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ISSN 19950780, Nanotechnologies in Russia, 2014, Vol. 9, Nos. 7–8, pp. 398–409. © Pleiades Publishing, Ltd., 2014. Original Russian Text © Yu.I. Svenskaya, N.A. Navolokin, A.B. Bucharskaya, G.S. Terentyuk, A.O. Kuz’mina, M.M. Burashnikova, G.N. Maslyakova, E.A. Lukyanets, D.A. Gorin, 2014, published in Rossiiskie Nanotekhnologii, 2014, Vol. 9, Nos. 7–8.

Calcium Carbonate Microparticles Containing a Photosensitizer Photosens: Preparation, Ultrasound Stimulated Dye Release, and In Vivo Application Yu. I. Svenskayaa, N. A. Navolokinb, A. B. Bucharskayab, G. S. Terentyuka, b, c, A. O. Kuz’minaa, M. M. Burashnikovaa, G. N. Maslyakovab, E. A. Lukyanetsd, and D. A. Gorina a

Chernyshevsky State University, ul. Astrakhanskaya 83, Saratov, 410012 Russia State Medical University, ul. B. Kazach’ya 112, Saratov, 410012 Russia c Ulyanovsk State University, ul. L. Tolstogo 42, Ulyanovsk, 432017 Russia d Organic Intermediates and Dyes Institute, ul. B. Sadovaya 1, korp. 4, Moscow, 123995 Russia email: [email protected] b Razumovsky

Received September 12, 2013; in final form, April 10, 2014

Abstract—Calcium carbonate microparticles with a size of 0.9 ± 0.2 µm containing a photosensitizer Pho tosens in a concentration of 2.0 ± 0.2 wt % were prepared by ultrasoundstimulated coprecipitation (20 kHz, 1 W/cm2). It is shown that the encapsulated photosensitizer can be released by ultrasonic irradiation (0.89 MHz, 1 W/cm2, 5 min) as a result of the destruction and recrystallization of calcium carbonate micro particles. It is established that the combined ultrasonic (0.89 MHz, 1 W/cm2) and light (670 nm, 10 mW/cm2) in vivo influence on the transferred PC1 strain tumors of rat liver containing intratumorally injected micro containers with a photosensitizer gives rise to dystrophic changes in tumor cells and to the appearance of extensive necrotic centers, pointing to the presence of the evident destructive effect. Such microcontainers are proposed for use in treating external tumors or tumors accessible for ultrasonic and optical irradiation. DOI: 10.1134/S1995078014040181

INTRODUCTION Studies on methods of treating oncological diseases such as photodynamic therapy (PDT) are now of great interest. The specified method consists in the applica tion of photosensitizers (PSs), also called photody namic dyes, the action of which is initiated by light irradiation [1]. Such therapy is based on the ability of PSs to accumulate selectively in a tumor tissue and to generate the singlet oxygen and free radicals that cause the destruction of tumor cells under the localized influence of light radiation with a wavelength corre sponding to its absorption maximum [2, 3]. PDT has been quickly adopted in clinical oncology and proven to be effective in the treatment of cancer in various stages and localizations [4–13], as well as for the treatment of a number of nontumor diseases in dermatology, contaminated surgery, otolaryngology, gynecology, ophthalmology, etc. [4, 11, 14–16]. This field continues to develop intensively [17–22]. The number of clinics offering treatment by the PDT method grows continually. Combining the fluorescent diagnosis and therapy itself in a single procedure is a considerable advantage of the PDT method [23–25]. The exploration of agents for the immobilization and addressed delivery of a photodynamic dye with the purpose of decreasing its therapeutic concentration

