작은 희토류 불화물 나노입자는 전기적 극성 상호작용을 통해 종양 세포 성장을 활성화합니다.

토론

암세포의 상대 성장률은 두 RE-NP의 더 높은 농도 수준에서 상승했습니다(그림 9a). 그러나 DMEM+FBS에서 RE-NP의 MHR 값은 직접 추적되었습니다(PrF₃ ) 및 반비례(LaF₃ ) 0.1–10 kg m ⁻³ 의 RE-NP 농도까지 (그림 1g, h). 따라서 평균 크기가 ~ 55 nm 이상인 RE-NP는 세포 성장에 영향을 미치지 않아야 하며 작은 크기의 NP만 종양 성장에 실제 역할을 할 수 있습니다.

RE-NP의 크기 및 구조

작은 RE-NP 식별

실험 데이터에서 RE-NP의 평균 크기, 분포 및 통계적 매개변수를 추출했습니다. t를 적용하여 건조된 PrF₃에서 NP의 "평균 동일 면적 원"의 "무효 가설"에 대한 테스트 통계 및 DMEM+FBS 정지, p 무작위로 선택한 두 AFM 이미지 사이의 NP 직경 값은 ~ 0.001(추가 파일 2)입니다. MEAC 직경(63 nm) 값은 AFM 데이터에서 확실하게 추출되었으며 이는 DLS 데이터의 MHR 값과 비슷했습니다(그림 1g).

반대로 무작위로 선택된 LaF₃에서 MEAC 직경의 값은 샘플은 더 높은 거부 확률 값(p)과 함께 26nm의 평균 MEAC 직경을 보여주었습니다. =0.07), LaF₃의 발산 동작을 나타냅니다. 액체 현탁액에서. MEAC 직경과 DLS(296 nm)의 MHR(그림 1) 사이의 불일치는 LaF₃의 상호 작용의 복잡성 때문입니다. 다시 정지. 실제로 2 × 2 μm ² 의 경우 AFM 팁 스캐닝 영역, 평균 z -높이는 ~ 140 nm였으며, 큰 크기의 LaF₃의 존재를 나타냅니다. NP는 기판의 액체 현탁액에서 옮겨집니다(그림 4). "무작위로 선택된 TEM 샘플의 동일한 MEAC 직경 값"의 "영무 가설"의 경우, p 값도 작았습니다(p =0.001). 두 RE-NP의 경우 PrF₃에 대해 결합된 TEM 및 XRD 데이터에서 추출된 평균 MEAC 직경 값 및 LaF₃ 높은 p 표시 값, p =0.29 및 0.06, 따라서 TEM과 XRD 데이터 사이의 상관 관계를 허용하지 않습니다. TEM, AFM만(PrF₃ ) 및 DLS 데이터는 MEAC 직경 및 코어-쉘 값을 추출하기에 충분히 신뢰할 수 있었습니다(추가 파일 2).

또한 Feret 직경의 비등방성 각도 분포는 두 PrF₃ 및 LaF₃ 이방성 각도 분포가 나노결정 사이의 강한 전기적 극성 상호작용을 나타내기 때문에 구조는 고도로 극성화된 유전체였다. LaF₃의 다양한 편광 상태 현탁액에서 응집 상태의 상대적 효율성을 축소하고 건조 샘플에서 표면 거칠기 매개변수를 확대하는 역할을 했습니다.

5μL 및 농도 0.1kg m ⁻³ 에 대한 무작위 AFM 이미지의 소프트웨어 입자 분석 15 nm 및 10 nm보다 작은 크기의 ~ 22 및 ~ 11 RE-NP를 식별했습니다(p =0.001), TEM 이미지의 ~ 60 RE-NP 수(p =0.001) ~ 4μm ² 의 영역 , 따라서 서스펜션에 작은 크기의 RE-NP가 존재함을 확인합니다(그림 3(c), 추가 파일 2). DLS에서는 감지되지 않습니다.

