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7Cellular Therapy and Transplantation (CTT). Vol. 12, No. 2, 2023 doi: 10.18620/ctt-1866-8836-2023-12-2-51-56 Submitted: 5 May 2023, accepted: 16 June 2023
Influence of the composition of doxorubicin delivery systems on the effectiveness of cancer therapy in transgenic FVB/N mice
Natalia N. Sudareva Dmitry N. Suslov 3, Olga М. Suvorova Galina Y. Yukina 2, Elena G. Sukhorukova 2, Natalia N. Saprykina Vladimir N. Anisimov 3
1 Institute of Macromolecular Compounds RAS, St. Petersburg, Russia
2 Pavlov University, St. Petersburg, Russia
3 N. N. Petrov National Medical Research Center of Oncology, St. Petersburg, Russia
Dr. Natalia N. Sudareva, Institute of Macromolecular E-mail: [email protected]
Compounds, 31 Bolshoi Ave, 199004, St. Petersburg, Russia
Citation: Sudareva NN, Suslov DN, Suvorova OM, et al. Influence of the composition of doxorubicin delivery systems on the effectiveness of cancer therapy in transgenic FVB/N mice. Cell Ther Transplant 2023; 12(2): 51-56.
Summary
The influence of doxorubicin (DOX) encapsulated into the delivery systems (DS) based on porous calcium carbonate vaterites doped with sodium dextran sulfate pol-yanions (CaC03+ DexS) was investigated in experimental model of FVB/N transgenic mice developing tumors associated with HER-2/neu overexpression. Doxorubicin (1 mg) was loaded in DS at different drug-to-DS ratios, then being administered intraperitoneally. It was demonstrated that the preparations with higher DOX/DS ratios suppressed tumor growth in female FVB/N mice more
efficiently. When injecting delivery systems of these compositions, the incidence of tumors was relatively lower, they developed at later terms and were smaller in size. In some cases, the tumors were not revealed until termination of the experiment (50 weeks).
Keywords
Doxorubicin, drug delivery system, CaCO3, sodium dextran sulfate, intraperitoneal administration, morphology.
Introduction
Doxorubicin (DOX), an anthracycline antibiotic which is successfully used in treatment of different malignancies, particularly, breast cancer. The main mechanisms of DOX action upon tumor cells include: (i) intercalation of DNA molecules, thus inhibiting DNA synthesis; (ii) generation of free radicals leading to DNA damage or lipid peroxidation [1]. However, the drug has several essential drawbacks: rapid clearance, dose-dependent cardiotoxicity, hepatic damage [1] and hematological toxicity [2]. The delivery systems that are able to eliminate these problems and increase DOX efficiency are discussed in the review [3], which is focused on delivery systems based on organic and inorganic compounds. Later publications describe complex multicompo-nent DOX delivery systems. For example, drug synergism observed upon combined use of DOX and ruthenium complex (A-Ru1/DOX) manifests as a decreased DOX cardiotoxicity [4]. Aiming for effective suppression of tumor growth,
DOX is introduced into the specially prepared conjugates of nanoparticles of poly(lactic-co-glycolic acid) with chondroi-tin sulfate A [5]. The authors used simultaneous encapsulation of DOX and photosensitizer Indocyanine Green, in order to enhance the effect of chemo-photothermal cancer therapy [6].
