Experimental and Theoretical Study of Doxorubicin Physicochemical

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C: Surfaces, Interfaces, Porous Materials, and Catalysis

Experimental and Theoretical Study of Doxorubicin Physicochemical Interaction with BN(O) Drug Delivery Nanocarriers Elizaveta S. Permyakova, Liubov Yu. Antipina, Andrey M. Kovalskii, Kristina Yu. Gudz, Josef Pol#ák, Pavel B. Sorokin, Anton M. Manakhov, and Dmitry V. Shtansky J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b07531 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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The Journal of Physical Chemistry

Experimental and Theoretical Study of Doxorubicin Physicochemical Interaction with BN(O) Drug Delivery Nanocarriers Elizaveta S. Permyakova,1* Liubov Yu. Antipina,1 Andrey M. Kovalskii,1 Irina Y. Zhitnyak,2 Kristina Yu. Gudz,1 Josef Polčak,3,4 Pavel B. Sorokin,1 Anton M. Manakhov,1 Dmitry V. Shtansky1* 1

National University of Science and Technology “MISIS”, Leninsky prospect 4, Moscow, 119049, Russia

2

N.N. Blokhin Medical Research Center of Oncology, Kashirskoe Shosse 24, Moscow 115478, Russia

3

Institute of Physical Engineering, Brno University of Technology, Technicka 2896/2, 616 69 Brno, Czech Republic

4

CEITEC-Central European Institute of Technology, Brno University of Technology, Technická 3058/10, 61600 Brno, Czech Republic; [email protected]

Research highlight - Chemical interaction of doxorubicin with BN(O) nanoparticles - Oxidation of BN nanoparticles significantly improves their interaction with DOX

- Formation of DOX-BN conjugates depends on electron density in HOMO of DOX - Protonated NH2 groups in DOX facilitate electron density transfer from DOXH+ to BNO

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ABSTRACT: Drug loaded nanocarriers have a great potential for tumor therapy. Such systems must have high drug loading efficacy in an alkaline medium and effectively release therapeutic agent in an acidic medium of endosomal/lysosomal compartments of tumor cells. Herein we experimentally and theoretically (using density functional theory) studied the chemical interaction of doxorubicin (DOX) with different BN surfaces depending on the degree of their oxidation. Three groups of hexagonal BN nanoparticles (BNNPs) obtained by boron oxide CVD process, i.e. (i) as-synthesized and those after (ii) repeated washing in water and (iii) hightemperature annealing, and their corresponding DOX-BN conjugates were studied. Oxidation of BNNPs significantly improved their interaction with DOX. As a result, the amount of immobilized DOX on the B2O3 surface was higher in comparison with the BNNPs containing little oxygen. The formation of stable DOX-BN conjugates mainly depended on the attraction of electron density in the area of aromatic rings in highest occupied molecular orbital of DOX. The presence of a protonated NH2 groups in DOX can facilitate electron density transfer from the DOXH+ to the boron oxide surface.

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1. INTRODUCTION Currently, chemotherapy (along with surgery and radiotherapy) is one of the main methods of cancer treatment. The main disadvantage of this approach is the huge dosage of therapeutic component that leads to severe side effects. A promising direction in the field of cancer therapy is the development of new drug delivery nanocarriers that can hide the drug inside and protect it from non-targeted interaction with eukaryotic cells and, thereby, reduce the required dose of therapeutic agent, increase safe circulation time in the body, improve drug uptake by cancer cells, selectivity, and, in the end, therapeutic efficacy. Boron nitride nanoparticles (BNNPs) were reported to be a promising biocompatible drug delivery system with pH-sensitive release of therapeutic agent aimed at a cancerous tumor.1-3 Moreover, the utilization of BN nanovehicles allow one to achieve a higher content of boron atoms in tumor cells in comparison with blood and other organs,4 which can be used for effective boron neutron capture therapy and diagnostics. Doxorubicin (DOX, adriamycin, C27H29NO11) is commonly used in various drug delivery systems due to its ability to effectively treat many types of cancer and the possibility of its detection by various methods, such as confocal microscopy, Fourier-transform infrared (FTIR) spectroscopy, UV–visible spectroscopy and others.5 DOX demonstrates chemotherapeutic effect by interacting with DNA, generating free radicals and direct exposure to cell membranes with suppression of nucleic acids production. This is achieved by intercalating between adjacent pairs of DNA bases and preventing replication, causing conformational changes in the DNA molecule. However, the DOX is also highly toxic, especially to the heart and kidneys, limiting its broad therapeutic applications. Thus novel DOX-delivery strategies are eagerly awaited in the medicine market for its more effective and safe application.6,7 It is amazing that many drug delivery nanoconjugates (DDNCs) demonstrate a strong pH-sensitive behavior (adsorbing drug at high pH and releasing therapeutic agent at low pH).4,8,9 For example, single-walled carbon nanotubes (SWCNTs) remained bound to DOX at pH > 7, yet readily released drug in acidic medium.10 The loading efficiency of DOX into BN nanotubes (BNNTs) increased with an increase in the pH value. The acid dissociation constant of DOX (pKa) that indicates at what pH the hydrogen is separated from the molecule (DOXH+)=DOX+H+) was reported to be 8.2.11 DOX is considered to be hydrophilic at lower pH due to the 3 ACS Paragon Plus Environment

