Hydroxyapatite and porphyrin-fullerene nanoparticles for diagnostic and therapeutic delivery of paramagnetic ions and radionuclides

Orlova Ma; Nikolaev Al; Nikolaev Al; Orlov Ap; Severin Av; Kalmykov Sn

HYDROXYAPATITE AND PORPHYRIN-FULLERENE NANOPARTICLES FOR DIAGNOSTIC AND THERAPEUTIC DELIVERY OF PARAMAGNETIC IONS AND RADIONUCLIDES

Orlova MA1,2 Nikolaev AL1, Trofimova TP1,3, Orlov AP1, Severin AV1, Kalmykov SN1

1 Faculty of Chemistry, Lomonosov Moscow State University, Moscow

2 Department of Biochemistry and Pharmacology,

Dmitry Rogachev National Medical Research Centre of Hematology, Oncology and Immunology, Moscow

3 Institute of Physiological Active Compounds of RAS, Chernogolovka

Nanoparticles for drug delivery are the subject of extensive research. Importantly, they can transform in size during synthesis or actual use, thereby changing their cytotoxic properties. The aim of the present work was to study the tendency of [®7Zn] porphyrin-fullerene nanoparticles (BFNP) to aggregate over time and to compare the properties of hydroxyapatite (HAP) nanoparticles obtained through 3 different techniques. We found that aggregation of BFNP nanoparticles does not affect their function but attenuates their cytotoxicity against leukemia cells. We were also able to obtain HAP nanoparticles with programmable properties (such as size, shape or the capacity to adsorb metal ions, ligands and chemical complexes) through enzymatic synthesis by varying its conditions. The synthesized HAP nanoparticles contain short-lived isotopes of zinc and copper (in the form of ions and complexes with pyrimidine or thiazine derivatives). These tumoricidal components (a radionuclide and a ligand or a complex) determine the diagnostic and therapeutic potential of the obtained radiopharmaceutical agents.

Keywords: hydroxyapatite, porphyrin-fullerene, thiazine and pyrimidine derivatives, HL-60, K-562, MOLT-4, zinc and copper

radionuclides

[XI Correspondence should be addressed: Marina A. Orlova

Leninskie gory 1, bl. 3, Moscow, 119992; orlova.radiochem@mail.ru

Received: 27.06.2018 Accepted: 20.09.2018

DOI: 10.24075/brsmu.2018.075

НАНОЧАСТИЦЫ НА ОСНОВЕ ГИДРОКСИАПАТИТА И ПОРФИРИНФУЛЛЕРЕНА ДЛЯ ДИАГНОСТИЧЕСКОГО И ТЕРАПЕВТИЧЕСКОГО ПРИМЕНЕНИЯ ПАРАМАГНИТНЫХ ИОНОВ И РАДИОНУКЛИДОВ

М. А. Орлова1,2 А. Л. Николаев1, Т. П. Трофимова1,3, А. П. Орлов1, А. В. Северин1, С. Н. Калмыков1

1 Химический факультет, Московский государственный университет имени М. В. Ломоносова, Москва

2 Отдел биохимии и фармакологии,

Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии имени Д. Рогачева, Москва

3 Институт физиологически активных веществ РАН, Черноголовка

Использование наночастиц как носителей лекарственных средств широко изучается. Одним из важных вопросов остается изменение размеров и цитотоксических свойств частиц в процессе их получения и применения. Целью работы было исследовать возможную агрегацию р^^порфиринфуллерен-наночастиц (BFNP) в зависимости от времени и провести сравнительный анализ свойств наночастиц гидроксиапатита (HAP), полученных различными способами. Оказалось, что агрегация BFNP качественно не влияет на функцию наночастиц, но количественно уменьшает их воздействие на лейкемические клетки. Варьирование способов получения и обработки наночастиц НАР позволяет менять их форму, размеры и сорбционную способность по отношению к ионам металлов, а также лигандам и комплексам. Используя ферментативный метод, мы получили НАР с заранее заданными свойствами путем варьирования условий синтеза. Полученные наночастицы НАР представляют собой радиопрепараты, содержащие короткоживущие изотопы цинка и меди (в виде ионов и соединений — производных тиазина и пиримидина). Эти наноконструкции содержат два антиопухолевых компонента (радионуклид и лиганд или комплекс), что определяет их фармакологический потенциал для диагностики и лучевой терапии.