RE-NP의 구조 및 기하학

RE-NP의 크기 분포는 에탄올과 DMEM+FBS 현탁액에서 다양합니다(그림 4). 다양성은 흡착된 단백질, 탄수화물, 전해질 및 RE-NP의 표면 사이의 서로 다른 분자 상호작용을 소유하여 RE-NP와 세포의 특정 상호작용을 조절하는 고도로 복잡한 유기 망토(코로나)의 형성을 초래합니다. DMEM+FBS 배지.

흡습성 RE-NPs와 DMEM+FBS에 갇힌 물 분자 사이의 상호작용도 코어-쉘 형성에 중요했습니다. 또한 준비 초기 단계에서 단백질과 중간 상호작용의 구조적 변화에 중대한 영향을 미쳤습니다. NPs의 표면 대 부피 비율이 현탁액에서 높은 값을 나타내면서 단백질을 흡수하는 능력을 포함하여 RE-NPs의 효과적인 안정성과 물리화학적, 기계적 및 흐름 특성이 매우 다양했습니다[39,40,41] .

AFM 및 TEM을 통한 액체(DLS)의 RE-NP 및 응고된 현탁액의 비교 크기 분포는 RE-NP가 RE-코어를 둘러싸는 단백질 쉘인 코어-쉘 유전체 구조를 형성하는 유기 형태 내부에 캡슐화되었음을 보여주었습니다. 유리 기판에 증착된 DMEM+FBS 현탁액의 응고된 RE-NP에서 이미지화된 AFM은 또한 다면적 RE-NP 및 단백질 코로나 복합체의 형성을 나타냅니다(그림 5). 건조된 매체가 결정 구조의 규칙적인 자기 조립 패턴을 형성하는 동안(그림 5a-d), 건조된 RE-NPs 현탁액은 AFM의 디지털 카메라로도 볼 수 있는 몇 개의 검은 반점이 있는 비정질 층 구조를 나타냈습니다(그림 5e-l). . 더 높은 광학 배율에서 배지 단독보다 작은 구형 모양의 개별 덩어리가 DMEM + FBS의 두 RE-NP와 수상 돌기 유형 구조에 대해 모두 감지되었습니다. 둘 다 상호 작용의 복잡성을 보여줍니다. 표면 매개변수 결과(그림 4). 가장 높은 AFM 분해능(1 × 1 μm ² 영역), 그림 5의 마지막 레인에서 분리된 RE-NP 응집체는 ~ 5 nm의 분해능 한계 내에서 두 RE-NP에 대한 건조 구조에서 확인되지 않았습니다. 광학 이미지에서 볼 수 있는 1-2 μm 길이의 검은색 구형 균사체는 코어-쉘 RE-NP의 큰 덩어리 덩어리였습니다. RE-NP와 DMEM+FBS 사이의 복잡한 반응은 순수한 DMEM+FBS에서 장기간 자가 조립된 긴 구조가 수상돌기 구조로 변형되는 것을 통해 시각화되었습니다.

결과는 단백질 껍질 내부에 캡슐화된 단일 RE-NP 코어 구조의 그림을 가리킵니다. 이러한 구조는 RE-NP와 교차 반응하는 유기물과 전해질로 둘러싸여 있기 때문에 감지할 수 없었습니다. PrF₃의 VUV 스펙트럼 140에서 170nm 사이의 일부 스펙트럼 피크를 보여줍니다(그림 8). 이온 전이는 최대 168nm에서 4f6s의 스펙트럼 서명만 최대의 전자 구성 132 및 127 nm에서 스펙트럼에 존재했습니다. However, these bands could evince the presence of water in the high hygroscopic PrF₃ suspensions. Water has a rich, structured absorption band in the VUV spectral range centred at 122 nm, revealing the presence of water molecules in the core-shell NPs.

Activation of Mechanosensors

Activation of Integrins by External Forces

The activation of oncogenic pathways by RE-NPs [24], besides the 3D structural nature of TSRs, is based on some Natural Evolution principles for sustaining the viability of cells. First, upon binding a specific external ligand in a LABS, conformational changes along the entire TSR spectrum underline a series of cascading pathways, triggering tumour cell growth (Fig. 9c, d). The transmission of signals advances through the plasma membrane via various protein chains. Signal transduction was via conformational transformations of integrins responding to a high affinity external force (Fig. 10a, b).