The porous particles of calcium carbonate (CaCO3) vaterites are used as delivery systems (DS) for various drugs. They draw attention of researchers due to their biocompatibility, biodegradability, non-toxicity, low cost, and ease of preparation. A comprehensive review [7] discusses the recent successful applications of the CaCO3 vaterites to the delivery (in vivo and in vitro) of various diagnostic agents and therapeutic drugs. Special attention to the use of CaC03 as carriers for antitumor preparations (in particular, with DOX) has been given elsewhere [8]. In the present work, DOX was encapsulated in the delivery systems on the basis of CaC03 cores doped with DexS polyanions (CaCO3+DexS). DOX in
the form of hydrochloride (thus creating acidic medium) is introduced into carbonate cores, thus causing re-crystallization of vaterites into non-porous calcites. Vaterites are then coated with polymers to protect their porous structure [9, 10]. Dextran sulfate is a bioavailable polymer [11]. It was shown that CaC03 doped with DexS by various methods (coating, co-precipitation) was virtually non-cytotoxic at concentrations below 0.5 mg/mL towards human dermal fibroblasts and human epidermal carcinoma A431 cells [12]. The prolonged release of DOX from delivery systems of this structure into rat blood (2-3 weeks) has been revealed in our previous publications [13] where the DS preparations were administered in two ways (intraperitoneally, or subcutane-ously). Of note, the carrier-free DOX was excreted from rats as early as 2 days after its intraperitoneal administration at similar dosage. In vivo experiments [14] demonstrated that intraperitoneal administration of 2 mg of DOX in DS (based on CaCO3 vaterites covered by DexS) in rats inoculated with Seidel hepatoma resulted in a two-fold increase of their life expectancy and a 1.5-fold decrease in ascites volume.
The purpose of this study was to demonstrate growth inhibition of breast adenocarcinomas in transgenic FVB/N mice after intraperitoneal administration of DOX loaded into the CaC03+DexS delivery systems at different component ratios.
Materials and methods
Reagents
Doxorubicin was purchased from Veropharm (Russia) as "Doxorubicin LENS" dosage form which contained 20% of doxorubicin hydrochloride (DOX) and 80% of mannitol. Inorganic salts (CaCl2 x 2H2O, Na2CO3), acetone, and sodium dextran sulfate (Mw = 9-20 kDa) were purchased from Sigma-Aldrich (St. Louis, MO).
Preparation of the delivery system and DOX loading
The preparation techniques for porous vaterites, methods of coating vaterites with DexS polyanions and introduction of DOX into these carriers are described in [15]. In brief, porous vaterites (CaC03 cores) were prepared by co-precipitation of equal volumes of 1 M aqueous solutions of CaCl2 x 2H2O and Na2CO3 during intensive mixing for 30 seconds. Then the suspension was filtered through Schott filter glass, washed with distilled water and acetone. The precipitate was dried in a thermostat at 40-50°C until a constant weight was achieved.
The core DS was coated with a polyanion (sodium salt of dextran sulfate, DexS). CaC03 cores (50 mg) were added to 10 mL aqueous solution of DexS (1 mg/mL). The suspension was stirred using a Multi Bio RS-24 rotor (Biosan, Latvia) for 1 h; the solid fraction was filtered off using a Schott glass filter, washed with distilled water and dried at 30°C.
DOX was loaded into DS under continuous stirring of the mixture containing the CaC03+DexS (DS) suspension and 1 mg/mL DOX solution for 24 hours. The weight-to-weight DOX/(CaCO3+DexS) ratios were equal to 0.40 and 0.25;
thus, two sets of DOX-containing delivery systems were formed. After mixing, the suspension was centrifuged at 8000 rpm for 3 min, and the DOX amount in supernatant was determined.
DOX load (L) was calculated using the following equation: L = (m{ - m)/mp, where m{ is the initial weight of DOX; ms is the weight of non-encapsulated DOX in supernatant solution; mp is the weight of DS particles. DOX concentrations were determined using the calibration curves obtained from optical density measurements at X=480 nm in the corresponding solvents. The mean error in triplicate measurement of DOX load under similar conditions was about 8%.
Scanning electron microscopy (SEM)
The DS samples were studied with Supra 55VP scanning electron microscope (Carl Zeiss, Germany) using secondary electron imaging. Before measurements, the samples were coated with a thin platinum layer.
In vitro experiments with DOX release
The in vitro release of DOX from both variants of DS was analyzed in human blood plasma. Incubation of DS in this media was carried out at 20°C and at constant mixing by means of programmable Multi Bio RS-24 rotator (Biosan, Riga, Latvia). Aliquots were collected from incubation mixtures every 24 hours after centrifugation of core suspension at 3000 rpm for 3 min. Content of DOX in supernatant aliquots was determined spectrophotometrically (see above). The supernatant obtained by incubating CaCO3+DexS cores without DOX under the same conditions was used as a reference solution. After the measurements, the aliquots were returned to the incubation medium. The release parameter was determined as a percentage of the of DOX contents present in the initial DS suspension. The average errors in triplicate determination of DOX release under similar conditions were about 10%.