and reducing the photosensitization effect on healthy tissues is one of the problems in modern cancer PDT [26–31]. Up to the now, it was suggested to solve this problem using liposomes [28], micelles [29], PS con jugates with nanoparticles [30, 32], monoclonal anti bodies [26], and proteins [27]. Also, calcium phos phate [33, 34] and silicon dioxide nanoparticles [35–37] were used as containers for the encapsulation of a pho tosensitizer. To solve the PS localization problem, we earlier suggested using calcium carbonate particles (CaCO3) in the vaterite polymorphic form [38]. The efficiency of loading the microsized and submicron vaterite particles was estimated and the photodynamic release of dye from them at various pH values of a dis persion medium was studied. The other studies on the incorporation of dyes and proteins in the bulk of cal cium carbonate particles are also known [39–43]. The effectiveness of using calcium carbonate submicron particles as containers for the endocellular delivery of a pharmaceutical substance was shown [44]. CaCO3 microparticles are promissory as containers for the encapsulation of biologically active materials (BAMs), since they show a porous structure and a number of advantages such as biocompatibility, mild decomposi tion conditions, preparation simplicity, and low pro duction cost [45].

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A number of studies showing an increase in the cytotoxicity of various anticancer drugs upon local ultrasonic irradiation have been published over the last decade [46–51]. The specified effect is essential in sonodynamic cancer therapy (SDT). The experimen tal data about the presence of a synergistic effect at the combined application of PDT and SDT for a number of foreign [52–54] and Russian drugs [55, 56] are of particular interest. The use of solidphase nanoinclu sions playing the role of ultrasound energy concentra tors in the ultrasonic therapy of oncological diseases is also of great interest [57]. Based on the above, the immobilization of a photo sensitizer into calcium carbonate microparticles and studies on the possibility of the controllable release of dye from them by ultrasonic treatment are urgent problems, as well as testing the obtained micocontain ers in tumor tissues upon the combined ultrasound and light irradiation. Such testing was implemented in the present work by analyzing the morphological changes in a tumor tissue of rats with a transferred PC1 strain of liver cancer. EXPERIMENTAL Materials The experimental samples were prepared from 1M aqueous solutions of calcium chloride dihydrate (CaCl2 · 2H2O, SigmaAldrich Chemie) and sodium carbonate (Na2CO3, SigmaAldrich Chemie). Photo sens® (Organic Intermediates and Dyes Institute Russia), a mixture of sodium salts of hydroxoalumi num di, tri, and tetrasulfophthalocyanines with a degree of sulphonation of about 3, was chosen as a photosensitizer. The drug shows the absorption maxi mum at a wavelength of 675 nm [58] and can be acti vated by a laser radiation source with a power density of 100 J/cm2 [59]. Photosens has been used in clinical practice in Russia since 2001 (registration certificate no. 000199.012001 issued by the Ministry of Health of the Russian Federation), and is effective both for the diagnosis [24, 25, 60] and therapy of lip, pharynx, throat, tongue, lung, esophagus, and stomach tumors [61–63]. There are also studies that evidence its suc cessful application as a sonosensitizer [56]. Photosens was used in the form of an aqueous solu tion with a concentration of 2 mg/mL. A 0.9% iso tonic solution of sodium chloride (physiological saline solution) produced by Biokhimik (Russia) and an ace tate buffer with a pH value of 5 prepared from acetic acid and sodium hydroxide (Ekros, Russia) were used as a dispersion medium for the preparation of suspen sions of microcontainers. Deionized water obtained by twolevel purification in a BS192 system (Russia) and double treatment in a Vodolei deionizer (Khimelektronika, Russia) was used in all experimental stages. NANOTECHNOLOGIES IN RUSSIA

