. CNTox administration did not show statistically significant changes in AST, ALT, and ALP levels. In addition, it should be noted that CNTox, Doxorubicin, and CNT-Dox slightly increased the level of total protein in the blood of experimental animals. So, we found that systematic introduction of CNTox have not influence on mice serum enzyme profile. And opposite, administration of CNT-Dox and Dox had toxicity influence and induced a chronic hepatic damage. Notably, symptoms of hepatitis had more manifestation after Dox treatment than CNT-Dox. To determine possible inflammatory processes and systemic effects of CNTox, CNT-Dox on the state of blood cells composition, it was analyzed the blood cell formula of the experimental animals. The results are shown in Table 3. Obtained data are in good agreement with the well-known manifestations of doxorubicin’s hematological toxicity:anemia (decreased red blood cells, hemoglobin, hematocrit), thrombopenia (platelet count), neutropenia (decrease in the number of granulocytes). Interestingly, that it was demonstrated the same direction of the changes of practically all hematological parameters in the 1 (CNTox), 2 (CNT-Dox), and 3 (Dox) groups of animals. However, the change in the 2 and 3 groups of animals was statistically significant accordingly to control and 1 group. In the 2 group (CNT-Dox), it was found pronounced decreasing of the monocyte content related to the system of phagocyte mononuclear cells and cell’s immune response (9.5% and 0.12 × 10 ⁹ /l in control, 4.6% and 0.06 × 10 ⁹ /l in 2 group, and 4.55 and 0.05 × 10 ⁹ /l in 3 group). Animals of 2 and 3 groups demonstrated reducing the content of granulocytes (neutrophils) (12.6 and 0.16 × 10 ⁹ /l in control and 8.5% and 0.2 × 10 ⁹ /l in 2 group and 8.05 and 0.16 × 10 ⁹ /l in 3 group). The number of lymphocytes, both in percentage and absolute, increased in 2 and 3 groups (77.9% and 0.97 × 10 ⁹ /l in control and 86.9% and 2.0 × 10 ⁹ /l in 2 group and 87.8% and 2.3 × 10 ⁹ /l in 3 group). The main function of the lymphocytes is recognition of the antigen and participation in the adequate immunological response of the body. T lymphocytes perform regulatory and effectors functions. B lymphocytes take part in humoral immunity, providing immunoglobulins in response to stimulation of other people’s antigens. So, it can be assumed that an increase in the index of lymphocyte content in experimental 1, 2, and 3 groups can be associated with the introduction of foreign antibodies (CNTs) and/or tissue distraction [44]. Some decrease in the absolute number of leukocytes in 2 (1.2 × 10 ⁹ /l) and 3 (0.85 × 10 ⁹ /l) groups compared with the 1 group of animals (2.0 × 10 ⁹ /l) can be regarded as the result of a gradual accumulation of the reversal of toxic effects of Dox. It is described the reaction of WBC in experimental animal groups as well-known toxic hematologic impact from doxorubicin in the development of leucopenia. The analysis of indicators reflecting the number and state of RBC (erythrocytes and hemoglobin) also showed the same trend of misbalance in the 2 and 3 groups of animals. Statistically significant thrombocytopenia and anemia are more indicative in animals of 2 and 3 groups:the number of erythrocytes (5.54 × 10 ¹² /l in control, 3.97 × 10 ¹² /l in 2 group, and 3.12 × 10 ¹² /l in 3 group), the amount of hemoglobin (78 g/dl for intact animals, 55.5 g/dl for 2 group, and 46.2 g/dl for 3 group), hemoglobin (25.25% for intact animals and 16.75% for 2 group and 15.12% for 3 group). The amount of erythrocytes decreased but the average content of hemoglobin in one erythrocyte did not decrease. And, as a result, concentration of hemoglobin per one erythrocyte increased. Analysis of the RBW counts let us suggest that in 2 and 3 group of animals, there are several processes:decreasing the formation of erythrocytes in the bone marrow, the acceleration of erythrocyte destruction, the violation of the structure of membranes of red blood cells, and molecular defects (oxidation) of hemoglobin. Thrombocytopenia (27 × 10 ⁹ /l in intact animals, 14.0 × 10 ⁹ /l in 2 group, and 12.05 × 10 ⁹ /l in 3 group), a dose-dependent reversible myelosuppression, leucopenia and granulocytopenia (neutropenia) are the predominant manifestations of doxorubicin hematologic toxicity and is the most common acute dose-limiting toxicity of this drug. The direction of changes in the platelet content in the blood of experimental animals treated with CNTox and CNT-Dox is the same; however, significantly more pronounced in the group of animals receiving free Dox.

Monitoring of clinical parameters following CNTox (1 group), CNT-DOX (2 group), free DOX (3 group) administration in Balb/2a mice. Mice were treated by CNTox, CNT-DOX, DOX every 3 days during 4 weeks, in concentrations:CNT—1.5 g/kg and Dox—20 mg/kg of weight. Each experiment was done in triplicate. Statistical significance:*р  ≤ 0.05 or **р  ≤ 0.01. ALT alanine transferase, AST aspartate transferase, AP alkaline phosphatase

Thus, according to our results from in vivo experiments, it was demonstrated that systematic introduction of CNTox have not influence on mice serum enzyme profile and protein turnover. And opposite, administration of CNT-Dox and Dox had toxicity influence and induced a chronic hepatic damage. More than that, dose-dependent reversible myelosuppression, leucopenia, and granulocytopenia (neutropenia) was shown for the group of animals receiving free Dox. This predominant manifestation of hematologic toxicity was less pronounced in the group of animals receiving free CNT-Dox and CNTs. So, we can assume that, in the case of parenteral administration of CNT-Dox under action of gastric juice peptidases, CNT-Dox particles breaks up into free CNT and Doxorubicin. After that, Doxorubicin enters to the stomach cells and partly in the bloodstream. That also causes the specified effect on blood cells composition. For modeling Dox release in vivo, trypsin were used in vitro.