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presence of positively charged amino –NH3+ groups. The increased deprotonation of -NH2 groups in DOX at higher pH 9.0 accounts for the increased hydrophobicity and decreased solubility of DOX,7 which leads to increased interaction between DOX and BNNTs. This feature has very important implication to regulate drug loading and leaching from the BNNP-based DDNCs since the physiological environment is weakly basic (pH 7.4), while intracellular lysosomes are acidic, hereby enabling high extracellular loading of DOX and its effective release inside tumor cells. The spontaneous encapsulation of various biomolecules such as single strand DNA,12-15 protein,16,17and anticancer drug molecules18 has been well established. Herein, we study the immobilization of DOX onto the surface of BNNPs depending on the degree of their oxidation and acidity of medium. Zeta potentials of DOX-BNNP complexes at different pH were determined to uncover the physicochemical regularities of the DOX-BNNPs interaction. Using X-ray photoelectron spectroscopy it was demonstrated that the presence of boron oxide in the BNNPs increased the amount of loaded DOX. In addition, the saturation of BNNP surface with DOX (i.e. the DOX adsorption on the surface of BNNP) depending on the protonation of the -NH2 groups in DOX and the presence of different defects on the BNNP surface was systematically investigated using density functional theory (DFT) calculations. It was well established that the adsorption plays an important role in loading and releasing of DOX to/from BNNP nanocarriers. 2. EXPERIMENTAL SECTION 2.1. Preparation of BN Nanoparticles. BNNPs were synthesized in a boron oxide CVD process using a vertical induction heating reactor, as described elsewhere.19 After the synthesis, the BNNPs formed agglomerates. In order to separate the agglomerates into individual nanoparticles, the BNNPs were ultrasonically treated in a distilled water solution (BNNPs concentration 2 mg/ml) using a Bandelin Sonoplus HD2200 unit (Germany) at a power of 80 W for 30 min. The resultant BNNPs were 200-300 nm in size. The energy dispersive spectroscopy analysis revealed that the BNNPs contained boron oxide. To estimate the influence boron oxide on the chemical interaction of BNNPs with DOX, nanoparticles were divided into three groups. Samples of the first group were washed in water three times during centrifugation at 9000 rpm for 15 min. The second group of BNNPs was annealed at 1580 ºC for 2 h. The remaining samples (third group) were not subjected to additional processing and were used in the as-synthesized state. 2.2 Saturation of BNNPs with DOX. 4 mg of BNNPs was suspended in 5 ml of DOX solutions (0.5 mg/ml) at different pH values (4.4, 5.4, 6.4, 7.4, 8.0, 9.0, 10.0). The suspensions were incubated at room temperature for 24 h. The precipitates were then washed out from the DOX in water 10 times under repeated 4 ACS Paragon Plus Environment

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centrifugation at 9000 rpm for 15 min. Dispersions of the BNNPs and DOX-BNNPs complexes were further investigated using zeta potential measurements. 2.3. Drug Loading Capacity. The amount of DOX loaded into BNNPs, BNNPswashed, and BNNPsannealed was determined by comparing the intensities of the absorption of DOX in the initial DOX solution used for loading of different types of BNNP with the supernatant DOX solution after removal of all DOX-BNNPs. 0.2 mL of samples were analyzed by a “Benchmark Plus” spectrophotometer (Bio-Rad) at the laser wavelength of 488 nm using “Microplate Manager 5.2.1” software. 2.4 Material Characterization. The morphology of synthesized BNNPs and DOX-BNNPs was studied using a scanning electron microscope JSM-7600F (JEOL) equipped with the energy-dispersive X-ray (EDX) detector. Transmission electron microscopy (TEM) studies, including high resolution TEM (HRTEM) and high-angular dark field scanning TEM (HADF-STEM) imaging, were carried out using a JEM 2100 microscope (JEOL) operated at 200 kV. The chemical and phase compositions were analyzed by energydispersive X-ray spectroscopy (EDS) using a 80 mm2 X-Max EDX detector (Oxford Instruments) and Fouriertransform infrared spectroscopy (FTIR) with a Vertex 70v vacuum spectrometer (Bruker) in the range of 400– 4000 cm−1 using the partial internal reflection device. X-ray photoelectron spectroscopy (XPS) studies of the BNNPs and DOX-BNNPs complexes were carried out on an Axis Ultra DLD instrument (Kratos Analytical Ltd) equipped with a monochromatic Al Kα X-ray source (hυ = 1486.6 eV). The maximum lateral dimension of the analyzed area was 0.7 mm. The spectra were fitted using CasaXPS software after subtracting Shirleytype background and using methodology described in our previous work.20 In addition, zeta potential measurements were carried out using a Zetasizer Nano ZS unit (Malvern Panalytical). 2.4 Atomistic Simulations. Ab initio calculations of the atomic structure and stability of the system were performed using DFT21,22 within the generalized gradient approximation (GGA)23 using the normalized Troullier-Martins pseudopotentials24 in SIESTA software package.25,26 Numerical pseudoatomic wave functions were used as a basis for atomic localized orbitals. Grimme DFT-D3 corrections27 were applied to include van der Waals interactions between DOX and h-BN surface. The system under consideration was modeled as a supercell with the sufficiently large vacuum gap (not less than 15 Å) to exclude the intermolecular interaction in the non-periodic direction. The structural relaxation was performed until the forces acting on each atom became less than 0.03 eV/Å. The real-space mesh cutoff was set to at least 175 Ry. To calculate the equilibrium atomic structures, the Brillouin zone was sampled according to the Monkhorst– Pack scheme;28 k-grid cutoff was equal to 6 Å. For calculation of electronic properties, the k-grid cutoff was equal to 24 Å.