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Synthesis and Characterization of Microcontainers Calcium carbonate microparticles were obtained by pouring together equimolar amounts of calcium chloride and soda solutions with simultaneous ultra sonic irradiation. For this purpose, 5mL portions of 1M solutions of the above salts were added simulta neously into 20 mL of deionized water and sonicated for 30 s by a Sonopuls ultrasonic homogeniser (Bande lin, Germany) at a frequency of 20 kHz and radiation intensity of 1 W/cm2. Then a suspension of the pro duced particles was settled for 1 min to complete the crystallization of the calcium carbonate. Next, the CaCO3 precipitate was washed twice with deionized water and once with a medical antiseptic solution (95% ethanol solution) and then dried at a tempera ture of 60°C for 30 min. The dried CaCO3 micropar ticles were stored in a closed container at ambient temperature. CaCO3 microparticles were loaded by Photosens in the course of their formation using the coprecipitation technique [40]. The same synthetic procedure was used for the formation of calcium carbonate micro particles, with the only difference that a solution of Photosens with a concentration of 2 mg/mL was used instead of deionized water. In order to estimate the concentration of the pho tosensitizer in the CaCO3 containers, their weighed portion was dissolved in a known volume of an aqueous solution of ethylenediaminetetracetic acid (EDTA, 0.2 M). The wavelength dependences of the optical densities of the obtained solutions were investigated on a Lambda 950 spectrophotometer (PerkinElmer, United States). The loading efficiency of the contain ers, i.e., the weight ratio of the encapsulated dye to the unloaded containers, was calculated from the mea surement results. The microcontainers were investigated by scanning electron microscopy using an MIRA II LMU device (Tescan) with an XL 30 ESEM FEG electron micro scope (Philips). Samples were prepared by drying a drop of an aqueous suspension of the microcontainers on a wafer coated with an electroconductive layer. The specific surface area of the calcium carbonate particles and size distribution of the particle pores were evaluated using a NOVA 1200e analyzer (Quan tachrome, United States) using the physical gas adsorption method with subsequent calculation of the specified parameters by means of a special software tool integrated into the device. A sample of the inves tigated substance was preliminarily degassed in vac uum at a temperature of 60°C for 4 h, and then the adsorption isotherm was obtained. Nitrogen was used as an absorbate. The Brunauera–Emmeta–Teller (BET) method was applied to calculate the specific surface areas. The size distribution of pores was defined using the Barrett–Joyner–Halenda (BJH) method. 2014

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Studies on the Destruction of Microcontainers under Ultrasonic Irradiation In Vitro The release of Photosens and the destruction of the microcontainers loaded with it under the influence of ultrasound irradiation were studied using a UZT 1.01 F device (Med TeKo, Russia) widely applied in the clin ical practice for ultrasonic therapy of diseases of the peripheral nervous system, locomotor apparatus, and viscera, as well as dermatoses. Suspensions of micro containers with a volume of 2 mL that contain 600 μg of a dry powder of CaCО3 particles were sonicated. A physiological saline solution and acetate buffer (pH = 5) were used as media for the preparation of suspensions. According to the standard procedure [64], a buffer solution was prepared by adding 73.4 mL of a 1 N solution of acetic acid and 50 mL of a 1 N solution of sodium hydroxide to 500 mL of deionized water. Ultrasonic irradiation was carried out in a con tinuous mode with radiation power densities of 0.05, 0.2, 0.4, 0.7, and 1 W/cm2. The duration of the expo sure in each case was 5 min with a radiation frequency of 0.89 MHz. Suspensions of the unloaded calcium carbonate microparticles dispersed in physiological and buffer solutions (Control 1), as well as blank media, i.e., physiological and buffer solutions (Control 2), exposed to ultrasound irradiation with a radiation power density of 1 W/cm2 were taken as reference samples. The suspensions were centrifuged after sonication, and then the precipitate was investigated by scanning electron microscopy on a MIRA II LMU device (Tes can) using a XL 30 ESEM FEG scanning electron microscope (Philips). Supernatant liquids were inves tigated by the spectrophotometric method on a Lambda 950 instrument (PerkinElmer, United States). Experimental Techniques for Investigating the Effect of Ultrasonic and Light Irradiation on the Transferred PC1 Strain Tumors of a Rat Liver Forty male white laboratory rats with an average weight of 160 ± 20 g, to each of which 0.5 mL of a 25% suspension of alveolar liver cancer cells of the PC1 strain from the cancer cell line repository of the Blokhin Cancer Research Center, Russian Academy of Medical Sciences, were implanted hypodermically into their scapular area, were used to investigate the effect of combined ultrasonic and light irradiation on the transferred tumors in the presence of calcium car bonate microparticles containing a photosensitizer. Upon reaching a tumor diameter of 1.0 ± 0.3 cm, the animals were divided into the following four groups using the random sampling technique with ten rats in each group: the first group was comprised of nonex posed rats (reference group), the second group with ultrasonic irradiation of the tumor, the third group