Ключевые слова: гидроксиапатит, порфиринфуллерен, производные тиазина и пиримидина, HL-60, K-562, MOLT-4, радионуклиды цинка и меди

[23 Для корреспонденции: Марина Алексеевна Орлова

Ленинские горы, д. 1, стр. 3, г Москва, 119992; orlova.radiochem@mail.ru

Статья получена: 27.06.2018 Статья принята к печати: 20.09.2018

DOI: 10.24075/vrgmu.2018.075

The evolution of contemporary medicine prompts researchers to seek new approaches to drug design and administration, especially when it comes to highly toxic tumoricidal agents whose delivery to a biological target must be strictly precise.

Attention is increasingly paid to monoclonal antibodies [1], biological transporters [2], nanostructures and nanoplatforms. Especially worth mentioning are such carriers as fullerenes [3], nanodiamonds [4], inorganic nanoparticles of different nature

[5, 6], liposomes [7], nanoporous silicon [8], and hydroxyapatites (HAP) [9]. There are a few important goals that novel drug delivery systems are intended to achieve. First, they are expected to reduce the toxic effect of a carried drug on healthy organs and tissues. A good example here is doxil, the liposomal formulation of doxorubicin [10], Second, they can enhance the tumoricidal effect through the synergy of the drug/carrier complex [11]. For example, synthetic Buckminsterfullerene (C60)-2-(butadiene-l-yl)-tetra(o-y-aminobutyryl-o-phthalyl) porphyrin (BFNP) nanoparticles can be used to deliver magnetic isotopes 25Mg and 67Zn, which have a biological activity of their own. Such complexes can alleviate metabolic acidosis induced by post-chemotherapy hypoxia [12]. Third, drug delivery systems aim to increase the bioavailability of the drug (which can be done by using, say, albumin as a carrier), ensure its sustained release (nanofullerenes, HAP) and enhance the tumoricidal effect by binding to the target specifically (monoclonal antibodies). Fourth, a carrier and a drug constituting a delivery system can have different tumoricidal effects. For example, fullerene derivatives capable of killing cancer cells are used as carriers of magnetic isotopes or radionuclides [13]. This principle is employed in designing radiopharmaceuticals for anticancer radiation therapy.

HAP nanoparticles are attractive drug and radionuclide carriers due to their biodegradability, biocompatibility and bioresorbability. These qualities are determined by their calcium-phosphate origin, which mimics the chemical composition of the human bone mineral fraction [14, 15].

The aim of this work was to study the properties of НАР obtained through different methods of synthesis (and therefore exhibiting different properties) and BFNP in relation to their use as bioactive metal ion carriers, including radionuclides, and ligands with tumoricidal activity.

METHODS

Zinc complexes

The N(5,6-dihydro-4H-1,3-thiazine-2-yl)benzamide (L1) ligand in the form of L1HBr was synthesized following a previously described technique [16]. To obtain the L1ZnCl2 (C1) complex,

)

L1HBr in the aqueous solution was converted to its basic form in the presence of NaOH (Fig. 1). A solution of zinc chloride in diethyl ether at a ratio of 1 : 1 was gradually added to the L1 base in diethyl ether. The solution was stirred for 0.5 h. The white crystalline precipitate was separated and washed with ether. The reaction yield was 64%. Ligand L2 was 2-aminopyrimidine (Sigma; USA), ligand L3 was 2-aminopyrimidine salicylate. Their complexes with zinc Zn(L2)2Cl2 (C2) and Zn(L3)2 (C3) were obtained as described in [17]. The composition of all complexes was characterized by element analysis and 1H-NMR (Bruker CXP-200 spectrometer; Germany).