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3. RESULTS AND DISCUSSION 3.1. Characterization of BNNPs. SEM images of the BNNPs are shown in Fig. 1a. Particle size analysis performed after ultrasonic treatment revealed that a major fraction of BNNPs falls within the range of 250-500 nm (Fig. 1a(inset)). The BNNPs had nearly spherical morphology, smooth surface and hollow central part with a shell thickness in the range of 50-70 nm (Fig. 1a(inset)). 3.2. FTIR Spectroscopy Analysis of BNNPs and DOX-BNNP Conjugates. Figure 1b compares the FTIR spectra of pure DOX, BNNPs and DOX-BNNP conjugates. The BNNPs show two characteristic features in their FTIR spectrum: an asymmetric peak at 769 cm−1 with a shoulder on low wave number side and a broad maximum with high intensity at 1359 cm−1, which correspond to out-of-plane B−N−B bending and in-plane B−N stretching vibrations, respectively. The FTIR spectrum of B2O3 was reported to contain two intense absorption bands located at approximately 1400 and 1265 cm-1, and a peak of lower intensity at 720 cm-1.29 Since the B2O3 peaks were located close to the BN peaks, their superposition led to broadening of the maxima. The DOX spectrum is characterized by a number of peaks that can be ascribed to NH2 or OH (3300 cm−1), C−H (2883 cm−1 and 1200−1400 cm−1), C−O stretching (1000−1200 cm−1 ), and out-of-plane O−H bending vibrations (700−900 cm−1 ).28,30 After BNNPs saturating with DOX, some of the DOX peaks were well seen in the FTIR spectrum of DOX-BNNPs sample, hereby indicating the presence of drug on the BNNPs surface (Fig. 1b(right panel)). 3.3. DOX Loading Efficiency of BNNPs. First, we studied the efficiency of DOX loading into different types of BNNPs (BNNPs, BNNPswashed, and BNNPsannealed ) in DOX solutions with pH 8.4 (Fig. 2). The spectrophotometric analysis of DOX solutions after BNNP saturation and their removal from solution showed that the concentration of free DOX significantly decreased with increasing B2O3, thereby demonstrating a more efficient DOX loading into BNNPs (244 μg/mg, 98%). After saturation of 2 mg/mL of BNNPsannealed in 1 mL of DOX solution (0.5 mg/mL), the DOX loading capacity was 17 μg/mg (5%). 3.4. Surface Zeta Potential Measurements. Dispersions of the BNNPs and DOX-BNNPs were examined by measuring their zeta potentials at different pH (Fig. 1c). In neutral and alkaline media the surface zeta potential decreased along the row BNNPs  BNNPswashed  BNNPsannealed. The addition of DOX led to a further decrease in zeta potential of the BNNPs. This is in good agreement with the available literary data.31 It should be remarked that the negative values of surface zeta potential are necessary for long-term nanoconjugate circulation in the body and their effective delivery to cells. Nanoparticles carrying a positive charge were reported to have a toxic effect on living cells.32 BNNPs efficiently interact with DOX via π-π stacking interaction between aromatic rings that contain π bonds.33 Note, however, that boron oxide 6 ACS Paragon Plus Environment

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significantly affects the interaction between DOX molecules and nanoparticle surface since the boron oxide can react with hydroxyl groups of DOX in alkaline conditions.3 DOX is considered to be hydrophilic at low pH because of the presence of positively charged amino –NH3+ groups. The increased deprotonation of –NH2 groups in DOX at higher pH accounts for the increased hydrophobicity and decreased solubility of DOX, which lead to increased interaction between DOX and BNNPs.4 The isoelectric point for all types of BNNPs and DOX-BNNPs conjugates was observed at pH 5.2. Thus a change in the surface charge leads to DOX leaching out from the DOX-BNNPs conjugates. 3.5. XPS Analysis of BNNPs before DOX Immobilization. The characteristic XPS B1s and N1s spectra of BNNPs and DOX-BNNPs conjugates obtained by different methods are presented in Figs. 3 and 4. The content of all elements on the sample surfaces are summarized in Table 1 and the percentage of different B environments is depicted in Table 2. The XPS B1s spectrum of the as-synthesized BNNPs was deconvoluted into three peaks located at 190.7, 191.7 and 192.8 eV that corresponded to the BN, BNO and B2O3 bonds, respectively (Fig. 3a). The content of BNO and B2O3 species was very high, whereas the contribution of BN component to the total peak intensity was low. The XPS N1s spectrum of the as-prepared BNNPs can be well divided into three components BN, N-C=O and NH3+ located at 398.6, 399.9 and 402 eV, respectively (Fig. 4a). The XPS data indicate that the content of oxygen and the B/N ratio in the as-prepared BNNPs was very high and it can be assumed that B environment was mostly represented by BO and BNO species.

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100 nm

Surface zeta potential, mV

100 nm

c)

Intensity (a.u.)

a)

Intensity (a.u)

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4000

3500

3000

1500

1000

500

Wave number (cm-1) b) 2000

1500

1000

500

Wave number (cm-1)

Figure 1. SEM and TEM image of BNNPs (a) with enlarged image, showing hollow central part and a shell thickness, and BNNPs size distribution obtained after ultrasonic treatment (insets), FTIR spectra (b) and surface zeta potentials of BNNPs and DOX-BNNPs complexes at the different pH (c). 1 – BNNPs, 2 – BNNPswashed, 3 – BNNPsannealed, 4 – DOX-BNNPs, 5 – DOX-BNNPswashes, 6 – DOX-BNNPsannealed, 7 – DOX.

