Kleine zeldzame-aarde-fluoride-nanodeeltjes activeren tumorcelgroei via elektrische polaire interacties

Discussie

De relatieve groeisnelheid van kankercellen nam toe bij hogere concentratieniveaus van beide RE-NP's (Fig. 9a). De MHR-waarden van RE-NP's in DMEM+FBS werden echter direct gevolgd (PrF₃ ) en omgekeerd evenredig (LaF₃ ) tot de concentratie van RE-NP's van 0,1–10 kg m ⁻³ (Fig. 1g, h). Daarom zouden RE-NP's met een gemiddelde grootte van meer dan ~ 55 nm geen enkel effect moeten hebben op de celgroei en alleen kleine NP's kunnen een daadwerkelijke rol spelen in tumorgroei.

Grootte en structuur van RE-NP's

Identificatie van kleine RE-NP's

Uit de experimentele gegevens werden de gemiddelde grootte, distributie en de statistische parameters van RE-NP's geëxtraheerd. Door t . toe te passen teststatistieken voor de "nulhypothese" van "gemiddelde gelijke oppervlaktecirkel" van NP's in gedroogde PrF₃ en DMEM+FBS-schorsingen, de p waarde van de diameter van NP's tussen twee willekeurig geselecteerde AFM-afbeeldingen was ~  0,001 (aanvullend bestand 2). Een waarde van de MEAC-diameter (63 nm) werd met vertrouwen uit de AFM-gegevens gehaald en dit was vergelijkbaar met de MHR-waarde uit de DLS-gegevens (Fig. 1g).

Integendeel, de waarde van de MEAC-diameter van willekeurig geselecteerde LaF₃ monsters vertoonden een gemiddelde MEAC-diameter van 26 nm met een hogere afwijzingswaarschijnlijkheidswaarde (p = 0.07), wijzend op een afwijkend gedrag van LaF₃ in vloeibare suspensies. De discrepantie tussen de MEAC-diameter en de MHR van DLS (296 nm) (Fig. 1) is te wijten aan de complexiteit van interacties in LaF₃ weer schorsingen. Inderdaad, voor een 2 × 2 μm ² AFM tip scangebied, de gemiddelde z -hoogte was ~ 140 nm, wat de aanwezigheid van groot formaat LaF₃ . weergeeft NP's, overgebracht van de vloeibare suspensies op het substraat (figuur 4). Voor de "nulhypothese" van "gelijke MEAC-diameterwaarden van willekeurig geselecteerde TEM-monsters", de p waarden waren ook klein (p = 0,001). Voor beide RE-NP's zijn de gemiddelde MEAC-diameterwaarden geëxtraheerd uit de gecombineerde TEM- en XRD-gegevens voor beide PrF₃ en LaF₃ aangegeven hoge p waarden, p =0,29 en 0,06, waardoor er geen correlatie tussen de TEM- en XRD-gegevens mogelijk is. Alleen TEM, AFM (PrF₃ ) en DLS-gegevens waren voldoende betrouwbaar om de MEAC-diameter en core-shell-waarden te extraheren (aanvullend bestand 2).

Ook gaf een niet-isotrope hoekverdeling van Feret-diameters aan dat zowel PrF₃ en LaF₃ structuren waren sterk gepolariseerde diëlektrica, omdat de anisotrope hoekverdeling een indicatie is van sterke elektrische polaire interacties tussen nanokristallen. Een diverse gepolariseerde toestand van LaF₃ was verantwoordelijk voor het verlagen van de relatieve efficiëntie van de agglomeratietoestand in suspensies en het opschalen van de oppervlakteruwheidsparameters in gedroogde monsters.

Een softwaredeeltjesanalyse van willekeurige AFM-beelden voor 5 μL en een concentratie van 0,1 kg m ⁻³ identificeerde een aantal ~ 22 en ~ 11 RE-NP's met een grootte kleiner dan 15 nm en 10 nm (p = 0,001), en een aantal ~ 60 RE-NP's van TEM-beelden (p = 0,001) in een gebied van ~ 4 μm ² , bevestigend dus de aanwezigheid van kleine RE-NP's in de suspensies (Fig. 3(c), Aanvullend bestand 2) niet gedetecteerd met DLS.

Structuur en geometrie van RE-NP's

De grootteverdeling van RE-NP's is divergent in ethanol- en DMEM + FBS-suspensies (figuur 4). De diversiteit is het bezit van verschillende moleculaire interacties tussen de geadsorbeerde eiwitten, koolhydraten, elektrolyten en het oppervlak van RE-NP's, wat leidt tot de vorming van zeer complexe organische mantels (corona), die de specifieke interacties van RE-NP's met cellen in DMEM+FBS-medium.