Spectrophotometry

Spectrophotometry was performed using the UV-1280 spectrophotometer (Shimadzu; Japan). Calibration curves were constructed for the ligands and the complexes in the aqueous, physiological saline and alcohol solutions.

Protonation and stability constants

The constants were determined by potentiometry using the automatic titrator Metrohm 848 Titrino plus (Metrohm AG; Switzerland). The stability constant was measured using the glass electrode. Computations were done in Hyperquad 2013.

HAP synthesis

Coprecipitation. There are different methods of HAP synthesis [18-20] producing end products with different parameters. We synthesized HAP1 at room temperature using the stoichiometric Ca/P molar ratio of 1.67 [21]. All reagents were taken in the amount sufficient to produce a 5% (solid mass content) suspension [22]. Trace amounts of calcium and zinc in the residual liquid were determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) on Optima 100 DV (Perkin Elmer; USA). To obtain HAP2, the suspension was continuously heated to 90 °C without boiling for 4 h.

Enzymatic hydrolysis. HAPE was synthesized through the enzymatic hydrolysis of calcium glycerophosphate in the presence of alkaline phosphatase (Merck; Germany). Reaction

(C2)

П

* ZnCh

-1 2

(C1)

Fig. 1. The structural formula of the compounds used In the present study

conditions were varied, including the enzyme concertation, the medium (water, glycerol buffer, tris buffer), pH (7 to 10), calcium concentration and the reaction time. The glycerophosphate hydrolysis reaction was triggered by adding the enzyme solution to the calcium glycerophosphate solution taken at a concentration of 0.02 mol/l. Working concentrations of the enzyme ranged from 0.02 to 0.4 pg/ml. Depending on the conditions, the reaction time was varied between a few hours and a few days.

Introduction of zinc and copper ions during HAP synthesis. Weighted amounts of zinc or copper oxides (chemically pure, C. P.) corresponding to the anticipated Zn(Cu)/Ca molar ratio of 5 (15 mol%) were dissolved in H3PO4. The obtained solution was introduced dropwise to the suspension of Ca(OH)2 under continuous stirring. The samples of HAPZn1, HAPZn2 and HAPCu were obtained. Some of those samples were dried, annealed in a muffle furnace in air at 900 °C for 3 h and subsequently studied with XPA (see below).

was carried out between the C1 complex in physiological saline and ethanol solutions (1 : 1) and a concentrated solution of carrier-free 69mZn eluted from a chromatography column. To obtain labeled C2 and C3 complexes, the ligands in the basic form were treated with 69mZnCl2 solution under continuous stirring and heating.

64>67Cu (T1/2 = 12.7 h and 61.8 h, respectively; Ey(64Cu) = 1345.8 keV, Ey(67Cu) = 184.5 keV) was produced by the reaction "atZn(y,np)64Cu; "atZn(n,P)67Cu. Copper was separated from zinc by extraction in the 0.001% dithizone solution in CCl4 followed by re-execration in 6M HCl and ion-exchange chromatography in the column with Cu-Resin (Triskem; France). Radioactivity of the reaction products was measured using a gamma-ray spectrometer with the GC 3020 HPGE detector (Canberra; USA). 67Zn and 25Mg (Isotope; Russia) are stable isotopes with s of -5/2 and +5/2, respectively (enrichment in 67Zn is 94.5%, isotope 25Mg frequency is 99.9%).

Electron microscopy of HAP samples

Size of nanoparticles

Some of the samples were sonicated in a sonic bath for 30 s at 22 kHz frequency and 50 W power. The samples were prepared for microscopy using a standard technique and then inspected under the JSM-6380LA microscope (JEOL; Japan) at the accelerating voltage of 20 kV.

Metal nuclides

69mZn (T1/2 = 13.7 h; Ey = 438.7 keV) was produced by the photonuclear reaction 71Ga(y, np)69mZn as described in [21]. To obtain a labeled compound, a reaction of isotope exchange

Nanoparticle sizes were measured by dynamic light scattering. Thin-layer chromatography (TLC)

For TLC we used Silufol plates with different eluents. The plates were developed with iodine vapors.

