Modulation of Induced Cytotoxicity of Doxorubicin by Using Apoferritin and Liposomal Cages

Int. J. Mol. Sci. 2014, 15, 22960-22977; doi:10.3390/ijms151222960 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Modulation of Induced Cytotoxicity of Doxorubicin by Using Apoferritin and Liposomal Cages Jaromir Gumulec 1,2, Michaela Fojtu 1,3, Martina Raudenska 1,2, Marketa Sztalmachova 1,2, Anna Skotakova 1, Jana Vlachova 2,4, Sylvie Skalickova 2,4, Lukas Nejdl 2,4, Pavel Kopel 2,4, Lucia Knopfova 5, Vojtech Adam 2,4, Rene Kizek 2,4, Marie Stiborova 6, Petr Babula 3 and Michal Masarik 1,2,* 1 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic; E-Mails: [email protected] (J.G.); [email protected] (M.F.); [email protected] (M.R.); [email protected] (M.S.); [email protected] (A.S.) 2 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-61600 Brno, Czech Republic; E-Mails: vlachova.ja[email protected] (J.V.); [email protected] (S.S.); [email protected] (L.N.); [email protected] (P.K.); [email protected] (V.A.); [email protected] (R.K.) 3 Department of Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic; E-Mail: [email protected]uni.cz 4 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-61300 Brno, Czech Republic 5 Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic; E-Mail: l.knop[email protected] 6 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12840 Prague 2, Czech Republic; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: ma[email protected]; Tel.: +420-5-4949-3631; Fax: +420-5-4949-4340. External Editor: Bing Yan Received: 3 September 2014; in revised form: 6 November 2014 / Accepted: 1 December 2014 / Published: 11 December 2014 Abstract: Doxorubicin is an effective chemotherapeutic drug, however, its toxicity is a significant limitation in therapy. Encapsulation of doxorubicin inside liposomes or ferritin cages decreases cardiotoxicity while maintaining anticancer potency. We synthesized OPEN ACCESS Int. J. Mol. Sci. 2014, 15 22961 novel apoferritinand liposome-encapsulated forms of doxorubicin (“Apodox” and “lip-8-dox”) and compared its toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen. The toxicity of the modified doxorubicin forms was compared to conventional doxorubicin using the MTT assay, real-time cell impedance-based cell growth method (RTCA), and flow cytometry. The efficiency of doxorubicin entrapment was 56% in apoferritin cages and 42% in the liposome carrier. The accuracy of the RTCA system was verified by flow-cytometric analysis of cell viability. The doxorubicin half maximal inhibition concentrations (IC50) were determined as 170.5, 234.0, and 169.0 nM for PNT1A, 22Rv1, and LNCaP, respectively by RTCA. Lip8-dox is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines similar to doxorubicin or epirubicin (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Apodox IC50 was determined as follows: 603.1, 1344.2, and 931.2 nM for PNT1A, 22Rv1, and LNCaP. Keywords: doxorubicin; liposome; apoferritin; cancer; cardiotoxicity; modification; encapsulation 1. Introduction The anthracycline antibiotic family comprises hundreds of analogues, but only a few are in actual clinical use. The best known and the most widely used are doxorubicin and epirubicin. Doxorubicin is an effective chemotherapeutic drug in a wide range of cancers, including both hematological and solid tumors [1]. The therapeutic activity of doxorubicin is achieved through the processes of intercalating into DNA, inhibiting topoisomerase II, and preventing DNA and RNA synthesis [2]. One of the biggest hurdles encountered in cancer therapy includes the problem of dose-dependent toxicity toward the non-cancerous cells because high doses of drug treatment were generally required owing to poor drug accessibility to the tumor site. The total lifetime dose of the doxorubicin is limited to 550 mg/m2. As the lifetime accumulative dose approaches 500 mg/m2 and beyond, life-threatening cardiomyopathy becomes more likely, which can lead to dilated cardiomyopathy (DCM) and congestive heart failure (CHF) in up to 20% of cases [3]. It has also been shown that doxorubicin can produce acute toxicity due to bone marrow attenuation, alopecia and oral ulceration [4]. The newer anthracyclines, such as epirubicin, and idarubicin have higher lipophilicity and putatively greater safety. However, the risk of inducing cardiomyopathy is not abated; the median cumulative