Simplified layout of integrin activation by NPs and signal transaction pathways. 아 Structure and conformational geometries of integrins at a low (A), medium (B) and high-affinity strengths (C). ㄴ AKT and ERK1/2 signal transaction pathways activated by RE-NPs via external integrin stimulation

Because of “life sustainability” and “survival laws” that prevents cancer cell growth by random “noise”, it is required that the strength of the external force should be within a bounded range of values and also the external strength stimulus should apply for a long period on a large number of mechanosensors in a cancer cell. The external strength that stimulates cancer must be slightly larger than the strength of the interatomic molecular forces under normal conditions. For a thermal energy of a ligand at room temperature (kT  = 0.025 eV, T  = 298 K), and for a regular thermal stress of molecular bonds of ~ 0.05 nm, the mean thermal force acting on the LABS stays for 1.2 × 10 ⁻¹² N. In principle, a force above ~ 10 × 10 ⁻¹² N acting coherently on the whole set of mechanosensors on a cell should activate signal transduction in tumour cells. Consequently, ignoring any thermal and mechanical stressing in the ECM normal conditions, integrin activation via electrical polar interactions between LABS and NPs has the potency to start signal transduction in cancer cells and to initiate tumorigenesis.

Integrin Structure and Geometry

An integrin receptor in the upright conformation state extends ∼ 20 nm upwards from the cell membrane [42] (Fig. 10a). For no contacts between the two α- and β-subunits, other than those in the headpiece near the ligand-binding pocket, the α- and β-subunits are well separated with their cytoplasmic tails extended out up to ∼ 8 nm [42]. A conic projection geometry (20 nm slant height, 5–10 nm diameter of its circular base), bounded by the α- and β-subunits, defines a projected area on the surface of cell’s membrane between ~ 19 and ~ 80 nm ² , for a typical mean radius of a tumour cell R _ㄷ  ≈ 5 μm (equivalent surface area of a spherical cell \( {S}_c=4\pi {R}_c^2=3.14\ \mathrm{x}\ {10}^8\ {\mathrm{nm}}^2 \)). By dividing the area S _ㄷ of a spherical tumour cell surface with the projected area of an integrin on a cell surface, an upper limit of the number of integrin receptors for these projected areas was n _int  = 1.6 x 10 ⁷ and 3.9 × 10 ⁶ 각기. These numbers are compared with the mean number of integrins on a cell \( {\overline{N}}_{int}\approx 2\ \mathrm{x}\ {10}^5 \) and for an average interspacing of 45 nm between adjacent integrin receptors [43]. Nevertheless, \( {\overline{N}}_{int} \) might be larger because of an uneven surface structure, different separating distances between integrins and variable size of tumour cells (Fig. 9c), but the number of integrins on a cell membrane stand between n _int and \( {\overline{N}}_{int} \).

Interaction of Mechanosensors with RE-NPs

ERK ½ and AKT Activation

The TEM images and the elemental mapping of F, La and Pr showed that RE-NPs were unable to penetrate inside the cell. They gathered around the A549 cell membrane (Fig. 11), confirming that an external force can stimulate cell growth because of TSRs activation [44]. The Pr atoms were distributed around the boundaries of the cell’s membrane. The small numbers of F, La and Pr identifications inside the cell were not associated with endocytosis of RE-NPs, but they were images of RE-NPs from the projections of the two cells hemispheres on cell’s equatorial cycle.