One of the goals of this investigation was to minimize the side effects of carbon vehicle on the body. Previously, the authors demonstrated that CNTs may be a potential threat to tumor development due to its ability to stimulate cell migration and support cells in suspension fraction [33]. In this case, CNTs themselves play a role of artificial extra cellular matrix. Another way CNTs can stimulate suspension cells to aggregation. If this process will be coincident with antitumor drug accumulation on CNTs surface, CNTs will attract substrate-independent cells and kill them. Simultaneously, it was found that pure CNTs did not have statistically significant cytotoxicity to tumor cells. So, CNTs alone are not dangerous as cytotoxic agents and can act as carrier for antitumor drugs to target cells. Then, studies were conducted to the functionalization of the CNTs by antitumor antibodies. It was demonstrated that CNTs are able to carry on the surface not only the Dox but also specific tumor antibodies—anti EGFr [42]. Under the action of trypsin, Dox was released and realized strong cytotoxic effect on tumor cells. Recent studies have shown the synergy cytotoxic effect of CNTs and Doxorubicin after release from CNT-Dox construct in the presence of trypsin. Such effect was not demonstrated by CNTs and Dox separately. Therefore, the benefits from the use of CNTs seems in the usage CNTs as vehicle for antitumor antibody and drug. On the other hand, the negative effects of Doxorubicin, like many other early antitumor drugs, are totally cytotoxic effects. Binding of the Dox to the CNTs partially blocks it. This effect allows ensuring local accumulation of the Dox in the focus of tumor activity with subsequent release and action. Thus, greater efficacy can be achieved with a smaller dose of administration. This effect can be achieved only if there are tumor-specific antibodies on the surface of the CNTs. The possibility of creating such construct was demonstrated by the authors in previous work. Undoubtedly, that CNTs dissemination, accumulation, and Dox effective should be tested on mixed culture in vitro and on a tumor model in vivo. For this purpose, synthesized CNT-FITC was created and conducted. The ability of the CNTs to act as an extra-cellular matrix and the significance of this process for the “tumor-body” system should be tested on the animal model. The effectiveness of the drug will be investigated on the model of Ehrlich carcinoma and colon rectal carcinoma [44]. The distribution of CNTs in the tissues of the body will be investigated using the construct created by the CST-FITC, as was mentioned in this work. The accumulation of CNTs in the tissues of the organs of the gastrointestinal system, namely the stomach, liver, and intestines, will be analyzed by histological assay. These studies are already conducted by the authors.

According to Shang-Lin Wang [4] and our data, free doxorubicin causes similar side effects, but more pronounced than CNT-Dox. Other authors reported that toxicity effect of CNTs depends on way of administration, dose, and time of exposure and varies according to the size and type of the cells [45,46,47,48,49]. Settling of CNTs in the tissues of the liver, kidneys, and stomach with prolonged exposure can cause oxidative stress, DNA damages, compromise cell proliferation, necrosis of tissues, and chronic inflammation [50]. According to Manna [51], CNTs can cause oxidative stress and compromise cell proliferation. In the case of in vitro studies, the cytotoxicity of CNT highly depends on the degree of CNTs purification, functionalization, size, and surface charge [52,53,54,55]. According to our results and previous data, the obtained CNTs did not demonstrate a significant cytotoxic effect [33]. More than that, our data let us suggest that there is a combined cytotoxic effect of CNTs and DOX that occurs as in vitro. That is why we assumed that obtained CNTs can be used against tumor cells, in case of the target accumulation of CNTs/CNT-Dox in tumor tissue. However, mechanisms of the cytotoxic effect of CNTs are not completely clear. Some authors suggested that CNT particles activate NF-kappaB pathway depending on the dose and that the mechanism of activation was due to activation of stress-related kinases [51]. Other authors reported that CNTs have a direct pro-apoptotic effect in vitro in different cancer cell lines and tumor cells obtained from surgical specimens [56], CNTs able to modify fatty acids in cell membranes [57] or erythrocyte membrane damage [58]. For further investigation of accumulation CNTs in cells and tissue, we plan to use CNT-FITC composite.

Other unclear questions are as follows:what is the prolonged impact of CNTs/CNT-Dox on organism level and how does it stimulate accumulation CNTs/CNT-Dox in tumor tissue? To investigate the influence of chronical introduction of CNTs/CNT-Dox, long-time studies on model of transplanted or initiated tumors of various localizations will be realized. Other authors analyzed the distribution of nanotubes throughout the body of mice after pulmonary exposure [59]. In results, accumulation of MWCNTs was documented in several organs, including notably the white pulp of the spleen and the bone marrow. The CNTs usually deposit in the liver, spleen, or lungs after they have served their purpose from where they are expelled gradually out of the body through the renal excretion route [60]. Zhao and Liu reported that accumulation of CNTs in the body can lead to granulomatous inflammation or alveolar septal thickening. Zhuang Liu et al. reported that SWNT-PTX affords higher efficacy in suppressing tumor growth than clinical Taxol® in a murine 4T1 breast-cancer model, owing to prolonged blood circulation and tenfold higher tumor paclitaxel (PTX) uptake by SWNT delivery likely through enhanced permeability and retention [61]. Accumulation of CNTs in the target tissue can be enhanced by placing on the CNTs surface specific antibodies which over expressed by tumor cells. The possibility of using antibodies for the targeted delivery of nanostructured preparations has been described by many authors [62, 63].