dose of epirubicin to the development of symptomatic CHF is 1134 mg/m2 [5]. Early studies found that encapsulation of doxorubicin inside liposomes decreases the cardiotoxicity associated with the free form of the drug, while maintaining anticancer potency [6–13]. Since the liposomal form of the drug is less toxic for the nondividing cells, increased drug dosages can be administered, ensuing in improved efficacy and an increase in the therapeutic index. Furthermore, Rahman et al. have shown that liposome-encapsulated doxorubicin modulates the multidrug-resistance (MDR) phenotype in cancer Int. J. Mol. Sci. 2014, 15 22962 cells by changing the function of p-glycoprotein. The liposomes bound to p-glycoprotein [14,15], which was associated with enhanced cellular drug accumulation and altered drug distribution inside the cells [14,16–18]. The modulation of MDR phenotype by liposomes has been also demonstrated in mice transfected with a functional human mdr-l gene [19]. Another promising delivery system for anticancer drugs seems to be apoferritin [20]. Apoferritin is the iron-free form of ferritin, a naturally-occurring iron-storage protein consisting of 24 protein subunits. Its protein subunits assemble to form a hollow cage into which diverse substances, such as drugs, can be placed. Furthermore, ferritin is internalized by some tumors, which can enable targeting to those tumorous tissues [20]. Using apoferritin as a nanocarrier has the potential to move undetected through the body without inducing any resistance from the immune system of the patient. An invention of a drug delivery system, which is biocompatible, stable, and non-toxic for healthy tissue, but still has high anticancer potency, is a great challenge in anticancer drug research. We synthetized novel apoferritinand liposome-coated forms of doxorubicin (“Apodox” and “Liposome-8”) and compared their toxicity with doxorubicin and Myocet on prostate cell lines. Three different prostatic cell lines PNT1A, 22Rv1, and LNCaP were chosen as a model of prostate cancer. Cell line 22Rv1 is derived from a primary tumor and LNCaP represents secondary tumor derived from lymph node metastasis. PNT1A cells are androgen-receptor positive and express wild-type p53. This line was used as a representative of healthy prostatic tissue, taking into account a limitation resulting from faster proliferative rate of PNT1A compared with low basal proliferative rate in normal prostatic glands [21]. The point of interest of this study was to compare the toxicity of the modified doxorubicin forms with commercially available forms using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, real-time cell impedance-based cell growth method, and flow cytometry. 2. Results 2.1. Characteristics of Modified Doxorubicin Forms First, we prepared encapsulated 100 µg/mL doxorubicin into 1 mg/mL liposome and 1 mg/mL of apoferritin as is shown in Figure 1A,B. Details of liposome and apoferritin preparation as well as doxorubicin encapsulation are described in caption 4.2 and 4.3. The size and zeta potential of the nanovesicles were measured using a zetasizer. According to the results, the light scattering measurement of liposome (Figure 1C) showed that the median size is 106 nm (40–250 nm) with a small population of liposomes of average size about 800 nm. To prove stability of liposomes, the measurement of the zeta potential (or charge density) was carried out. The high value of zeta potential (−54 ± 0.15 mV) shows high stability as a result of its permanent negative charges on the surface of liposomes. On the Figure 1D is microphotograph of used liposome (highlighted in circle). Subsequently, we performed a light scattering measurement of apoferritin (Figure 1E). Our results proved that the median size of particles is 10 nm (5–15 nm) which is in a good agreement with other publications [1]. High stability of apoferritin and its negative charge a results in a high value of zeta potential (−7.09 mV). Int. J. Mol. Sci. 2014, 15 22963 Figure 1. Scheme of encapsulation of doxorubicin into (A) the liposome modified by cholesterol and (B) apoferritin. (C) Size distribution of liposome and (D) microphotography of liposomes. Liposomes are highlighted with a circle (red circle = 100 nm and orange circle = 200 nm); (E) Size distribution of apoferritin. Absorbtion spectra in the range 230–800 nm of (F) 100 µg/mL Myocet; (G) Epirubicin; (H) Liposome (a), Doxorubicin (b), Encapsulated Doxorubicin into the liposome (c); (I) Apoferritin (a), Doxorubicin (b), Encapsulated Doxorubicin into the apoferritin (c); Emission spectra in the range 520–820 nm of (J) 100 µg/mL Myocet; (K) Epirubicin; (L) Liposome (a), Doxorubicin (b), Encapsulated Doxorubicin into the liposome (c); (M) Apoferritin (a), Doxorubicin (b), Encapsulated Doxorubicin into the apoferritin (c). The excitation wavelength was 510 nm. Spectrophotometric characterization of epirubicin, myocet, and doxorubicin encapsulated into the liposome was carried out by measuring the absorbance spectra of studied variants of doxorubicin in the range from 230 to 800 nm of studied variants of doxorubicin. Figure 1E shows the absorbance maximum 0.1 absorbance units (AU) of (100 µg/mL) myocet at 496 nm. Epirubicin shows higher absorption maximum 0.52 AU at 490 nm in comparison with myocet (Figure 1F). Subsequently, we compared the absorbance spectra of Liposome (a), Doxorubicin (b), and encapsulated Doxorubicin into the Int. J. Mol. Sci. 2014, 15 22964 liposome (c). The results indicate that the liposomes do not show the highest absorbance. Absorbance of doxorubicin 0.49 AU at 490 m is comparable with epirubicin. Absorbance of encapsulated doxorubicin into the liposome shows a decreasing trend with a small peak at 590 nm and with an absorbance maximum of 0.23 AU (Figure 1G). In addition, from the graph in Figure 1H, the low absorbance of apoferritin is evident. In the case of encapsulated doxorubicin into apoferritin, the absorbance maximum of 0.23 AU at 590 nm is higher in comparison with doxorubicin encapsulated into liposome. Subsequently, the fluorescence spectra of anticancer drugs in the range 520–820 nm at λ = 510 nm of anticancer drugs were measured. Myocet (Figure 1I) shows the fluorescence maximum of 9654 AU at 596 nm, while this value for epirubicin is 75,675 AU at 596 nm, representing an 87% decrease of fluorescence caused by encapsulation of anticancer drug into the liposome. The same trend was estimated for doxorubicin and liposomal doxorubicin. The liposome alone does not have any fluorescence properties, after encapsulation of doxorubicin there is an increase of fluorescence to 22,686 AU at 596 nm what is in comparison with doxorubicin alone enhancement by 70%. In the case of apoferritin encapsulated doxorubicin (27,601 AU at 596 nm), the absorbance is decreaseing by 63% in comparison with doxorubicin and apoferritin showing no fluorescence. From obtained results we estimated the total 72.4 µM (42 µg/mL) doxorubicin was encapsulated into the total amount of 20 mg of liposome carriers. Consequently, cholesterol content was quantified in liposomal particles. Total 0.1 µg/mL was determined. Thus, the prepared liposomes contained 210 µg of doxorubicin per micromole of cholesterol. With regard to apodox, 56 µg/mL of doxorubicin (96.5 µM) was encapsulated into apoferritin cages. Final concentration of apoferritin was 1 mg/mL. Initial concentration of doxorubicin was 100 µg/mL (172.4 µM) in both cases. It follows that the efficiency of doxorubicin entrapment was 42% in case of liposome carrier and 56% in case of apoferritin. 2.2. Stability of Nanocarriers We determined doxorubicin in concentration range from 0 to 2 mM in solutions of various concentration of serum (in PBS), Figure 2A. We choose 5% serum phosphate buffer as a model condition for the following experiment, because of sufficient sensitivity for such determinations. Next we tested the stability of complex doxorubicin-liposome and doxorubicin-apoferritin in phosphate buffer pH 7.4 (Figure 2B). Here the difference of releasing amount was obvious for both carriers. Liposome seems to be more sensitive to the phosphate buffer pH 7.4. Apoferritin is stable in the time period from 0 to 60 min. Effect of serum presence was tested in time interval from 0 to 60 min (Figure 2C). Here the decrease of signal observed is related to the determinations in PBS only. Change in relative releasing amount in apoferritin was about 2% and in liposome about 8%. Both carriers seem to be relatively stable in the phosphate buffer pH 7.4 with 5% serum in the time period from 0 to 60 min. Liposome structure was more sensitive to the presence of 5% serum phosphate buffer at pH 7.4. Int. J. Mol. Sci. 2014, 15 22965 Figure 2. (A) Calibration curves of doxorubicine determined in phosphate buffer pH 7.4 with various content of