Autoradiography (ARG)

After TLC, the plates with the obtained radioactive compounds were analyzed using Cyclone Plus, the storage phosphor system

Table 1. Lethal concentrations (LC50) of Zn-BFNP and 67Zn-BFNP complexes for leukemic cell lines and healthy lymphocytes depending on the nanoparticle size

Complex LC50, Mg/ml

B-ALL AML HD*

Zn- BFNP 60 ± 8 64 ± 3 81 ± 9

67Zn- BFNP 16 ± 3 63 ± 8 79 ± 9

Average size of nanopartlcles and LC50

Zn- BFNP 25 nm 55 nm

67Zn- BFNP 37 ± 3 Mg/ml 78 ± 6, Mg/ml

Average size of nanopartlcles and LC50

Zn- BFNP 50 nm 80 nm

67Zn- BFNP 10 ± 2 Mg/ml 23 ± 3 Mg/ml

Note: *HD — lymphocytes obtained from healthy donors.

Table 2. Survival (LC50) of different cells in the presence of the studied zinc salts, chelators and HAPs

Compounds LC50, Mmol/ml

HD cells K-562 MOLT-4 MOLT-4 (res) HL-60 B-ALL*

ZnCl2 ** 1.6 ± 0.3 0.52 ± 0.05

ZnSal2(H2O)2 ** 0.68 ± 0.06 0.41 ± 0.05

C3 1.0 ± 0.2 0.25 ± 0.04

L1 10.6 ± 0.5 1.0 ± 0.3

C1 4.5 ± 0.4 1.1 ± 0.2

C2 0.24 ± 0.04 0.12 ± 0.02 0.062 ± 0.008 0.040 ± 0.009 0.039 ± 0.009 0.061 ± 0.007

HAP1 >> 5 • 10-3 mol/l 3.5 • 10-3 mol/l > 5 • 10-3 mol/l

hap2 >> 5 • 10-3 mol/l 4 • 10-3 mol/l > 5 • 10-3 mol/l

HAPZn1 >> 5 • 10-3 mol/l >> 5 • 10-3 mol/l > 5 • 10-3 mol/l

HAPZn2 >> 5 • 10-3 mol/l >> 5 • 10-3 mol/l 6,5 • 10-3 mol/l

hapcu >> 5 • 10-3 mol/l >> 5 • 10-3 mol/l 5 ^10-3 mol/l

Note: * — B-ALL designates BM cells of patients with B-cell acute lymphoblastic leukemia; ** — according to [17].

for digital ARG (Perkin Elmer; USA), and storage phosphor screens coated with BaFBr:Eu by the same manufacturer.

X-ray diffraction phase analysis (XPA)

The analysis was performed on the automated X-Ray diffractometer DRON-3 (Innoscope; Russia) with a Cu-Ka anode using the software supplied by the manufacturer. The size D of crystallites was determined by the Sherrer equation:

dhkl

л

ßcose

Measurements of adsorption

Measurements were done using the ASAP 2000 analyzer (Micromeritics; USA). The specific surface area was calculated in the Micromeritics software supplied by the manufacturer.

MTT-assay

The protocol of the assay is described in [13]. The cell lines used in the present study included HL-60, K-562, MOLT-4, and MOLT-4 (res) (res. means the cells were resistant to asparaginase, one of the main drugs used in children with acute leukemia). The cells were cultured following the standard protocol. Statistical analysis included the Mann Whitney U test. Each series of measurements consisted of at least 5 tests carried out in 3 replicates.

Isolation of mononuclear cells from bone marrow and peripheral blood

Bone marrow (BM) cells collected from patients with B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (TALL) were kindly provided by D.Rogachev National Research Center of Pediatric Hematology, Oncology and Immunology. Blood tests demonstrated that the proportion of blast cells in the peripheral blood mononuclear fraction was at least 80%.

Cell morphology was inspected under the fluorescence microscope LEICA DM6000B (Leica Microsystems; Germany) or the confocal laser scanning microscope LSM 710 (Carl Zeiss; Germany). The images were captured and saved by a digital camera.