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Figure 2. Efficiency of DOX loading into various types of BNNPs in sodium acetate buffer solutions at pH 8.4. Data are presented as a percentage of DOX content in DOX-BNNPs, DOX-BNNPswashed, and DOXBNNPsannealed (loaded DOX) or in solution (free DOX) to total amount of DOX in the DOX solution used for loading (0.5 mg/ml) (a) and an amount of loaded DOX per 1 mg of nanoparticles (b). Washing of BNNPs led to significant changes both in their elemental (decrease of O and B concentrations, see Table 1) and phase compositions. In discussing the XPS results we should first note that the content of BN significantly increased at the expense of BNO and B2O3 (Fig. 3b). The B2O3 species completely disappeared and replaced by the boron suboxides BOx (binding energy (BE) ~ 192.3 eV). The XPS N1s signal from the BNNPswashed sample was fitted using two components, i.e. BN and BNO located at the BE of 398.3 and 398.6 eV, respectively (Fig. 4b). It should be noted that the positions of both environments are very close and, therefore, the deconvolution of the XPS N1s peak may lead to large errors. However, it is important to highlight that the BNNPswashed sample had the B/N ration close to 1, hereby indicating the presence of almost stoichiometric BN. The annealing of BNNPs led to a significant decrease in boron oxide environment (oxygen content decreased to 4.4 at.%). Since the B/N ratio was about 0.95, some nitrogen vacancies on the BNNPsannealed surface could be expected. The content of B and N environments were determined using the same XPS B1s curve fitting procedure as described above for the BNNPswashed sample and the obtained results are presented in Table 2 and 3. 3.6. XPS Analysis of BNNPs after DOX Immobilization. The immobilization of DOX onto the surface of asprepared BNNPs led to significant changes in the elemental composition (increase in carbon content) and in the B1s, C1s and N1s environments. Curve fitting of the XPS B1s peak suggests that B exists in the DOX-BNNPs sample as two different environments: BOx and BN (Fig. 3d). It is also likely that oxygen in the BOx was also bound to carbon forming a B-O-C bridge; but this assumption cannot be confirmed by XPS. The nitrogen environment was also modified by DOX immobilization, as NH3+ and N-C=O components made a significant contribution to the overall concentration (Table 3). 9 ACS Paragon Plus Environment

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Figure 3. XPS B1s spectra and their curve fitting. (a) BNNPs, (b) BNNPswashed, (c) BNNPsannealed, (d) DOXBNNPs, (e) DOX-BNNPswashes, (f) DOX-BNNPsannealed. Table 1. Atomic Compositions Derived from XPS Data Sample BNNPs DOX-BNNPs BNNPswashed DOX-BNNPswashed

Concentration, at.% [B] [C] 31.8 14.3 5.5 65.1 39.2 8.2 29.0 23.3

B/N [N] 19.0 4.7 38.1 28.2

[O] 34.9 24.7 14.5 19.5 10

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1.67 1.17 1.03 1.03

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BNNPsannealed DOX-BNNPsannealed

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39.8 25.3

14.4 36.8

41.7 24.8

4.2 13.2

0.95 1.02

Figure 4. XPS N1s spectra and their curve fitting. (a) BNNPs, (b) BNNPswashed, (c) BNNPsannealed, (d) DOXBNNPs, (e) DOX-BNNPswashed, (f) DOX-BNNPsannealed.

Table 2. Contents of B Environments Derived from XPS B1s Curve Fitting Sample BNNPs

B environments, % BN BNO 6.8 41.33

BOx 0.0

B2O3 51.9 11

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DOX-BNNPs

45.6

0.0

54.4

0.0

BNNPswashed DOX-BNNPswashed

65.6 67.9

23.5 22.8

10.9 9.4

0.0 0.0

BNNPsannealed DOX-BNNPsannealed

74.3 79.2

22.0 15.8

3.7 5.0

0.0 0.0

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Table 3. Contents of N Environments Derived from XPS N1s Curve Fitting Sample BNNPs DOX-BNNPs