In vivo experiments with tumor-bearing mice
Transgenic FVB/N mice characterized by overexpression of oncogenic HER-2/neu proteins were used in the experiments. The body weight of mice ranged from 23 to 26 g. The in vivo experiments involved fourteen 16 weeks-old FVB/N female mice divided into three groups. Two experimental groups included 5 mice each. NaCl solution (0.9%, 1 mL) containing various DS compositions was administered in-traperitoneally (IP) to the experimental animals. The load characteristics are given in Table 1. Each sample contained 1 mg of DOX. The mice tolerated the administration satisfactorily. The third (control) group consisted of four FVB/N mice that did not receive the treatment. 29-weeks old mice (13 weeks after administration of the DS, one animal of each group) were sacrificed for morphological study. The total experimental period lasted 50 weeks.
All manipulations with animals were performed under general anesthesia: Sol. Zoletil 50 (0.05 mL per 0.1 kg of body mass), Sol. Rometarum 20 mg/mL (0.0125 mL per 0.1 kg of body mass, intramuscularly). The animals were caged (5 individuals in a cage), had free access to water and food. The animals were fed the standard diet for laboratory mice used
in the vivarium (4R F18 prolonged keeping formula for rodents, Macedonia, Italy). The animals of experimental and reference groups were examined daily; consumption of water and food was registered, body temperature and weight were measured. Behavior and life expectancy of animals were estimated for 44 weeks.
All the manipulations with animals were performed in accordance with State Standard 33216-2014 "Regulations for work with laboratory rodents and rabbits".
Characteristics of the process of tumor growth
The process was characterized using the "growth inhibition" parameter. This parameter describes antitumor effect of a tested compound at a given moment of time before or during treatment.
Growth inhibition GI (%) = (V - V/ V) x100, where V is the average volume of a tumor in the control group, and V is the average volume of a tumor in the experimental group [16].
Morphological studies
Tumor nodules were not observed in animals of the experimental group. However, the nodules were visible in the mice from the non-treated reference group.
The biological material (mammary glands, liver, lung, small intestine) were fixed in 10% neutral formalin in phosphate buffer (pH=7.4) for, at least, 24 hrs, dehydrated using a series of ethanol solutions at increasing concentrations, and enclosed in paraffin blocks according to the standard histological technique. To obtain comparable results, the samples were treated simultaneously under similar conditions. The paraffin sections (5 ^m thick) were prepared by means of an Accu-Cut SRT 200 microtome (Sakura, Japan) and stained with Mayer hematoxylin and eosin (BioVitrum, Russia). Microscopic analysis was performed using a Nikon Eclipse E200 light microscope (Nikon, Japan) with a 10x ocular and 4, 10, 20, and 40x objectives. Digital images were recorded with a Nikon DS-Fi3 camera (Nikon, Japan).
Results
Tumor incidence
The study was performed in FVB/N transgenic mice which develop spontaneous mammary tumors associated with overexpression of HER-2/neu proteins. HER-2 is a human epidermal growth factor receptor. Super-expression of activated HER-2/neu in a female transgenic FVB/N mouse leads
to malignant transformation of epithelial cells of the breast followed by development of several breast adenocarcino-mas [17]. In this work, we compared antitumor activities of DOX-containing delivery systems at various compositions administered intraperitoneally into mice; the amount of injected DOX was 1 mgper animal.
The drug carriers were based on CaC03 vaterites doped with DexS polyanions, by coating the carbonate core surface with the DexS polymer. The two used sets of DOX carriers differed in the DOX load per unit weight of a carrier. Their characteristics are given in Table 1. Antitumor activities of various DOX-containing DS were compared using the number, time of appearance and size of the detected tumors. These characteristics are also given in Table 1.