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RESULTS

Relationship between the size of porphyrin-fullerene nanoparticles and their cytotoxicity

Some findings suggest that increased aggregation of nanoparticles (in particular, fullerene-based) promotes survival leading to a rise in LC50, i.e. attenuates the cytotoxic effect of a drug [23-26]. However, there is counterevidence to that, mainly for nanoC60. So, we decided to study the effect of aggregation in BFNP.

Table 1 presents data on the survival of leukemia cell lines and BM cells of untreated patients with acute leukemias in the presence of BFNP loaded with a naturally occurring zinc isotope or enriched in magnetic 67Zn. The table demonstrates the relationship between LC50 and the nanoparticle size. In all cases, cell survival improved with increased nanoparticle aggregation, but the specificity of the studied complexes did not change.

Sonication caused the average nanoparticle size to shrink to 15-20 nm. However, a week after the average nanoparticle size reached 20-30 nm; 2 weeks after, 30-40 nm; one month after it was as big as 50-60 nm.

Morphological examination revealed that not only nanoparticles but also cells treated with 67Zn-loaded BFNP tended to aggregate increasingly (these data are not provided in the table).

Effect of ligands and HAP-ligand compositions on the cells

Both ligands and their complexes with metal ions, such as zinc or copper (which can be substituted with radionuclides to produce a radiopharmaceutical), can be regarded as potential tumoricidal chelating agents and used for HAP doping. Ligands

А

B

Fig. 2. MOLT-4 cells treated with L3 (A) and C3 (B) under the confocal laser scanning microscope

0.7 0.6 0.5 m 0.4

-Q <

0.3 0.2 0.1

200

250

B

300 Л (nm)

350

400

0.1

0

200

250

300 Л (nm)

350

400

Fig. 3. Absorption spectra of 10-2 mg/ml L1 solution that was in contact with HAP2 (A) and HAP1 (B) for 0 min (1), 5 min (2), 10 min (3), 15 min (4), 30 min (5), and 60 min (6)

0

and metal ions can be loaded to HAP together as complexes or separately. In both cases, the complex and its components will produce either synergistic or independent effects.

Table 2 compares the survival of K-562 cells and lymphocytes of healthy donors (HD) in the presence of L1 and complexes. Both healthy lymphocytes and MOLT-4 u MOLT-4 (res) cells demonstrated very high survival rates when treated with different HAP samples, including those that contained zinc and copper ions.

The studied chelators and complexes displayed a tendency to increased (decreased) toxicity against healthy lymphocytes and cancer cells. The lymphocytes of healthy donors demonstrated the following pattern of survival rate decline: L1 > C1 > ZnCl2 > C3 > ZnSal2(H2O)2 > C2. With chronic myeloid leukemia cells (K-562), the pattern changed: C1 > L1 > ZnCl2 > ZnSal2(H2O)2 > C3 > C2. The therapeutic window (or the therapeutic index TI = LC50 (HD)/LC50 (leukemia cells)) declined in the following fashion: L1 > C3 ~ C1 > ZnCl2 > C2 > ZnSal2(H2O)2. This leads us to conclude that all 3 studied complexes have a potential to be considered as tumoricidal agents.

When comparing the toxic effects of C2 on leukemia cell lines and BM cells obtained from patients with B-ALL, we observed the following pattern: HD cells > K-562 > HL-60 ~ MOLT-4 > MOLT-4(res) ~ BM (B-ALL). Importantly, C2 exhibited higher toxicity towards MOLT-4 (res) than to MOLT-4 cells, meaning it has specificity to the cells with the most resistance to chemotherapy. The complexes tended to exhibit higher cytotoxicity than the ligands, perhaps due to the development of necrosis in addition to apoptosis (Fig. 2).

Stability of ligands and their complexes in aqueous/ physiological saline solutions is their important property. Unfortunately, much more common are bioactive chelating ligands and complexes that are poorly soluble. They need a special shell or a carrier to be delivered to a target. HAP meets this requirement only partly.