with the intratumoral injection of a suspension con taining unloaded calcium carbonate particles and the subsequent ultrasonic irradiation of the tumor, and the fourth group with the intratumoral injection of a sus pension containing microcontainers loaded with Pho tosens and the subsequent combined irradiation of the tumor by ultrasound and light. The intratumoral injection of the investigated sus pensions in the in vivo experiments was carried out in the following manner: the volume of the tumor was determined and then the investigated solution in a vol ume of 30% from the tumor volume introduced into the tumor from two opposite points with a rate of 0.1 mL/min; 15 min after the injection, ultrasonic irradiation in a continuous mode by means of a UZT 1.01 F device (Med TeKo, Russia) was performed for 10 min at a radiation power density of 1 W/cm2 (according to the in vitro experiments, the destruction and recrystallization of calcium carbonate particles accompanied by the release of a dye take place during this period); after the next 15 min, the tumor was irra diated for 10 min by means of an AFS phototherapeu tic lightemitting diode (Polironik, Russia), the wave length of which, corresponding to the maximum emis sion intensity, is 660 nm and the radiation power density is 10 mW/cm2. In order to estimate the tumor pathomorphism 1 day after irradiation, all the animals were deduced from the experiment and the tumor tis sues were taken away for histological studies with the use of standard hematoxylin and eosin coloring stains. Morphometric studies on the histological samples were performed using a μVizo103 medical microvi sion system for the digital image analysis. RESULTS AND DISCUSSION The SEM image of the obtained calcium carbonate microparticles and their size distribution are shown in Figs. 1a and 1b. The average size of microparticles is 0.9 ± 0.2 μm. Upon an analysis of the image given in Fig. 1a, it can be concluded that ultrasonic irradiation during the synthesis of calcium carbonate particles allows one to reduce the size of the formed particles and also their degree of polydispersity, unlike the tra ditional method of agitation with a magnetic stirrer, where the size of the prepared calcium carbonate microparticles varies from 2 to 7 μm, depending on the synthesis conditions (concentration of the salt solu tions, stirring rate, and settling time) [65]. For the preparation of microcontainers loaded with Photosens, the method of coprecipitation with cal cium carbonate microparticles was chosen. When using such a technique, a substance incorporated into the structure of porous microparticles is added directly in the course of their formation. The choice of this particular method is determined by the fact that it allows one to encapsulate five times more biologically active material in the bulk of the microparticles than

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Number of particles 60 50 40 30 20 10 0 (a)

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Number of particles 60 50 40 30 20 10 0 (c)

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Fig. 1. (a, c) SEM images and (b, d) size distribution diagrams of (a, b) unloaded calcium carbonate microparticles and (c, d) microparticles loaded with Photosens; the scale bar size corresponds to 5 µm.

