시험관 내 종양 세포 및 생화학적 매개변수, 생체 내 세포 혈액 조성에 대한 탄소 나노튜브 및 그 유도체의 영향

결과 및 토론

MWCNT의 주사 전자 현미경

프로토콜[38]에 따르면 CNT의 평균 직경은 10~20nm이고 아르곤 탈착에 의해 결정된 비표면적은 200~400m ² 입니다. /G. 20–40g/dm ³ 이내의 부피 밀도 . 특히 20~500μm 크기의 얽힌 튜브 형태의 덩어리는 CVD 방법을 적용한 산업용 CNT 생산 과정에서 얻어진다(그림 3).

CNT의 SEM 이미지, 1) 스케일 0.5 μm; 2) 스케일 5μm

나노튜브 현탁액의 제타 전위

CNTox 및 CNT는 응집에 대한 상당한 안정성을 나타내며, 이는 제타 전위 값에 직접적으로 의존합니다. CNT-DOX는 CNTox 및 CNT보다 제타 전위가 작고 측정의 재현성이 높아 입자의 균질성을 나타냅니다(표 1). CNT-FITC의 제타 전위의 작은 값은 측정 시 높은 노이즈 비율에서 알 수 있듯이 상당한 입자 크기 또는 작은 농도를 나타낼 수 있습니다.

CNT, CNT-Dox 및 CNT-FITC의 FTIR 분광법

CNTox와 독소루비신(DOX) 및 플루오레세인(FITC) 간의 결합은 그림 4에 나타난 바와 같이 적외선 스펙트럼 데이터를 기반으로 추정되었습니다.

CNTox(CNT-O), CNT-DOX(CNT-D), CNT-FITC(CNT-F)의 적외선 스펙트럼

υ =3311 cm ⁻¹ 에서 강한 밴드의 존재 (CNT-O) 및 υ =3451cm ⁻¹ (CNT-D) CON-H 커플링 변동과 υ =1634 cm ⁻¹ 에서 강한 밴드의 존재로 인한 것 (CNT-O) 및 υ =1631cm ⁻¹ (CNT-D)는 CNT와 Dox 사이의 아미드 결합 유형을 명확하게 나타내는 O-CNH 결합 진동에 기인합니다. 또한 IR은 2969–2834cm ^-1 에 위치한 C-H 결합의 흡수를 나타냈습니다. 및 1460–1407cm의 광대역 ⁻¹ C-O-H 단편에서. 따라서 우리는 화학 반응의 결과로 CNT가 항종양 약물(doxorubicin)과 형광 표지(FITC)로 기능화되었다는 결론을 내릴 수 있습니다.

단층 및 회전타원체 세포 성장 모델에 대한 다양한 기능화 단계에서 CNT의 세포독성

다음 단계는 산화된 CNT(CNTox), 독소루비신으로 기능화된 CNT(CNT-Dox) 및 형광 표지(CNT-FITC)의 세포독성을 결정하는 것이었다. HT29의 단층 배양에 대한 MTT 테스트 결과는 그림 5에 나와 있습니다.

CNT 및 그 유도체(CNT-Dox 및 CNT-FITC)와 함께 48시간 동안 배양한 후 단층 배양에서 종양 세포 HT29의 생존력. 통계적 유의성:*р ≤ 0.05 또는 **р ≤ 0.01

그 결과, CNTox, CNT-Dox, CNT-FITC는 12.5μg/ml(81%) 농도에서 중간 정도의 세포독성 효과를 나타내는 것으로 나타났다. CNTox 농도를 25에서 50으로, 100μg/ml로 증가시키면 종양 세포의 생존 능력이 대조군과 비교하여 71.8-69.6-62.5%로 용량 의존적으로 감소했습니다. 최대 200μg/ml의 CNTox 농도에서 HT29의 생존율은 39.2%로 감소했습니다. 그 당시 저농도(12.5-50μg/ml)의 CNT-Dox는 CNTox와 비교하여 통계적으로 유의한 세포독성을 나타내지 않았습니다. 고농도(200μg/ml)에서 CTN-Dox는 CNTox(50%)보다 세포 독성이 훨씬 적습니다. 동시에, 플루오레세인으로 기능화된 CNT는 종양 세포에 대해 상대적으로 더 높은 세포독성 효과를 나타냈다. 25μg/ml 농도의 CNT-FITC와 함께 배양한 후 HT29 세포 생존율은 55%로, 100–200μg/ml에서는 각각 23% 및 7%로 감소했습니다. 따라서 이 경우 CNT는 오히려 불활성 세포 기질의 역할을 합니다. 그 이상으로, CNT는 Dox를 고정화하고 Dox 세포독성을 감소시켰습니다. 이전 연구[33]에서 우리는 1차 나노튜브가 소수성 특성, 중간 정도의 세포독성 효과를 가지며 많은 수의 세포 응집체 형성을 자극한다는 것을 입증했습니다. 문헌[39]에 따르면, CNT의 표면 전하는 산화 동안 변한다. CNT는 더 친수성이 되어 더 작은 응집체를 형성하고 CNT의 세포독성 효과를 증가시킵니다. 이전 연구에서 얻은 데이터는 이러한 경향을 확인시켜줍니다. CNTox는 대조군, CNT-Dox 45%, CNT-FITC 97%(200μg/ml)에 비해 종양 세포의 생존을 최소 60% 감소시켰습니다. 얻은 데이터에 대한 설명은 기능적 리간드가 대부분의 경우 CNT의 표면 제타 전위를 변경하고 CNT의 용해도를 자극하며 CNT의 세포 침투를 증가시킨다는 다른 저자의 보고서에 있을 수 있습니다[40]. 산화 및 기능화 후에 응집체를 형성하는 CNT의 능력은 감소하는 반면, 세포막을 통한 투과성은 증가하고 세포 소기관의 반응성은 증가합니다. 그 이상으로 CNT-Dox는 고농도에서 CNTox보다 세포독성 효과가 낮습니다. 동시에 얻은 데이터에 따르면 펩타이드 결합이 CNT 표면에 독소루비신을 유지한다고 가정할 수 있다. CNT와 Dox 사이의 펩타이드 결합은 세포 배양 조건에서 충분히 안정적입니다. 그것은 분해되지 않으며 비활성 형태의 독소루비신을 취합니다. 동시에, 플루오레세인-나트륨 분자(C₂₀ H₁₂ O₅ ) 332.311g/mol 크기의 리간드는 CNT의 표면에서 매우 쉽게 해리되어 세포로 들어갑니다. 그 결과 DNA 구조, 핵 및 미토콘드리아에 돌이킬 수 없는 침해가 발생했습니다[41].