serum as follows: (a) 0%, (b) 5%, (c) 7.5%, and (d) 10%; (B) Current signals of the released doxorubicin (related to the enclosed concentration) from liposome (a) and apoferritin (b) in phosphate buffer at pH 7.4 over a 60-min period; (C) Current signals of the released doxorubicin from liposome (a) and apoferritin (b) in 5% serum phosphate buffer at pH 7.4 over a 60-min period. Displayed as a mean ± S.E. 2.3. Comparison of Toxicity of Modified Doxorubicin First, toxicity of the modified forms of doxorubicin was analyzed. Factorial analysis of variance (ANOVA) was conducted to compare the effect of modified forms of doxorubicin on prostatic cell lines, all other factors were adjusted. There was a significant effect of the form of doxorubicin used on cell lines F(4, 68) = 20.49, p < 0.0001. Post-hoc comparisons using Bonferroni test indicated the highest toxicity of the epirubicin and doxorubicin, with mean half-maximal lethal dose (inhibition concentrations, IC50) 118.0 and 268.9 nM, respectively. Toxicity of Apodox and Lip-8-dox was significantly lower; IC50 = 1095.0 and 1014.5 nM, respectively. Myocet showed the lowest toxicity with mean IC50 = 1915.4 nM (Table 1). Consequently, the effect of cell lines was analyzed in order to assess, if the sensitivity of individual cell lines to cytostatics differs significantly. After adjustment to all other variables, there was no significant effect of cell line, F(2, 68) = 0.46, p = 0.62 (Table 1). Despite the statistical insignificance, the highest sensitivity to cytostatics was observed in 22Rv1 cell line and the lowest sensitivity was observed in non-tumor PNT1A cell line. To reveal, whether this trend in cell lines is common for all cytostatics, a combined effect of cytostatics and cell lines was analyzed. Using this test, significant differences were observed, F(8, 68) = 3.08, mp = 0.005. However, the only significant trend between combined effect of cytostatics and cell lines was determined in Myocet; sensitivity of LNCaP cell line to this cytostatic was significantly lower compared to the non-tumor PNT1A cell line (IC50 = 3216.1 and 1259.9 nM, respectively). Results demonstrated in previous paragraphs did not take into concern type of viability assay. To check whether these assays provide identical results, MTT and real-time cell impedance-based cell growth method (RTCA) IC50 values were analyzed after adjustment of all other variables. Impedance-based RTCA IC50 values are on average 1.6-fold lower compared to metabolic-based MTT assay, F(1, 68) = 6.48, p = 0.013, (Table 1). Int. J. Mol. Sci. 2014, 15 22966 Table 1. Comparison of half-maximal inhibition concentrations (IC50) for particular cell lines, cytostatics and used assay. S.E., standard error. Differences were considered significant, when p < 0.05. * Significantly different from doxorubicin (Analysis of variance (ANOVA) followed by Bonferroni post hoc testing) † Significantly different from epirubicin (ANOVA followed by Bonferroni post hoc testing) ‡ Significantly different from lip-8-dox (ANOVA followed by Bonferroni post hoc testing) § Significantly different from apodox (ANOVA followed by Bonferroni post hoc testing) ǁ Significantly different from myocet (ANOVA followed by Bonferroni post hoc testing) $ Significantly different from MTT (ANOVA followed by Bonferroni post hoc testing). Factor Level of Factor N IC50 (Mean ± S.E.) Significant Difference (At p < 0.05) Cytostatic F(4, 68) = 20.49, p < 0.0001 Epirubicin 21 118.0 ± 27.2 ‡, §, ǁ Doxorubicin 18 268.9 ± 43.6 §, ǁ Apodox 22 1095.0 ± 163.8 *, †, ǁ Lip-8 16 1014.5 ± 209.8 †, ǁ Myocet 21 1915.4 ± 417.3 *, †, ‡, § Cell line F(2, 68) = 0.46, p = 0.47 PNT1A 36 936.3 ± 167.9 22RV1 32 834.5 ± 161.5 LNCaP 30 915.1 ± 299.6 Method F(1, 68) = 6.48, p < 0.013 MTT 31 1200.1 ± 316.1 RTCA 67 756.1 ± 98.1 $ Taken together, these results suggest that doxorubicin modified by a liposome or apoferritin is less toxic to prostatic cell lines. The commercially available liposomal modification Myocet exhibited the lowest cytotoxicity among all the studied antineoplastic agents; it also showed a less toxic effect on LNCaP derived from the secondary site compared to non-tumor PNT1A. However, differences between assays were revealed, therefore, the type of the assay has to be taken into account when analysing the cytotoxic effects of individual doxorubicin modifications on individual cell lines. Therefore, detailed results