with the adsorption on their surface [40]. The SEM image of the synthesized microcontainers is shown in Fig. 1c. The size of the obtained structures is 0.9 ± 0.2 μm. The size distribution of the containers is given in Fig. 1d. As was estimated by the photometric method, the loading efficiency of microcontainers is 2.0 ± 0.2 wt %, which agrees with the literature data on the encapsula tion of biologically active compounds in CaCO3 parti cles [40]. The specific surface area of microcontainers esti mated by the BET method is 22 m2/g. The data on the size distribution of pores are given in Table 1. The average pore size of the sample is 55 ± 15 nm. Next, the in vitro experiment on the destruction of microcontainers by ultrasound and subsequent release of a photosensitizer immobilized in them was per formed. A physiological solution and acetate buffer with pH = 5, which simulate the environment in the affected area, were chosen as a dispersion medium for the preparation of suspensions. It is known that near the centers of pathological processes, in particular in the internal space of solid tumors, a decrease in the pH NANOTECHNOLOGIES IN RUSSIA

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values to a range from 6.5 to 7.2 [66, 67] is observed; the pH value falls to a range from 5.0 to 6.5 at the endocytosis of particles into a cell, transferring them into the endosome compartment, and it reaches values from 4.5 to 5 in lysosomes [68, 69]. Since the dissolu tion process for calcium carbonate microparticles starts at pH ≤ 5.5 [38, 70] and it is accompanied by the carbon dioxide formation, similar processes can be expected upon incorporating such containers into a tumor. At a certain intensity of ultrasonic irradiation on the medium that is called the cavitation threshold, the phenomenon of the acoustic cavitation connected with the formation of cavitation bubbles and their col lapse, which is accompanied by the generation of Table 1. Size distribution of pores in the calcium carbonate particles Diameter of pores, nm

Concentration of pores, %

10–20 30–40 60–80

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Table 2. Results of ultrasonic action with different power density on the microcontainer suspensions in physiological saline (PSS) and buffer solutions (BS) and on the Control 1 and Control 2 reference samples; the bubble cavitation mode is denoted by “++” and “+++” During irradiation Immediately after One hour after Before irradiation, Power density irradiation irradiation initial sample 1–2 min 3–5 min Solution of ultrasonic type irradiation Precipi Precipi Precipi Precipi Precipi Bubbles Bubbles Bubbles Bubbles Bubbles tate tate tate tate tate 0 W/cm2 0.05 W/cm2 0.2 W/cm2 0.4 W/cm2 0.7 W/cm2 1 W/cm2 Control 1 Control 2

PSS BS PSS BS PSS BS PSS BS PSS BS PSS BS PSS BS PSS BS

+ + + + + + + + + + + + + + – –

– – – – – – – – – – – – – – – –

+ + + + + + + + + + + + + + – –

– + – – – + + + ++ +++ ++ +++ ++ +++ + +

microjets and shock waves, is observed [71]. Such effects applied to a tumoral tissue are capable of break ing its integrity [71], which can give rise to a local decrease in the pH values down to 4.5. This fact prede termined the choice of the above specified buffer solu tion for carrying out the experiments. In order to create thin walls of easily deformable material more approaching the actual conditions in soft tissues (when compared to the glass or plastic cyl inders in which samples are usually investigated in vitro), polyethylene bags 6 × 8 cm in size were used as containers in the experiment. The results of the exper iment are summarized in Table 2 and the foremost characteristic images are shown in Fig. 2. As was seen from Table 2, a weighed sample of microcontainers containing Photosens retains integ rity for 1 h in the absence of ultrasonic radiation in a medium of physiological solution. Ultrasonic irradia tion of such a system with a radiation power density from 0.2 to 0.4 W/cm2 leads to microcontainers dis persing, even though the precipitate is partially pre served (Fig. 2b, Table 2). An increase in the color intensity of a physiological solution due to the release of PS, as well as the evolution of gas, was observed visually during ultrasonic irradiation. The specified effects are amplified upon reaching an ultrasound power density value of 1 W/cm2 (Fig. 2b, Table 2). Also, the disappearance of the precipitate (Table 2)