N environments, % BN BNO C-N 0.0 81.2 0.0 59.5 0.0 0.0

NH3+ 9.2 12.5

N-C=O 9.6 28.0

BNNPswashed DOX-BNNPswashed

93.3 86.8

0.0 0.0

6.7 10.8

0.0 0.0

0.0 2.4

BNNPsannealed DOX-BNNPsannealed

56.5 81.0

32.5 0.0

11.0 15.0

0.0 0.0

0.0 4.0

The high-resolution XPS C1s spectra of the BNNPs and DOX-BNNPs samples varied significantly (Fig. 5a,b). The XPS C1s spectrum of the as-prepared BNNPs was fitted with three components, i.e. CHx (285.0 eV), -C-O (286.5 eV), and -C=O/N-C=O (288 eV), which reflects the natural surface contamination (Fig. 5a). In case of the DOX-BNNPs sample, the XPS C1s spectrum was deconvoluted into four peaks positioned at 285.0, 286.5, 288, and 290.0 eV, which could be attributed to the CHx, -C-O, -C=O/N-C=O and – C(O)O groups, respectively (Fig. 5b). Additionally, the shake-up satellite observed at approximately 291.5 eV suggests the aromatic nature of carbon on the sample surface. The content of all carbon environments are summarized in Table 4. Comparison of C environment content in the DOX-BNNPs with that in a DOX molecule (calculated from the chemical formula) showed that they almost coincide. Thus it is reasonably to assume that, after the BNNPs were saturated with DOX, almost all C1s signal originated from DOX molecules. Considering that DOX is described by the formula C₂₇H₂₉NO₁₁ and the carbon content in the DOXBNNPs sample was estimated to be 65 at.%, one may conclude that the whole BNNPs surface was covered with DOX. The curve fitting of XPS C1s spectra obtained from the DOX-BNNPswashed and DOX-BNNPsannealed samples was performed using three components: CHx, (285.0 eV), -C-O (286.5 eV) and C=O/N-C=O (288.2 eV). No shake-up satellite was detected, suggesting low content of the aromatic carbon. Importantly, that the amount of immobilized DOX on the surface of BNNPswashed and BNNPsannealed samples appeared to be lower in comparison 12 ACS Paragon Plus Environment

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with the as-synthesized BNNPs counterpart because their C contents were significantly lower (two to three times, see Table 1). Note that the carbon environments after DOX saturation were close to a DOX molecule for all three samples.

Figure 5. XPS C1s spectra and their curve fitting. (a) BNNPs, (b) DOX-BNNPs, (c) DOX-BNNPswashed, (d) DOX-BNNPsannealed.

Table 4. Contents of C Environments Derived from XPS C1s Curve Fitting Sample

C environments, % CHx C-O N-C=O/C=O

C(O)O

BNNPs DOX-BNNPs

82.1 54.0

13.5 28.7

4.4 11.2

0.0 3.5

Shake-up sattelite 0.0 3.5

DOX (based on chemical formula) DOX-BNNPswashed DOX-BNNPsannealed

51.9

37.0

11.1

0.0

-

57.6 56.1

32.8 35.6

9.6 8.3

0.0 0.0

0.0 0.0

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3.7. Simulation of DOX Chemical Interaction with Pure and Oxidized BN Surface. DOX has three aromatic hydroxyanthraquinonic rings and, therefore, its π orbitals delocalize over the planar aromatic rings. A few intramolecular hydrogen O/H–O bonds stabilize its conformation. The tetrahydropyran ring has a chair configuration in which the hydroxyl and NH2 groups are in staggered positions. In order to simulate acidic medium in endosomal/lysosomal compartments of tumor cells, protonated DOX (DOXH+) was also considered in the theoretical model. Taking into account the relatively large size and small curvature of BNNPs, in a simplified model, a BN nanoparticle was considered as an infinite h-BN sheet. The absence of electron transfer between physically bonded adjacent h-BN layers allows us to consider only a few atomic planes. The atomic structure of DOXBNNP complex is presented in Fig. 6a. Figure 6b displays the frontier molecular orbital (MO) of neutral DOX and DOXH+. The highest occupied molecular orbital (HOMO) of DOX delocalizes mainly on three aromatic rings of tetracyclic anthracycline part of molecule with pronounced shift to oxygen-contained functional groups, while for the lowest unoccupied molecular orbital (LUMO) of DOX the contribution mainly comes from three aromatic rings of anthracycline part. The molecular orbitals in DOX and DOXH+ are rather similar, mainly centering over the aromatic rings. Figure 6b(insets) shows the difference in the structures of HOMOs and LUMOs between the freestanding DOX and DOXH+. The protonation of DOX molecule leads to only slight orbital modification, mainly occurring in the areas of hydrogen bonds (HOMO) and three aromatic rings (LUMO).

Figure 6. Atomic structure of DOX-BNNP conjugate [top and side views] (a). HOMO and LUMO of freestanding neutral and protonated DOX (b). Insets in (b) show the differences in HOMO and LUMO spatial distributions between freestanding DOX and DOXH+ (charge loss and gain are denoted by bluish and yellowish colors, respectively). B, N, C, O and H atoms are marked by green, blue, grey, red and cyan, 14 ACS Paragon Plus Environment

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respectively. The isosurface cutoff was set to 10-3 e/Å3. When calculating MO differences (insets), the cutoff was set to 10-4 e/Å3.

Our experiments described above indicated that the DOX binds more strongly to the oxidized BNNP surface when compared the pure one. This can be explained by the fact that the B2O3 surface layer has uncoupled bonds, whereas on the BNNP surface all bonds are balanced. The uncoupled bonds on the B2O3 surface interact with different groups in DOX (=O, -OH and –NH2 group) by transferring hydrogen atom from the –NH2 group to the B2O3 surface (Fig. 7). The grafting of DOX to the pure BN and unpassivated (001)B2O3 (space group Cmc21) surfaces is shown in Fig. 7a and b, respectively. It is important to note that we have limited our theoretical study to two extreme cases (perfect h-BN and B2O3 surfaces) because the annealing of BNNPs in vacuum led to almost complete removal of B2O3 surface layer and a significant decrease in total oxygen concentration. Indeed, Table 1 shows decreasing of oxygen content in BNNPs more than 3 times to 4.2 at.%. It is also likely that some oxygen was inside the BNNPs. Therefore, after annealing, it is reasonably to consider the system as perfect h-BN. The O-doped BN model is rather complicated because it requires the introduction of O-containing groups and structural defects. Since the surface structure of BNO nanoparticles (in terms of defects and impurities) is difficult to control experimentally, the O-doped theoretical BN model can give inaccurate results.