Spectrophotometric measurements of stability of the studied complexes in water, ethanol and physiological saline solutions revealed that C1 was the least stable complex: it tended to hydrolyze over time producing thiazine that, however, has mild tumoricidal and strong radioprotective properties.

Other complexes were quite stable when dissolved, which makes them more suitable for clinical purposes.

As determined by the potentiometric titration of the ligand L1 performed in aqueous and physiological saline solutions with varying pH (at C (L1) = 1 • 10-3 mol/l, I = 0.15 NaCl (0.1 mol/l KNO3)), the values of protonation constants (log K) were 5.1 ± 0.1 (aqueous solutions) and 5.3 ± 0.2 (physiological solutions). Attempts to potentiometrically determine the stability constant of C1 failed. This might have been due to the production of zinc hydroxide that interfered with titration. For C2 the stability constant logK (C2) was 10.4 ± 0.5.

Behavior of nanoHAP doped with ligands, metal ions and/or complexes

Fig. 3 (A, B) shows sorption of the ligand L1 by HAP1 and HAP2. Changes occur when sorption starts and are probably due to the interaction with calcium released as nanoHAP dissolves. It means that the ligand L1 does not bind to hydroxyapatite. The same behavior was observed for L2 and L3.

The morphology of nanocrystals can be visualized using electron microscopy, while the composition of the solid phase (biohydroxyapatite) can be inferred from the results of X-ray diffraction phase analysis. For HAP1, results of these analytical modalities are presented in Fig. 4. When subjected to heating, both zinc-loaded and "pure" nanoparticles shrank in size. This transformation was considerable for "pure" samples and almost insignificant for those doped with zinc (Table 3).

The major phase of HAPE (Fig. 5A) was hydroxyapatite (Fig. 5B); HAPE nanoparticles tended to have a spherical shape and formed aggregates. The specific surface area of the studied sample determined by nitrogen absorption was 300 m2/g. Pore sizes varied considerably (2-300 nm). The size of crystallites in the samples with crystal structure was calculated using the Sherrer equation (12-14 nm). Crystallites and aggregates significantly varied in size; their average dimensions are shown in Fig. 6.

We synthesized 6 different types of HAP: 3 without metal ions (HAP1, HAP2, HAPe) and 3 with metal ions (HAPZn1, HAPZn2, HAPCu). HAPE were big-sized hollow spheres that

20 25 30 35 40 45 50 55 60 65 70

Fig. 4. Electron microscopy (A) and X-ray diffraction phase analysis (B) of HAP1 synthesized from the suspension of calcium oxide and phosphorus acid Table 3. Average sizes of HAP nanoparticles obtained through precipitation and HAP nanoparticles doped with zinc

Parameter HAP1 HAPZ1 Zn1 HAPZn2 hap2

Length, nm 120 ± 5 110 ± 5 98 ± 4 56 ± 2

Width, nm 36 ± 3 23 ± 2 26 ± 2 19 ± 1

А

цг

Р р 00 032 1 рл

Fig. 5. Laser scanning electron microscopy (A) and X-ray diffraction phase analysis (B) of HAPE synthesized from the aqueous solution of calcium glycerophosphate (0.2 mol/l) at the alkaline phosphatase concertation of 0.1 Mg/ml

D(jm)

Fig. 6. Differential function of nanoparticle diameter distribution in the sample synthesized in the tris buffer (pH = 9.2) in the presence of alkaline phosphatase taken at a concentration of: 1) 0.02 jg/ml (the average nanoparticle size was 1.57 |jm); 2) 0.1 jg/ml (the average nanoparticle size was 2.22 |jm)

exhibited increased sorption capacity. Averages sizes of other nanoparticles were as follows: HAP1 > HAPZ 1 > HAPZ 2 ~ HAPCu > hap2.

We measured specific adsorption of zinc/copper ions by HAP1 and HAPE and calculated adsorption isotherm constants using Langmuir and Freundlich methods. Adsorption of copper ions corresponded to the Langmuir model, while adsorption of zinc ions (with equal correlation coefficients) could be described by both Langmuir and Freundlich models.