+ + + + + + + – – – – – – – – –

– + – + + + + + + + + + + + – –

+ – + + + + + – – – – – – – – –

– – – – – – – – – – – – – – – –

+ – + – + – + – – – – – – – – –

– – – – – – – – – – – – – – – –

was observed visually at similar values of the radiation power density. As was established by the SEM method, ultrasonic irradiation with an intensity of 1 W/cm2 leads to the destruction of the CaCO3 containers dis persed in a physiological solution, as was indicated by the presence of spherical particle fragments (Fig. 3a), and also accelerates the recrystallization process of calcium carbonate microparticles (cubic particles in Fig. 3b). It should be noted that both processes give rise to the release of the encapsulated PS. The possibil ity of recrystallization of calcium carbonate particles under the influence of ultrasound radiation is also confirmed by other works [72]. In the absence of ultrasonic influence, the micro containers are dissolved in a buffer solution (pH = 5) in 5 min, which is accompanied by the release of Pho tosens (see Fig. 2d and Table 2). It is in full agreement with the results obtained in [38], where it was shown that CaCO3 microcontainers loaded by PS dissolve in just 5 min in an acetate buffer solution with pH = 5, which gives rise to the encapsulated dye releasing. Ultrasonic treatment with a radiation power density of more than 0.2 W/cm2 accelerates the process of disso lution of CaCO3 particles (Figs. 2e, 2f). Bubbles were produced in such systems, since the reaction of cal cium carbonate dissolution in an acidic medium pro ceeds with the formation of CО2 (Table 2). However, the


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Fig. 2. Photographic images of suspensions of the calcium carbonate microcontainers loaded with Photosens dispersed in (a–c) physiological and (d–f) buffer solutions in the course of ultrasonic irradiation with a power density of (a, d) 0.2, (b, e) 0.4, and (c, f) 1 W/cm2; calcium carbonate microparticles not loaded with a photosensitizer (Control 1) in (g) physiological and (h) buffer solutions at the ultrasonic influence with a power density of 1 W/cm2; (i) physiological solution (Control 2) at the ultra sonic influence with a power density of 1 W/cm2.

1 µm

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10 µm

Fig. 3. SEM images of microcontainers loaded with Photosens dispersed in a physiological solution after the sonication for 5 min with a power density of 1 W/cm2: the fragments of (a) spherical and (b) cubic particles of calcium carbonate. NANOTECHNOLOGIES IN RUSSIA

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effect of ultrasonic irradiation on calcium carbonate particles in the buffer medium intensifies the bubbling process, as is seen from the photographic images pre sented in Figs. 2e and 2f (the intensive bubbling mode is denoted by “++” or “+++” in Table 2). In the reference suspension containing calcium carbonate microparticles without a dye (Control 1), the same effects were observed under the influence of ultrasound as in the case of microcontainers loaded with a dye (Figs. 2g, 3h; Table 2). When studying the influence of ultrasound on a physiological solution and a buffer solution without microcontainers (Control 2), the formation of small bubbles was observed at the maximum irradiation power (1 W/cm2), but they were less mobile than in the presence of calcium carbonate microparticles and the processes of the intensive formation of bubbles and their coalescence were not observed (Fig. 2i, Table 2). Thus, it is shown that CaCO3 microcontainers dis persed in a physiological solution can be destroyed and recrystallized upon 5min ultrasonic irradiation (0.89 MHz, 1 W/cm2) and PSs incorporated in them can be released; it is also shown that the ultrasonic influence in an acidified medium (pH = 5) immedi ately dissolves calcium carbonate particles. The cavitation threshold in an aqueous suspension of calcium carbonate microparticles is reduced when compared with a medium without particles. That is caused by the simultaneous action of several factors. First, the suspension of microparticles dispersed in a physiological solution is a heterogeneous system. It is known that the nucleationrate threshold in a hetero geneous system is lower than in a homogeneous system [73]. The nucleationrate threshold of nanobubbles, which serve as nucleation centers for cavitation bub bles, decreases in this case; as a result, their formation rate increases and, hence, the probability of cavitation processes at the specified power density of ultrasound also increases. Since calcium carbonate particles are mesoporous, the nucleation of nanobubbles can be implemented in the pores and its threshold is lower than the threshold of a heterogeneous nucleation rate. Second, as was mentioned above, calcium carbonate microparticles are dissolved in an acidic medium (pH = 5) with the formation of CO2. An increase in the concentration of gases dissolved in a medium sub jected to ultrasonic irradiation leads to a decrease in the cavitation threshold. The cavitation threshold for water under the standard conditions is 0.3 W/cm2 [71]. It was shown using the example of liver cells of rats that abnormalities in the lysosome membranes are observed under the ultrasonic irradiation with an intensity of 2.5 W/cm2 and frequency of 1 MHz for 5 min [71]. A decrease in the nucleation threshold leads to an increase in the number of cavitation bub bles, which also can be generated on the surface of tumor cell membranes with a certain probability and give rise to their destruction. It is shown in [74] that