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Figure 7. The structure and charge distribution of conjugated DOX to pure BN (a) and oxidized (B2O3) surface. The bottom panels show the part of structure without charge redistribution on the isosurface to display the bonds between DOX and carrier surface. The charge loss and gain are denoted by bluish and yellowish colors, respectively. B, N, C, O, and H atoms are marked by green, blue, grey, red and cyan, respectively. The isosurface cut off was set to 10-3 e/Å3.

The binding energy (BE) values of DOX and DOXH+ conjugated to BN carriers were calculated as the b difference between system energy with an attached molecule ( Etot ) and each freestanding constituent ( E DOX

and Ecar , indicating the total energy of DOX and carrier (BN or B2O3), respectively): n Ebn  Etot   Em .

The BE values in plane position on the surface of different carriers are summarized in Table 5. The vertical position does not show any significant bonding (less than 2 and 10 eV/molecule for BN and B2O3 carrier, respectively). Both freestanding neutral DOX and protonated DOX are grafted to the h-BN surface via van der Waals interaction with the binding energies -11.47 and -11.96 eV/molecule. The average binding energy is -0.2 eV/atom, which is of the same order as the van der Waals bonding. But the unprotonated DOX binds strongly, because the total binding energy difference between the DOX-h-BN and DOXH+-h-BN systems is 0.49 eV/molecule (Table 5). The bonding of DOX to the B2O3 surface is much stronger due to the formation of a chemical bond between the DOX and the oxidized BN surface (-36.46 eV/molecule). In case of DOXH+ B2O3 bonding, there is an additional proton transfer from the –NH3+ group to the B2O3 surface. Thus the conjugation of DOX to the B2O3 surface is stronger when the DOX is in protonated form. Note that the B2O3 is not a stable phase and rapidly dissolves in water or physiological solution. This can provide additional benefits for the oxidized BN nanocarriers because boric anhydride (B2O3) and boric acid (H3BO3) were reported to possess anti-inflammatory, antiseptic, and antitumor properties.34-37 Table 5. Binding Energy of in Plane-Bound DOX and DOXH+ with Different Freestanding Parts (h-BN and B2O3) and Binding Energy Difference between DOX and DOXH+ Grafted to a Certain Carrier Type. BE, eV/molecule (kcal/mol) h-BN B2O3 + DOXH -11.47 -42.35 (-264.43) (-976.53) 16 ACS Paragon Plus Environment

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DOX BE difference (DOX – DOXH+)

-11.96 (-275.92) -0.49 (-11.49)

-36.46 (-840.89) 5.89 (135.63)

To uncover the mechanism of DOX conjugation with the perfect h-BN surface, the differences in HOMO and LUMO spatial distributions between the freestanding and conjugated DOX were considered (Fig. 8). The results indicated that after the DOX to h-BN conjugation, the LUMO and HOMO were redistributed in the areas occupied by three aromatic rings and hydrogen O/H-O bonds, respectively. In case of the DOXH+ there is an inverse: after DOX conjugation, the outflow of HOMO electrons from three aromatic rings area leads to a weakening of the DOX-BN bonds. Thus, the negative electron density destabilizes the DOXH+/h-BN system.

Figure 8. Atomic structure of conjugated DOX and DOXH+ showing the difference in HOMO and LUMO spatial distribution between freestanding and conjugated DOX (charge loss and gain are denoted by bluish and yellowish colors, respectively). Insets show the total HOMO and LUMO spatial distributions. B, N, C, O and H atoms are marked by green, blue, grey, red and cyan, respectively. The isosurface cutoff was set to 10-3 e/Å3. When calculating MO differences (insets), the cutoff was set to 10-4 e/Å3.

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4. CONCLUSIONS Chemical interaction of doxorubicin (DOX) with different BN surfaces (depending on the degree of their oxidation) was thoroughly studied using Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy (XPS), surface zeta potential measurements and density functional theory calculations. Three groups of hexagonal BN nanoparticles (BNNPs) obtained by boron oxide CVD process, i.e. (i) as-synthesized and those after (ii) repeated washing in water and (iii) high-temperature annealing, and their corresponding DOX-BN conjugates were investigated. Normal DOX and protonated DOX (DOXH+) were considered in the theoretical model. The results indicated that the formation of stable DOX-BN conjugates mainly depends on the attraction of electron density in the area of aromatic rings in highest occupied molecular orbital of DOX. The presence of a protonated NH2 group can facilitate electron density transfer from the DOXH+ to the boron oxide surface. The negative electron density destabilizes the DOXH+/h-BN system. It was demonstrated that boron oxide significantly improves interactions between the BN surface and DOX. XPS results confirmed that the amount of immobilized DOX on the B2O3 surface was higher in comparison with BNNPs containing little oxygen. Changing surface charge from negative to positive at the isoelectric point (pH 5.2) leads to the DOX leaching out from the DOX-BNNPs nanocarriers.  AUTHOR INFORMATION Corresponding Authors * E.S. Permyakova, E-mail: [email protected] Tel: +79162780199 * D.V. Shtansky, E-mail: [email protected] Tel: +74992366629 ORCID E.S. Permyakova: 0000-0003-2581-0803 L.Yu. Antipina: 0000-0003-1176-317X I.V. Sukhorukova: 0000-0003-2458-164X J. Polčak: 0000-0002-6571-6291 A.M. Kovalskii: 0000-0002-3822-8102 A.M. Manakhov: 0000-0003-4517-1682 D.V. Shtansky: 0000-0001-7304-2461 Notes The authors declare no competing financial interest. 18 ACS Paragon Plus Environment

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 ACKNOWLEDGMENTS The work was supported by the Ministry of Education and Science of the Russian Federation (Increase Competitiveness Program of NUST “MISiS” No. K2-2018-012). Authors are grateful to the supercomputer cluster provided by the Materials Modelling and Development Laboratory at NUST "MISIS" (supported via the Grant from the Ministry of Education and Science of the Russian Federation No. 14.Y26.31.0005) and to the Joint Supercomputer Center of the Russian Academy of Sciences.