The values of tumor volumes in mice were used to determine characteristics ofthe process of disease development, i.e., inhibition of tumor growth GI (%) = (159.3-52.33)/159,3=67.1% for set 1 and GI (%) = (159.3-23)/159.3=85.5% for set 2. This parameter characterizes antitumor effect of a studied preparation. The data presented in Table 1 show that the efficiency of treatment with higher DOX load in DS (set 2) seems to be more pronounced than with delivery system from the set 1. A decrease in the average tumor volume correlates with an increase in the growth inhibition index. Hence, the inhibitory trend is more pronounced in the set 2. These results suggest that the DOX-containing DS administered intraperito-neally in both experimental sets exerts a moderate antitumor action in the FVB/N mice.
Morphological studies
Visual inspection of animals from the control group (non-treated mice) revealed breast tumors; microscopic studies showed a typical pattern of infiltrative ductal cancer: cystic formations were lined with tumor cells displaying moderate nuclear atypia and mitoses. Invasion into fatty tissue and a moderate nuclear polymorphism were also visible. When examining mice of the both experimental groups (DS-treated animals), no tumors were visually observed, but microscopic studies revealed focal atypical ductal hyperplasia, tumor cells with high nuclear atypia and pathological mitoses. Thus, we observed cytostatic effect of the administered DOX-containing DS.
Histological analysis of the studied material did also reveal morphological changes in liver and lungs, while the morphological picture of small intestine remained unchanged. In the liver, changes in cytoarchitectonics of hepatic lobules (manifested as disturbance of the normal structure) are observed. Sinusoidal capillaries were dilated, central veins are
Table 1. Influence of DS composition on development of tumors in transgenic FVB/N mice
Object DOX load, pg/mg Mice number Number of tumors per mouse Manifestation of detectable tumor, weeks Average tumor volume, mL
Control - 4 3.2 27 159±13
Set 1 250±20 5 1.0 41 52±6
Set 2 380±30 5 0.4 42 23±5
collapsed. Pronounced vacuolar dystrophy in the cytoplasm of the majority of hepatocytes was observed only in experimental group (Fig. 1 A, B). All vessels of the lung are varicose and plethoric; hemorrhages are visible. Interalveolar partitions are thickened and contain increased amounts of macrophages. The described morphological changes are possibly caused by toxic action of the injected preparation.
Figure 1. Fragment of a liver of an animal from the control group (A) and an animal from the experimental group (set 2) (8) studied 29 weeks after the beginning of the experiment. Staining: hematoxylin, eosin; magnification: "200
SEM patterns of different structures in the drug carriers
Figure 2 shows SEM photos of the parent CaCO3 vaterites (Fig. 2A) and vaterites doped with sodium dextran sulfate polyanion (Fig. 2B). The set 1 and set 2 delivery systems with different DOX loads are presented in Figs. 2C and 2D, correspondingly. Comparison of Figs. 2A and 2B shows that doping calcium carbonate cores with DexS causes some slight changes in the structure of carriers. DexS molecules penetrate rather deeply into the porous structure of calcium carbonate (SEM photo of doped vaterite sample chip is not presented).
Analysis of SEM images of set 1 and set 2 delivery systems with different DOX loads reveals films of different areas that
cover individual particles of the carriers (Fig. 2C, two particles are covered here together), or form coatings of larger areas (Fig. 2D). One should mention again that in our work doxorubicin was used in the form of the LENS preparation, which contains mannitol, and the DOX/mannitol ratio is equal to 1/4. Mannitol is an excipient widely used as a filler/ binder in pharmaceuticals due to its chemical stability, solubility and low hygroscopicity. In addition to this function, mannitol is used to prevent kidney-related side effects that arise during use of some antitumor drugs [2]. Since the DOX load in the set 1 sample is 250 ^g/mg (i.e. almost 1.5 times less than that in the set 2 samples), the set 1 preparations contain less mannitol, as is evident from the SEM images (Figs. 2C and 2D). Perhaps, higher amounts of mannitol in the delivery systems of set 2 may interfere additionally with DOX release, thus prolonging its action.