Optical density, rel. units 0.9 0.8 0.7 0.6 0.5 A 0.4 0.3 B 0.2 0.1 0 550 600 650 700 750 Wavelength, nm

800

Fig. 4. Absorption spectra of (a) physiological and (b) ace tate buffer solutions in 5 min after dispersion in them of the microcontainers loaded with Photosens under ultrasonic influence.

the hydrophobization of the substrate surface results in the significant reduction of the nucleation threshold and, as a consequence, breaking the integrity of the substrate surface under the influence of the energy lib erated upon the collapse of cavitation bubbles formed on it. Thus, it can be concluded that cavitation pro cesses occur in a suspension upon ultrasonic irradiati ation, which gives rise to the observable effects of destruction of microcontainers and the intensification of recrystallization processes accompanied by the release of an immobilized dye. We assume that an increase in the number of cavitation bubbles can lead to additional effects in the therapy of tumors, specifi cally, to the destruction of tumorcell membranes. The dye release in this series of experiments is evi denced by the coloration of the dispersion medium, which is confirmed by a change in the optical density of supernatant liquors, i.e., physiological and acetate buffer solutions, namely, by the appearance of charac teristic peaks for Photosens at 608 ± 2 and 676 ± 2 nm. Figure 4 shows the typical absorption spectra of a physiological solution (Fig. 4a) and acetate buffer with pH = 5 (Fig. 4b) 5 min after the ultrasonic dispersion in them of microcontainers loaded with Photosens. Thus, it can be concluded that ultrasonic irradia tion promotes the release of a photosensitizer from microcontainers and its distribution over a suffered region, allowing one to increase the effectiveness of the further photodynamic treatment. In order to estimate the effect of the combined influence of ultrasonic and light radiations upon the use of CaCO3microcontainers containing a photo sensitizer, the in vivo experiment on the irradiation of a rat liver with the transferred PC1 tumor strain was performed. Morphological studies on the tumor microsamples showed that the tumor of rats from the 1st group (ref erence group) has a segmental structure, where the segments are separated by thin layers of connective tis sue. The tumor cells have a roundish oval form with an


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Fig. 5. Microphotographs of the tumor tissue of rats from the (a, b) 1st and (c, d) 2nd groups at various magnification rates; thick ening of the connective tissue partitions and the bleeding centers (indicated by arrows) are registered in the tissues of rats from the 2nd group; coloration by hematoxylin and eosin; the scale bar sizes correspond to (a, c) 200 and (b, d) 20 µm.