TEM images and elemental analysis of RE-NPs at the surface boundaries of A549 cells. 아 TEM image of small size LaF₃ NPs surrounding the A549 cells. ㄴ Elemental analysis of F atoms in RE-NPs distributed around the cell. ㄷ Elemental analysis of La atoms. The low concentration of La atoms was associated with a rather small scattering efficiency of the X-rays. d –f The same as for (a –ㄷ ) for PrF₃ RE-NPs

It was also evident that both RE-NPs were able to enhance AKT phosphorylation, especially in A549 cells (Fig. 9d), where the steady-state level of AKT pathway activity was higher for the SW837 cell line. The phosphorylation level for the MCF7 cell line was below the detection limit, in agreement with the relatively low levels of growth. High phosphorylation levels of ERK1/2 [36] and AKT were detected in A549 and SW837 cell lines. Cell growth was started once NPs with a proper size interact with the mechanosensors of the cells to provide the correct force for initiating cell growth [45, 46]. ERK and AKT pathways were frequently active in several cancer cell types via extracellular springing, as they were stimulated by the TSRs, upon a selective binding with various mitogenic ligands, or via the activation of the mechanosensory group. The interaction was responsible for a continuous intracellular stimulation that, according to the cell’s phenotype, driven the cancer cells to uncontrolled and endless growth. Viability tests were also run for 48 and 72 h, but the growth of all cell lines was saturated at 48 and 72 h after the initial moment of Cell plating.

Interaction of Cells with Ions

Likewise, as fluoride anions are the most reactive electronegative elements and, the mean radii extension of the unscreened 4f electronic configuration of La and Pr trivalent ions are relatively large, high electric surface charges could be developed via electric dipole interactions [47].

One crucial question stands whether a single ion binding on a specific site can activate tumour cell growth. Because the projected area of the 4f electronic configuration of a single RE ion is S _4f  = 0.040 and 0.043 nm ² (for an approximated spherical geometry of the 4f electronic configuration and a 4f mean orbital radii ~ r _4f =0.113 and 0.117 nm for Pr and La ions, respectively), a typical upper limit number of single RE ions, or other equivalent size ions, over the whole area of the cell membrane was ~ S_c / S _4f  = N _4f ~7.9 × 10 ⁹ RE ions; a number which is at least two orders of magnitude above the upper limit of the mean number of integrins on a tumour cell. As the relative overgrowth of cells was ascending with rising concentration (Fig. 9a), it is unlikely that tumour cell growth is triggered by a specific binding of single trivalent RE ions [48] on the ligand sites [49,50,51]. Indeed, the large number of RE ions should have saturated the cell’s growth and thus the viability of cells should have remain independent from the concentration of the RE ions.

Interaction of Integrins with RE-NPs

Within the requisite force range of few pN, and for efficient activation of integrins from NPs, the interaction between NPs and LABS should activate a large fraction of integrins of the cell for a long time. In the most extreme favoured case for cell growth, the number of NPs had to remain equal with the number of integrins on the cell’s surface, and the interactive force between LABS and NPs has to be attractive for obtaining a constant (long-term) action. A thin spherical shell of spherical NPs surrounding a tumour cell occupied a volume\( {V}_{sc}\approx 4\uppi {R}_c^2x \), where R _ㄷ  = 5 μm is the cell radius and x  ≈ 20 nm is half the separating distance between adjacent integrin receptors and V _sc  ≈ 6.3 x 10 ⁹ nm ³ . For justifying the requirement that each integrin receptor interacts only with one NP, a first estimation of the size of NPs to meet the above requirements for the whole set of integrins on a cell is obtained by dividing the volume of the spherical shell V _sc with the number of integrins. A simple calculation for a cell radius 5 μm shows that the limits of radii of NPs activating the whole set of integrins within the spherical shell volume V _sc  ≈ 6.3 x 10 ⁹ nm ³ covering the cell is obtained by divided the volume V _sc with the number of integrins \( {\overline{N}}_{int}\approx 2\ \mathrm{x}\ {10}^5 \) and n _int  ≈ 1.6 x 10 ⁷ . The volume of the spherical NPs stands for 3.15 × 10 ⁴ and 3.93 × 10 ² nm ³ 각기. Therefore, the radii of the NPs interacting with an integrin lay between ~ 20 and 5 nm. Allowing for one order of magnitude variations in the number of integrins \( {\overline{N}}_{int} \), the radii of the NPs interacting with integrins is between ~ 27 and ~  3 nm respectively.