Characteristics of carriers. In vitro experiments
The further questions concerns possible causes of differential therapeutic effects observed upon administration of the same DOX dose (1 mg) by differently loaded delivery systems. Let us compare compositions of the two used sets of delivery systems. The DOX load in set 1 DS is equal to 250 ^g/mg, and in the second DS it is equal to 380 ^g/mg. Therefore, the reason for the therapeutic differences may be found in the role of carriers. We have compared the in vitro behavior of DOX delivery systems. Fig. 3 shows release profiles of DOX into blood plasma from delivery systems CaC03+DexS at different DOX load values.
One may see from Figure 3 that an increase in DOX load in the delivery systems leads to prolonged release of the drug into the ambient medium. For example, Fig. 3B (curve 1) shows almost complete release of DOX already 10 days after the beginning of the experiment. Appearance of tumors in the in vivo model can be expected at a later time. Thus, the appearance of tumors at later stages in animals treated with high-load (set 2) DOX-containing delivery systems may be explained by more prolonged DOX release.
Figure 2. SEM photos: A, parent CaCO3 vaterites; B, CaCO3 vaterites covered by DexS polyanion; C, set 1 delivery system; D, set 2 delivery system. Marker bars, 300 nm
0 2 4 6 8 10 12 14 0 2 4 6 8 10
Time, days Time, days
Figure 3. Profiles of DOX release into blood plasma from DS at different load values. А, DOX loading in water. Load: set 1 - 250 pg/mg; set 2 - 380 pg/mg. B - DOX loading in phosphate buffer. Load: set 1 - 100 pg/mg; set 2 - 360 pg/mg. Abscissa, incubation terms, days.
Conclusions
It was demonstrated that the delivery systems (DS) for antitumor doxorubicin (DOX) preparation based on porous calcium carbonate vaterites doped with dextran sulfate pol-yanion exert a cytostatic effect upon intraperitoneal administration of equivalent amounts of DOX. In female FVB/N transgenic mice, these DS suppress tumor growth more efficiently in the case when higher DOX/(CaC03+DexS) ratios are used. Taking these results into account, one may recommend usage of delivery systems with higher DOX load in further experiments.
Financial support
The study was performed within the framework of budget-supported research project №, 122012100171-8 Institute of Macromolecular Compounds, RAS.
Conflict of interests
None declared.
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Резюме
Исследовали воздействие доксорубицина (ДОХ), инкапсулированного в системы доставки (СД) на базе пористых карбонатно-кальциевых ватеритов, допи-рованных полианионом декстрансульфатом натрия (СаСО3+ DexS), на организмы трансгенных мышей линии БУВ/М. Внутрибрюшинно вводили по 1 мг ДОХ в СД с разным соотношением компонентов. Показано, что при большем соотношении ДОХ/СД эффективность контроля над ростом опухолей у самок мышей БУВШ повышается. При этом опухоли меньшего размера и в меньшем количестве проявляются позднее при введении СД именно такого состава. В ряде случаев опухоли не выявляются вплоть до окончания эксперимента в течение 50 недель.
Ключевые слова
Доксорубицин, системы доставки лекарств, СаСО3, декстран сульфат натрия, внутрибрюшинное введение, морфология.
Влияние состава систем доставки доксорубицина на эффективность лечения спонтанных опухолей у мышей линии FVB/N
Наталия Н. Сударева и, Дмитрий Н. Суслов 3, Ольга М. Суворова Галина Ю. Юкина 2, Елена Г. Сухорукова 2, Наталия Н. Сапрыкина Владимир Н. Анисимов 3
1 Институт высокомолекулярных соединений РАН, Санкт-Петербург, Россия
2 Первый Санкт-Петербургский государственный медицинский университет им. акад. И. П. Павлова, Санкт-Петербург, Россия
3 Научный медицинский исследовательский центр им. Н. Н. Петрова, Санкт-Петербург, Россия