eccentrically positioned nuclei. A significant part of cytoplasm is occupied by large vacuoles containing mucus. Single mitoses are revealed. Agglomerations of mucous substance along with the fragments of destroyed cells are found in small necrosis zones (less than 5% of the total area). Mucous substance is present also in the intercellular spaces (Figs. 5a, 5b). As was established from the investigation of tissue histological sections of rats from the 2nd group by only ultrasoound, the tumor preserves a segmental struc ture. Small necrosis zones are observed (5–10% of the total area of the tissue), and tumor cells with necrotic changes are found. Single mitoses are revealed. Vessels are plethoric, thickening in the connective tissue par titions, and their infiltration by leucocytes are observed; small bleeding areas are present (Figs. 5c, 5d). According to morphological studies, the lobularity of the tumor structure in the 3rd group (ultrasonic influence with the preliminary injection of unloaded calcium carbonate microparticles) is impaired, necrotic areas in the tumor tissue are increased (10–20% of the total area of the section), and the connective tissue partitions in some spots are thickened and infiltrated by leucocytes (Figs. 6a, 6b). NANOTECHNOLOGIES IN RUSSIA

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The lobularity of the tumor structure in the 4th group (ultrasonic and light irradiation with the prelim inary injection of Photosenscontaining microcon tainers) is deteriorated, and more uttered changes in the tissue are observed in the tumor center; specifi cally, the area of necrotic zones is ranged from 40 to 60% of the section’s total area, tumor cells have dys trophic changes and pycnotic nuclei, connective tissue partitions are infiltrated by lymphocytes, and exten sive bleeding centers are observed. In the periphery of the tumor, the small necrotic centers are also observed, the area of which makes 10–20% of the total area of the tissue sections, and the connective tissue partitions are also infiltrated by lymphocytes (Figs. 6c, 6d). It can be concluded from the results of the above described in vivo experiment that pathomorphism in the liver tumor at the combined ultrasonic and light irradiation with the preliminary injection of CaCO3 microcontainers with a photosensitizer is revealed in the form of marked dystrophic changes in tumor cells and extensive necrotic zones, pointing to the presence of the expressed destructive effect. The destructive effect on the tumor tissue is also observed upon the ultrasonic influence in the presence of unloaded con 2014

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Fig. 6. Microphotographs of the tumor tissues of rats from the (a, b) 3rd and (c, d) 4th groups at various magnification rates; thick ened connective tissue partitions infiltrated by leucocytes in the tissues of rats from the 3rd group and extensive necrotic zones and bleeding centers in the tissues of rats from the 4th group are denoted by arrows; coloration by hematoxylin and eosin; the scale bar sizes correspond to (a, c) 200 and (b, d) 20 µm.

tainers, but the area of necrosis is markedly smaller in this case. CONCLUSIONS Using the method of coprecipitation with ultra sonic irradiation (20 kHz, 1 W/cm2), calcium carbon ate microparticles with a size of 0.9 ± 0.2 μm that con tain the photosensitizer Photosens with a loading effi ciency of 2.0 ± 0.2 wt % have been prepared. It is found that ultrasonic treatment with a frequency of 0.89 MHz and radiation power density of W/cm2 for 5 min leads to the destruction and recrystallization of microsized calcium carbonate particles dispersed in a physiological solution and the dissolution of micro particles dispersed in a buffer solution (pH = 5), which is accompanied by the release of the immobilized PC. It is shown that ultrasonic irradiation assists the release of a photosensitizer from the microcontainers and its distribution in the affected area. The energy liberated as a result of the collapse of cavitation bubbles can give rise to an additional effect in tumor therapy, specifi cally, to the destruction of the tumor cell membranes.

The effect of the in vivo ultrasonic and light irradi ation on the transferred PC1 strain tumors of the rat liver in the presence of CaCO3 microcontainers con taining the photosensitizer Photosens has been inves tigated. Employing such an approach leads to the appearance of dystrophic changes and extensive necrotic zones in the tumor cells, pointing to the pres ence of a marked destructive effect. The application of such microcontainers seems promising for the treatment of external tumors, or tumors accessible to ultrasonic and optical irradiation. ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research within the research project no. 120333088 mol_a_ved and by the U.M.N.I.K. Program within the R&D project no. 01201265349, as well as by the Russian Federation Government grant no. 14.Z50.31.0004 supporting scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institu tions of higher education.


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Translated by O. Kadkin

2014