By also applying similar simple calculations and within the experimental limits of concentration levels of RE-NPs (0.1–10 kg m ⁻³ ), the maximum numbers of PrF₃ with MHR 55–83 nm and LaF₃ with MHR 296–100 nm NPs (Fig. 1g, h) covering the surface of a tumour cell V _sc stood for 4.1 × 10 ⁴ –2.1 × 10 ⁴ and 17.1 × 10 ² –1.5 × 10 ⁴ NP. These values are placed well below the number of integrins on the cell surface. For rising concentrations of PrF₃ and LaF₃ from 0.1 and 10 kg m ⁻³ , the number of PrF₃ and LaF₃ NPs in the suspensions must go up for either descending or ascending size of NPs. As viabilities of cancer cells are raised at higher concentration levels, it is unlikely that 55–296 nm sized RE-NPs are responsible for cancer cell mitosis under the current experimental configuration.

Also, from the DLS data, the size of both RE-NPs between 10 and 20 nm remained constant (10.6 nm) at different RE concentrations. The number of RE-NPs with this size covering the cell surface is between 3.7 × 10 ⁵ and 1.5 × 10 ⁶ . This number is comparable with the mean number of integrins \( {\overline{N}}_{int}\approx 2\ \mathrm{x}\ {10}^5 \) on a cell surface. Therefore, only small size RE-NPs have the potency to stimulate cancer cell growth by stimulating all the integrins on a cell surface, in agreement with the experimental observations (Figs. 1g, h and 9a).

The number of tiny sizes RE-NPs with MEAC diameter (TEM) from 2 to 10 and 10 to 15 nm on the cell surface (S _ㄷ  = 314 μm ² ) stands for 1.3 × 10 ⁴ and 1.8 × 10 ⁴ RE-NPs, respectively. Those values stayed one order of magnitude below \( {\overline{N}}_{int}\approx 2\ \mathrm{x}\ {10}^5 \) and therefore tiny size RE-NPs had also the potency to justify the experimental results of rising viability values with concentration (Fig. 9a). Also, the rough surface of tumour cell (Fig. 9c) is able to form cavities, where small size RE-NPs are trapped, triggering thus cell’s mechanosensors. Most important, only tiny size RE-NPs have the potency to activate integrin receptors via electrical dipole interactions (vide infra).

Interaction of EGFR with RE-NPs

An upper limit of small size NPs capable of stimulating cell’s overgrowth via the EGFR was set previously to 14 nm [52], but a realistic size of NPs stimulating the EGFR should be < 5 nm [53] (Fig. 12). The area number density of EGFR on the surface of tumour cells stands for ~ 1.4 × 10 ⁻⁴ nm ⁻² and the total number of EGFR on the surface S _ㄷ of cells remains between ~ 4.2 x 10 ⁴ and 10 ⁵ [54,55,56]. RE-NPs with 5–10 nm size stayed for a number of 34 NPs (Fig. 3). Extrapolating this number to the surface of a cell S _ㄷ , the total number of RE-NPs remained at ~ 10 ⁴ NPs, a number which matches the number of EGFR receptors on a A549 cell. Therefore, the EGFR have the potency to be activated synergistically also by a number of tiny size RE-NPs.

AKT and ERK1/2 signal transduction pathways activated by RE-NPs via EGFR stimulation. EGFR is activated only by tiny size ~ 5 nm NPs

Electric Dipole Interaction Between RE-NPs and LABS

The above experimental results are supported by the hypothesis of cancer cell growth from LABS stimulation by tiny size core-shell RE-NPs via electrical dipole interactions, Appendix.