 REFERENCES (1) Ciofani, G. Potential Applications of Boron Nitride Nanotubes as Drug Delivery Systems. Expert Opin. Drug Deliv. 2010, 7, 889–893. (2) Emanet, M.; Şen, Ö.; Çulha, M. Evaluation of Boron Nitride Nanotubes and Hexagonal Boron Nitrides as Nanocarriers for Cancer Drugs. Nanomedicine 2017, 12, 797-810. (3) Sharker, S. M.; Alam, M. A.; Shill, M. C.; Rahman, G. M. S.; Reza, H. M. Functionalized hBN as Targeted Photothermal Chemotherapy for Complete Eradication of Cancer Cells. Int J Pharm. 2017, 534, 206212. (4) Li, X.; Zhi, C.; Hanagata, N.; Yamaguchi, M.; Bando, Y.; Golberg, D. Boron Nitride Nanotubes Functionalized with Mesoporous Silica for Intracellular Delivery of Chemotherapy Drugs. Chem. Commun. 2013, 49, 7337–7339. (5) Collins, Y.; Lele, S. Long-Term Pegylated Liposomal Doxorubicin Use in Recurrent Ovarian Carcinoma. J. Natl. Med. Assoc. 2005, 97, 1414–1416. (6) Park, J.; Fong, P. M.; Lu, J.; Russell, K. S.; Booth, C. J.; Saltzman, W. M.; Fahmy, T. M. PEGylated PLGA Nanoparticles for the Improved Delivery of Doxorubicin. Nanomedicine Nanotechnol. Biol. Med. 2009, 5, 410–418. (7) Steiniger, S. C. J.; Kreuter, J.; Khalansky, A. S.; Skidan, I. N.; Bobruskin, A. I.; Smirnova, Z. S.; Severin, S. E.; Uhl, R.; Kock, M.; Geiger, K. D. et al. Chemotherapy of Glioblastoma in Rats Using Doxorubicin-Loaded Nanoparticles. Int. J. Cancer 2004, 109, 759–767. (8) Liu, Z.; Fan, A. C.; Rakhra, K.; Sherlock, S.; Goodwin, A.; Chen, X.; Yang, Q.; Felsher, D. W.; Dai, H. Supramolecular Stacking of Doxorubicin on Carbon Nanotubes for In Vivo Cancer Therapy. Angew. Chem. Int. Ed. 2009, 48, 7668–7672. (9) Wang, Y.; Xu, Z. Interaction Mechanism of Doxorubicin and SWCNT: Protonation and Diameter Effects on Drug Loading and Releasing. RSC Adv. 2016, 6, 314–322. 19 ACS Paragon Plus Environment

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Page 20 of 23

(10) Kamiya, K.; Okada, S. Energetics and Electronic Structure of Encapsulated Single-Stranded DNA in Carbon Nanotubes. Phys. Rev. B 2011, 83, 155444. (11) Xing, Y.-F.; Yang, C.-L.; Mo, Y.-F.; Wang, M.-S.; Ma, X.-G. Dynamic Mechanism of SingleStranded DNA Encapsulated into Single-Wall Carbon Nanotubes: A Molecular Dynamics Simulation Study. J. Phys. Soc. Jpn. 2014, 83, 024801. (12) Li, L.; Tao, R.; Song, M.; Zhang, Y.; Chen, K.; Wang, H.; Gong, R. Fabrication of Self-Assembled Folate-Biotin-Quaternized Starch Nanoparticles as Co-Carrier of Doxorubicin and siRNA. J. Biomater. Appl. 2017, 32, 587-597. (13) Muralidharan, R.; Babu, A.; Amreddy, N.; Basalingappa, K.; Mehta, M.; Chen, A; Zhao, Y.D.; Kompella, U.B.; Munshi, A.; Ramesh, R. Folate Receptor-Targeted Nanoparticle Delivery of HuR-RNAi Suppresses Lung Cancer Cell Proliferation and Migration. J Nanobiotechnology 2016, 14, 47-63. (14) Alshehri, M. H.; Cox, B. J.; Hill, J. M. Determination of Optimal Nanotube Radius for Single-Strand Deoxyribonucleic Acid Encapsulation. Micro. Amp. Nano Lett. 2014, 9, 113–118. (15) Zheng, B.; Yang, S.; Wang, M.; Yang, X.; Teng, L.; Xie, J.; Teng, L.; Lee, R.J. Non-covalent Nanocomplexes of Folic Acid and Reducible Polyethylenimine for Survivin siRNA Delivery. Anticancer Res. 2015,35, 5433-5441. (16) Kang, Y.; Wang, Q.; Liu, Y.-C.; Shen, J.-W.; Wu, T. Diameter Selectivity of Protein Encapsulation in Carbon Nanotubes. J. Phys. Chem. B 2010, 114, 2869–2875. (17) Chen, Q.; Wang, Q.; Liu, Y.-C.; Wu, T.; Kang, Y.; Moore, J. D.; Gubbins, K. E. Energetics Investigation on Encapsulation of Protein/Peptide Drugs in Carbon Nanotubes. J. Chem. Phys. 2009, 131, 015101 (18) Mejri, A.; Vardanega, D.; Tangour, B.; Gharbi, T.; Picaud, F. Encapsulation into Carbon Nanotubes and Release of Anticancer Cisplatin Drug Molecule. J. Phys. Chem. B 2015, 119, 604–611. (19) Zhitnyak, I.Y; Bychkov, I.N.; Sukhorukova, I.V.; Kovalskii, A.M.; Firestein, K.L.; Golberg, D.; Gloushankova, N.A.; Shtansky, D.V. Effect of BN Nanoparticles Loaded with Doxorubicin on Tumor Cells with Multiple Drug Resistance, ACS Appl. Mater. Interfaces 2017, 9, 32498-32508. (20) Permyakova, E.S.; Sukhorukova, I.V.; Antipina, L.Y.; Konopatsky, A.S.; Kovalskii, A.M.; Matveev, A.T.; Lebedev, O.I.; Golberg, D.V.; Manakhov, A.M.; Shtansky, D.V. Synthesis and Characterization of Folate Conjugated Boron Nitride Nanocarriers for Targeted Drug Delivery J. Phys. Chem. C 2017, 121, 28096-28105 (21) Hohenberg, P.; Kohn, W. Inhomogeneous Electron Gas. Phys Rev B 1964, 136, 864–871.