The sorption capacity of HAP towards copper ions was significantly higher than towards zinc ions. Introduction of zinc into the reaction during HAP synthesis did not seriously affect the size of nanoparticles. However, average sizes of a thermally processed HAP2 were smaller than those of HAP1. HAP2 particles acquired a more regular isometric shape growing in thickness, as compared to HAP1, and therefore had a smaller specific surface area. As a result, the maximum sorption capacity of HAP2 decreased.

Complexes with zinc and copper radionuclides

Complexes 69mZnC1 (I), 69mZnC3 (II) and [(L2)264,67CuClJ (III) were obtained through isotope exchange and analyzed by TLC, ARG and y-spectroscopy. The time of isotope exchange was selected experimentally to be sure that the Rf values of radioactive complexes and those that did not contain a radionuclide were the same and also to prevent formation of side products. The obtained compounds were sorbed onto

HAP and physical and chemical measurements were then repeated. The ready pharmaceuticals had similar properties, but the copper complex disintegrated during HAP1 doping. HAP1 absorbed only copper ions. Perhaps, this problem can be solved by loading HAP with the copper complex during HAP synthesis.

DISCUSSION

Previously, it was demonstrated that the magnetic isotope 25Mg (amounting to 11% in the natural isotope mixture) hyper-activates magnesium-dependent regulation of ATP synthesis, which makes the delivery of this isotope to hypoxic tissues/ cells an ambitious pharmacological task [12]. Here, BFNP nanoparticles capable of sustained release of Mg2+ and Zn2+ ions can be used as carriers. However, the major constraint for the clinical application of fullerene derivatives is their possible uncontrolled aggregation accompanied by changes in their initial cytotoxicity and physiological properties. Our study demonstrates that size transformations do not lead to critical changes in the properties of nanoparticles but attenuate their cytotoxic effect. The fundamental finding of this study is that survival of BM cells of untreated patients with B-ALL decreases 6-7-fold in the presence of 67Zn-BFNP, as compared to natZn-BFNP. Aggregation of nanoparticles can be slowed down by sonication.

HAP nanoparticles have good prospects as drug delivery systems. There are a lot of methods for their synthesis that

produce particles of different sizes, shapes and sorption capacity. HAP nanoparticles are also lowly toxic. Introduction of ligands and complexes into the reaction during HAP synthesis is a clever trick that helps the components of the complex to retain their properties. Enzymatic synthesis seems to be especially promising as it allows varying reaction conditions and, therefore, the parameters of the end product. This significantly expands the area of the potential application of nanoparticles.

Zinc and copper are essential micronutrients [27, 28]. They are cofactors for the majority of regulatory and antioxidant enzymes; they also are involved in DNA repair and the work of transcription factors. Complexes of short-lived zinc and copper radionuclides can be used to solve a number of diagnostic and therapeutic tasks, either simultaneously or consecutively. HAP can serve as a scaffold for a drug ensuring a synergistic sustained effect. To achieve it, a vector is needed, monoclonal antibodies being the most optimal. Importantly, HAP itself has an ability to integrate into bone tissue and become a vector. This matters in the therapy of bone and blood cancers aimed at eliminating malignant stem cells.

In our experiments, the specific activity of the obtained radioactive agents was not high, because the experiments were preliminary and aimed to demonstrate that such complexes can be synthesized in reasonable time for the use in the clinical setting. We have sufficient resources to improve their specific activity.

CONCLUSIONS

Transformation of 67Zn-BFNP sizes following their aggregation does not affect their function but attenuates their cytotoxicity against leukemic cells. By applying different methods of HAP synthesis and processing, one can alter the sorption capacity of HAP towards metal ions, ligands and complexes. HAPE with preprogrammed properties can be synthesized by varying reaction conditions. We have synthesized HAP nanoparticles containing short-lived zinc/copper isotopes in the form of ions and compounds. These nanostructures have a good potential to solve a number of diagnostic and therapeutic tasks in patients with cancer.

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