Indeed, the mean electrical dipole force \( \left\langle {\overrightarrow{F}}_{V_2}\right\rangle \)acting on LABS from a core-shell RE-NP includes two terms (Fig. 13d and Appendix, Eq. A22). The first radial term is inversely proportional to the forth power of separating distance r ₁ between the RE-NPs and LABS and is also proportional to the size of NP. The second polar term is inversely proportional to both the separating distance r ₁ and the square power of the size of NP,

아 Electrical dipole interaction between one core-shell RE-NP and one LABS. ㄴ , ㄷ RE core-shell NP near a MIDAS (b ) and ADMIDAS (c ) adhesion sites. d Locus area (green) of the size of RE-NPs and separating distance between a LABS and a core-shell RE-NP for two electrical charging states

식에서 1, G 그리고 G ₁ are the geometrical factors of NPs, describing either core-shell or core spherical structures, Appendix, Eqs. A6 and A14; 아니 ₁ , N ₂ are the numbers of surface electrons on the a RE-NPs and LABS surfaces; d 그리고 b are the effective characteristic spatial extension of atomic orbitals of LABS, ~ 0.1 nm, and the radius of RE-NP; 이 그리고 ε ₀ are the electron charge and the vacuum permittivity and \( \theta =\frac{d}{r_1}<0.01\ rad \). Because the core of the RE-NPs is a crystalline semiconductive material, an inherent large number of surface and volume defective sites were accountable for a high density of pseudo-electron energy levels that allowed the electrons to move freely within the core volume [46]. Consequently, a core-shell structure had the potency to be highly polarised. Therefore, LABS can be activated efficiently by core-shell RE-NPs via electrical dipole interactions at close separating distances. The high polarised efficiency of the core nucleus was confirmed experimentally via the selective orientation of NPs along two distinct directions (Fig. 2(a4–d4) and Fig. 3(a4, b4)).

The polar interaction force is also proportional to the geometrical factor G ₁ , Appendix, Eq. A14. Typical values of dielectric constants of the culture media, shell configuration and RE core components stand for ε ₁  = 78, ε ₂  = 10 and ε ₃  = 15. When the ratio of core-shell to core radii b/a sets within 1 and 50, the geometrical factors G , G ₁ retain almost constant values (G  = 0.2, G ₁  = 0.01) and they are self-same for both a spherical core (b/a  = 1) and a spherical core-shell. Any permanent or induced polarisation of an open or closed a-I-MIDAS domain forming the LABS domain has its origin on six coordinated water oxygen atomic orbitals with Mn ²⁺ or Mg ²⁺ ions, arranged in a spherical geometric configuration [7] (Fig. 12a–c).

As the electrical dipole force in Eq. 1 stands for the vector sum of a radial (first term) and a polar component (second term), the last term prevails over the first one provided that

In this case, a LABS is activated from the polar force component for all (b/a) ratios and, most important this term is inversely proportional to the second power of the size of NPs, in agreement with the experimental results that only tiny or small size LaF₃ NPs activated cancer cell proliferation.

The prevailed polar force term for different r ₁ 그리고 b values and for different Ν ₁ , Ν ₂ charging states activating the LABS/MIDAS stay within the limits [57,58,59,60].

Inequality 3 relates the size b of the RE core-shell NPs, the separating distance r ₁ and the number of the bound or free electrons Ν ₂ , Ν ₁ on the surface of the two dipoles. The locus of points (r ₁ , b ) satisfying the inequality 3 for different surface charge states Ν ₁ , Ν ₂ is bounded by the black, red and blue lines (Fig. 13c). As there was no specific assumptions for the type of RE-NP, results can be equally applied for any type of polarised NPs.

When the algebraic product of the number of the surface electrons N ₁ 및 N ₂ (bound or free) on the LABS and the RE-NP, respectively, was N ₁ 아니 ₂  = 2, the locus of RE-NPs size and separating distance for integrin activation was < 1 nm. At higher charging states, N ₁ 아니 ₂ =10 ⁴ , the locus area spans a wider RE-NPs size and separating distance area set of values, from 0.5 nm–19 nm to 2.5–15 nm, respectively.

From the above analysis, it is found that only tiny or small size NPs can activate LABS at a certain separating distance r ₁ and the electrical dipole interaction strength decays inversely proportional to the second power of the size of NPs. From Fig. 13c and for a charging state with N ₁ 아니 ₂  = 5 x 10 ⁴ , the size of NPs capable to activate LABS is bounded by the limits