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(22) Kohn, W.; Sham, L. J. Self-Consistent Equations Including Exchange and Correlation Effects. Phys Rev A 1965, 140, 1133–1138. (23) Perdew, J. P.; Burke, K.; Ernzerhof, M. Generalized Gradient Approximation Made Simple. Phys. Rev. Lett. 1996, 77, 3865–3868. (24) Troullier, N.; Martins, J. L. Efficient Pseudopotentials for Plane-Wave Calculations. Phys. Rev. B 1991, 43, 1993–2006. (25) Ordejón, P.; Artacho, E.; Soler J.M. Self-Consistent Order-N Density-Functional Calculations for Very Large Systems. Phys. Rev. B 1996, 53, 10441-10444. (26) Soler, J.; Artacho, E.; Gale, J.; Garcia, A.; Junquera, J.; Ordejon, P.; Sánchez-Portal, D. The SIESTA Method for Ab Initio Order-N Materials Simulation. J. Phys. Cond. Matter. 2002, 14, 2745–2780. (27) Grimme, S. Semiempirical GGA-Type Density Functional Constructed with a Long-Range Dispersion Correction. J. Comput. Chem. 2006, 27, 1787–1799. (28) Monkhorst, H. J.; Pack, J. D. Special Points for Brillouin-Zone Integrations. Phys. Rev. B 1976, 13, 5188–5192. (29) Ramachandran, R.; Jung, D.; Bernier, N.A.; Logan, J.K.; Waddington, M.A.; Spokoyny, A.M. Sonochemical Synthesis of Small Boron Oxide Nanoparticles. Inorg Chem. 2018 57, 8037-8041. (30) Rudra, A.; Deepa, R. M.; Ghosh, M. K.; Ghosh, S.; Mukherjee, B. Doxorubicin-Loaded Phosphatidylethanolamine-Conjugated Nano liposomes: in Vitro Characterization and Their Accumulation in Liver, Kidneys, and Lungs in Rats. Int. J. Nanomed. 2010, 5, 811−823. (31) Feng, S.; Zhang, H.; Yan, T.; Huang, D.; Zhi, C.; Nakanishi, H.; Gao, X.-D. Folate-Conjugated Boron Nitride Nanospheres for Targeted Delivery of Anticancer Drugs. Int. J. Nanomed. 2016, 11, 4573– 4582. (32) Honary, S.; Zahir, F. Effect of Zeta Potential on the Properties of Nano-Drug Delivery Systems - A Review (Part 1). Trop. J. Pharm. Res. 2013, 12, 255-264. (33) Peak, D.; Luther III, G.W.; Sparks, D.L. ATR-FTIR Spectroscopic Studies of Boric Acid Adsorption on Hydrous Ferric Oxide. Geochim. Cosmochim. Acta 2003, 67, 2551–2560. (34) Nielsen, F.H. Dietary Fat Composition Modifies the Effect of Boron on Bone Characteristics and Plasma Lipids in Rats. Biofactors 2004, 20, 161-171. (35) Benderdour, M.; Bui-Van, T.; Dicko, A.; Belleville, F. In Vivo and in Vitro Effects of Boron and Boronated Compounds. J. Trace. Elem. Med. Biol. 1998, 12, 2-7. (36) Luan, Q.; Desta, T.; Chehab, L.; Sanders, V.J.; Plattner, J.; Graves, D.T. Inhibition of Experimental Periodontitis by a Topical Boron-Based Antimicrobial. J. Dent. Res. 2008, 87, 148-152. 21 ACS Paragon Plus Environment

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(37) Benderdour, M.; Van Bui, T.; Hess, K.; Dicko, A.; Belleville, F.; Dousset, B. Effects of Boron Derivatives on Extracellular Matrix Formation. J. Trace Elem. Med. Biol. 2000, 14, 168- 173.

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