of these assays were analysed separately by the assay type in the next step. Comparison of MTT and Real-Time Cell Impedance-Based Cell Growth Method (RTCA) Viability Assays In this step, results obtained only by MTT were analyzed. Results are not in agreement with previous overall results, that did not take into account viability assay, since these data do not show significant effect of cytostatics and cell lines on cell toxicity, F(8, 16) = 1.32, p = 0.30. Further, results obtained by RTCA were analyzed. The combined effect of cytostatics and cell line affected inhibition concentrations significantly; F(8, 52) = 2.24, p = 0.039. In contrast to MTT and overall results, RTCA-based results show that the Lip-8-modified form of doxorubicin is less toxic on the non-tumor cell line PNT1A compared to doxorubicin, while still maintaining the toxicity to tumorous cell lines (Table 2) (IC50 = 2076.7 nM for PNT1A vs. 935.3 and 729.0 nM for 22Rv1 and LNCaP). Int. J. Mol. Sci. 2014, 15 22967 To identify the relations between MTT and RTCA, Spearman correlation was calculated on mean IC50 values. Significant, but relatively weak positive correlation was observed (rSp = 0.48, p = 0.03). Taken together, since there is only the moderate correlation between methods used, the partial disagreement between the results of ANOVAs needs further elucidation. Therefore, the accuracy of the RTCA-based viability analysis was verified using another method, flow-cytometry. 2.4. Flow-Cytometric Viability Analysis To assess, whether the RTCA system determines viability exactly, flow-cytometric analysis of cell viability (% propidium-iodide-positive, i.e., dead) was employed (Figure 3). Cell lines were treated with concentrations of cytostatics defined as IC50 by RTCA. Concentrations are shown on Table 2. After the treatment, the portion of propidium-iodide-positive cells was determined as follows: 40.14% ± 11.86%, 38.83% ± 4.61%, and 35.97% ± 7.71% for PNT1A, 22Rv1, and LNCaP, respectively. These values were compared against a theoretical value of 50% using one sample t-test. None differed significantly, suggesting RTCA analysis accurately estimated the portion of necrotic cells (Figure 4D). Figure 3. Analysis of prostatic cell line growth treated with modified forms of doxorubicin using RTCA xCELLigence and determination of viable cell numbers by propidium iodide exclusion (inset). Cell lines shown in columns, modified doxorubicin forms in rows. Cell growth expressed as “cell index” on y-axis is based on impedance measurement. Increasing concentrations of antineoplastic agents in range 0–40 µM are depicted by green color gradient. Inset: viable cell numbers analyzed after treatment with IC50 concentrations of individual cytostatics on individual cell lines as determined by RTCA. FSC-A, forward scatter. Grey line indicate gating threshold for propidium iodide (PI)-positive (upper–left) and -negative cells (lower–right). Percentages in subsequent gating regions indicate number of PI-positive and -negative cells. Int. J. Mol. Sci. 2014, 15 22968 Table 2. Comparison of RTCA-based half-maximal concentrations (IC50). S.E., standard error. Differences were considered significant, when p < 0.05; * Significantly different from lip-8-dox PNT1A (ANOVA followed by Bonferroni post hoc testing); † Significantly different from myocet 22Rv1 (ANOVA followed by Bonferroni post hoc testing). Factor Cytostatic Cell Line NIC50 (Mean ± S.E.)Significant Difference (At p < 0.05) F(8, 52) = 2.24, p < 0.039 epirubicin PNT1A 3 12.2 ± 3.5 * 22RV1 6 49.2 ± 10.5 *, † LNCaP 6 107.9 ± 57.0 *, † doxorubicin PNT1A 5 170.5 ± 40.0 *, † 22RV1 3 234.0 ± 82.1 * LNCaP 3 169.0 ± 32.9 * apodox PNT1A 6 603.1 ± 256.8 * 22RV1 5 1344.2 ± 342.4 LNCaP 5 931.2 ± 274.4 Lip-8 PNT1A 4 2076.7 ± 353.6 22RV1 3 935.3 ± 113.5 LNCaP 3 729.0 ± 44.1 myocet PNT1A 8 1047.1 ± 279.5 22RV1 4 1657.1 ± 662.9 LNCaP 3 1440.0 ± 164.6 Figure 4. Determination of viability of prostatic cell lines treated with modified forms of doxorubicin using MTT assay (A–C); (D) verification of real-time cell impedance-based cell growth method (RTCA)-determined IC50 value. Red line indicates theoretical 50% viability. No significant difference was observed using one sample t-test. Int. J. Mol. Sci. 2014, 15 22975 Conflicts of Interest The authors declare no conflict of interest. References 1. Blum, R.H.; Carter, S.K. 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