Het samenspel tussen watermoleculen gevangen in hygroscopische RE-NP's en DMEM + FBS was ook van vitaal belang voor de vorming van kernschil. Het had ook een diepgaand effect op eiwit- en conformationele veranderingen in de tussenliggende interacties tijdens de beginfase van de voorbereiding. Omdat de oppervlakte-tot-bulkverhouding van NP's hoge waarden in de suspensies ontwikkelde, waren de effectieve stabiliteit en de fysisch-chemische, mechanische en stroomeigenschappen van RE-NP's, inclusief het vermogen om eiwitten te absorberen, buitengewoon gevarieerd [39,40,41] .

Vergelijkende grootteverdeling van RE-NP's in vloeistof (DLS) en gestolde suspensies via AFM en TEM toonde aan dat RE-NP's waren ingekapseld in organische vormen die diëlektrische kern-schaalstructuren vormden, waar een eiwitachtige schaal de RE-kern omringt. De AFM afgebeeld van gestolde RE-NP's in DMEM + FBS-suspensies die zijn afgezet op glassubstraten, wijzen ook op de vorming van veelzijdige RE-NP's en eiwitcoronacomplexen (Fig. 5). Terwijl gedroogde media een regelmatig zelf-geassembleerd patroon van kristalstructuren vormden (Fig. 5a-d), vertoonden gedroogde RE-NP's-suspensies een amorfe gelaagde structuur met verschillende zwarte vlekken, zelfs zichtbaar met de digitale camera van de AFM (Fig. 5e-l) . Bij hogere optische vergroting werden discrete agglomeraties van bolvormige vormen, kleiner dan die in het medium alleen, ook gedetecteerd voor beide RE-NP's in DMEM + FBS, samen met dendrietachtige structuren, die beide de complexiteit van interacties laten zien, in overeenstemming met de oppervlakteparameterresultaten (Fig. 4). Zelfs met de hoogste AFM-resolutie (1 × 1 μm ² gebied), de laatste baan van Fig. 5, werden geen geïsoleerde RE-NP's-aggregaties, binnen de resolutielimiet van ~ -5 nm, geïdentificeerd in gedroogde structuren voor beide RE-NP's. De zwarte bolvormige mycelia, 1-2 m lang, getoond in de optische afbeeldingen, waren grote agglomererende formaties van core-shell RE-NP's. De complexiteit van reacties tussen de RE-NP's en DMEM+FBS werd gevisualiseerd via de transformatie van de zelf-geassembleerde langwerpige structuren op lange termijn in pure DMEM+FBS naar dendrietstructuren.

De resultaten wijzen op het beeld van een enkele RE-NP-kernstructuur ingekapseld in een eiwitomhulsel. Deze structuren waren niet detecteerbaar omdat ze waren omgeven door organisch materiaal en elektrolyten, die beide een kruisreactie vertoonden met RE-NP's. Het VUV-spectrum van PrF₃ toont enkele spectrale pieken tussen 140 en 170 nm (Fig. 8). De ionische overgangen worden overlapt door een VUV-waterabsorptieband die is verlengd van 145 tot 180 nm met een maximum bij 168nm. Alleen de spectrale handtekeningen van de 4f6s elektronische configuratie met maxima bij 132 en 127 nm waren in het spectrum aanwezig. 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. een Structure and conformational geometries of integrins at a low (A), medium (B) and high-affinity strengths (C). b 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 _c  ≈ 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 _c 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 respectievelijk. 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. een TEM image of small size LaF₃ NPs surrounding the A549 cells. b Elemental analysis of F atoms in RE-NPs distributed around the cell. c 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 –c ) 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 _c  = 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 respectievelijk. 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's. 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 _c  = 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 _c 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 _c , 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,

een Electrical dipole interaction between one core-shell RE-NP and one LABS. b , c 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

In verg. 1, G en G ₁ are the geometrical factors of NPs, describing either core-shell or core spherical structures, Appendix, Eqs. A6 and A14; N ₁ , N ₂ are the numbers of surface electrons on the a RE-NPs and LABS surfaces; d en b are the effective characteristic spatial extension of atomic orbitals of LABS, ~ 0.1 nm, and the radius of RE-NP; e en 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 en 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 en N ₂ (bound or free) on the LABS and the RE-NP, respectively, was N ₁ N ₂  = 2, the locus of RE-NPs size and separating distance for integrin activation was < 1 nm. At higher charging states, N ₁ N 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 N ₂  = 5 x 10 ⁴ , the size of NPs capable to activate LABS is bounded by the limits