CNT 및 그 유도체의 세포독성과 종양 세포의 접착 특성에 미치는 영향을 확인하기 위해 HT29 세포 2D 집락의 면적을 분석했습니다. 그 결과를 그림 6에 나타내었다. CNT와 그 유도체는 단층 배양에서 종양 세포의 접착력과 종양 세포 콜로니 형성에 영향을 미치는 것으로 나타났다. 12.5, 50.0 및 200μg/ml 농도의 CNTox(샘플 # 1)와 함께 종양 세포의 배양은 대조군과 비교하여 55.2%, 57.8% 및 78.3%에서 2D 세포 집락의 용량 의존적 감소를 초래했습니다. 동시에 동일한 농도의 CNT-Dox 샘플(샘플 #3)에서는 이러한 효과가 발생하지 않았습니다. 2D 콜로니의 면적은 각각 34.8%, 61.6%, 82.4% 감소했습니다. CNT-FITC(샘플 #5)는 단층 배양에서 종양 세포에 가장 큰 영향을 미치는 것으로 나타났습니다. CNT-FITC 배양 후 2D 집락의 면적은 각각 59.8%, 85.2% 및 89.8%로 감소했습니다. 얻어진 결과는 MTT-assay와 동일한 경향을 갖는다는 점에 유의해야 한다. 그러나 MTT 분석의 결과는 그러한 유의한 세포독성 효과를 나타내지 않았다. 결과적인 불일치는 CNT가 세포 콜로니에 미치는 비세포독성 효과와 부분적으로는 항접착제 영향의 결과일 수 있습니다. 이전에 우리는 CNT가 항부착보다 세포독성 능력이 더 낮다는 것을 입증했습니다. 우리의 데이터에 따르면, CNT는 종양 세포의 현탁액 분획으로의 이동 및 다세포 종양 스페로이드(MTS)의 형성을 자극합니다. 단층 및 회전 타원체 배양에서 항종양제에 대한 세포의 민감도는 다릅니다. 그래서 우리는 다음 단계에서 CNTox, CNT-Dox 및 CNT-FITC가 있는 상태에서 종양 세포 HT29에 의한 MTS 형성을 조사했습니다. CNT의 농도는 이전 실험과 동일하였다. 그림 7에서 CNTox(샘플 # 1)가 MTS의 형성을 자극할 수 있음이 입증되었습니다. CNTox 농도의 증가는 MTS의 중간 부피의 용량 의존적 증가를 동반합니다. 12.5, 50, 200μg/ml의 CNTox 농도에서 MTS의 중간 부피는 1.79에서 2.18 및 10.98mm ³ 로 증가합니다.; 거의 10배입니다. CNT의 유도체는 종양 세포에 이러한 효과를 일으키지 않습니다. CNT-Dox(샘플 #3)는 2.83배에서 MTS의 형성을 자극합니다. 동시에 3D 종양 세포 배양과 CNT-FITC(샘플 # 5)를 배양하면 MTS 부피가 2.4배 감소합니다.

CNTox(#1), CNT-Dox(#3), CNT-FITC(#5)와 함께 배양된 단층 배양에서 HT29 종양 세포의 콜로니 면적. 통계적 유의성:*р ≤ 0.05 또는 **р ≤ 0.01

다세포 종양 회전 타원체 부피의 중앙값. MTS는 CNTox(샘플 #1), CNT-Dox(샘플 #3), CNT-FITC(샘플 #5)의 존재하에 생성되었습니다. 통계적 유의성:*р ≤ 0.05 또는 **р ≤ 0.01

대부분의 실험에서 CNT-Dox는 단일 독소루비신의 효과와 비교할 수 있는 2D 및 3D 배양 모두에서 종양 세포에 대해 이러한 세포 독성 효과가 없다는 점에 주목해야 합니다. 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].