Received date: October 15, 2015; Accepted date: January 20, 2016; Published date: January 27, 2016
Citation: Colone M, Kaliappan S, Calcabrini A, Tortora M, Cavalieri F, et al. (2016) Redox-active Microcapsules as Drug Delivery System in Breast Cancer Cells and Spheroids. J Mol Genet Med 10:200. doi:10.4172/1747-0862.1000200
Copyright: © 2016 Colone M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The purpose of our study was to develop new delivery systems for drugs effective against breast cancer by using biodegradable and biocompatible capsules. Redox-active microcapsules based on thiolated polymethacrylic acid (PMA) were employed. The interaction of these PMASH capsules with breast cancer cells and the mechanism of their internalization was investigated. PMASH biocompatibility was evaluated by MTT assay. To analyze their potential as drug carrier, we incorporated doxorubicin into the capsules. Confocal microscopy observations showed the presence of capsules inside the cells. Although some drug molecules still appeared co-localized with PMASH capsules, strong doxorubicin fluorescence was observed both in the cytoplasm and nucleus, indicating the disassembling of the capsule into PMASH-drug conjugate after internalization. These results were confirmed by both flow cytometry (time course of capsule uptake) and scanning electron microscopy. PMASH capsules were also internalized in 3D cell structures (spheroids) suggesting their potential use as drug delivery system for treatment of human diseases.
CLSM: Confocal Laser Scanning Microscopy; DOX: Doxorubicin; ECM: Extracellular Matrix; GSH: Glutathione; HBSS: Ice-cold Hank’s Balanced Salt Solution; MCB: Monochlorobimane; MTT: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS: Phosphate Buffer Saline; PMASH: Thiolated Poly (methacrylic acid) Capsules; SEM: Scanning Electron Microscopy
Breast cancer is the most frequent type of malignancy and the leading cause of cancer deaths of women in developed countries; it constitutes 23% of total cancer cases and 14% of the cancer deaths . Approximately up to 7% of breast cancers are being diagnosed in women less than 40 years old years and less than 4% in women below the age of 35 years . Breast cancer is a heterogeneous disease that can be classified into several subtypes, having different histological, molecular expression signature and clinical outcomes .
Hormonal therapy, surgery, radiation and chemotherapy represent the treatment options for breast cancer patients. Chemotherapeutic agents currently approved for breast cancer treatment include several drugs (anthracyclines, alkylating agents, platinum drugs, taxanes, vinca alkaloids) [NIH, National Cancer Institute, http:// www.cancer.gov/about-cancer/treatment/drugs/breast). Unfortunately, they often show lack of selectivity or specificity toward cancer cells, resulting in several side effects .
Recently, nanomedicine has received increasing attention for its ability to improve the efficacy of cancer therapeutics. Nanosized polymer therapeutic agents offer the advantage of prolonged circulation in the blood stream, targeting to specific sites, improved efficacy and reduced side effects. Various drug delivery systems such as nano/microparticles, liposomes and implants have been demonstrated to significantly enhance the preventive/therapeutic efficacy of many drugs by increasing their bioavailability and targetability. Furthermore, their administration would become less expensive in the near future . The achievement of a local, controlled delivery of the drug at the target site would allow keeping the systemic concentration of the drug low. In this regard, other Authors reported that "ideal smart nanoparticles" for redox and pH-responsive drug delivery system (DDS) should enhance therapeutic efficacy thus reducing side effects due to bioaccumulation [6,7].
The purpose of our study was to verify a drug delivery system for breast cancer cells using a microcarrier for doxorubicin (DOX) which disassembles into a polymer-DOX conjugate after internalization. To this aim, we decided to use redox sensitive degradable microcapsules. It is well known that in tumor tissues and in cancer cell cytosol, biological reducing agents have a 4-fold higher concentration than that in the normal tissues . Based on this finding, redox shell-sheddable capsules possess great prospective for anticancer drug release because they are stable in the extracellular environment but able to rapidly respond to the intracellular GSH, releasing drugs into the cytosol and nuclei.
Redox-active microcapsules based on thiolated polymethacrylic acid (PMASH) have been extensively studied over the past few years. PMASH are assembled by the alternate deposition of PMASH and poly(vinylpyrrolidone) (PVPON) onto silica particles, followed by subsequent cross-linking of the thiol groups in the PMASH to form stable disulfide bonds and dissolution of the sacrificial silica core. The disulfide-bonded capsules are designed to be stable in oxidizing conditions (such as the bloodstream) but to disassemble in reducing environments such as the cytoplasm of the cell [9-12]. The potential of PMASH as an in vitro therapeutic delivery system of encapsulated proteins and peptides, oligonucleotides as well as small molecule drugs, has been demonstrated [13,14]. Previous studies reported the cytotoxic effect induced in human colorectal cells by DOX loaded into PMASH capsules by an oil phase or by pH labile hydrazone linkages [9,15]. Herein we report the loading of PMASH microcapsules with DOX using no hydrolysable amide bonds. The interaction of these PMASH capsules with a cancer cell model (SKBR3, human breast cancer cell line) as well as the mechanism of their internalization were then investigated. In addition, in order to predict more accurately in vivo results, PMASH capsules were assessed as delivery system in SKBR3 cells grown as spheroids in 3D culture model to analyse their efficacy in a system that mimics in vivo condition.
The human breast cancer cell line SKBR3 was from American Type Culture Collection (ATCC, Rockville, MD). Cells were grown in DMEM (EuroClone S.p.A., Milan, Italy) supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2 and sub-cultured prior to confluence using trypsin/EDTA.
Three-dimensional (3D) cell culture
SKBR3 cells were also cultured as multicellular spheroids. To this aim, 3D ON-TOP assay was performed . Eight-well glass chamber slides were pre-coated with undiluted growth factor-reduced Matrigel™ (10 mg/ml) (BD Biosciences, Franklin Lakes, NJ, USA). After Matrigel solidification, 3 × 103 SKBR3 cells (resuspended in 100 μl growth medium) were seeded on each well and incubated for 10-30 min at 37°C. A mixture of complete medium containing 10% Matrigel was added to the plated culture (100 μl/well). Cultures were maintained for 12-15 days, replacing Matrigel-medium mixture every 2 days. To analyse the effects of DOX-loaded PMASH, once spheroids were formed (10 days), the particles (1:25) were added to this mixture (3D cultures) and their effects analysed after 72 h.
Preparation of PMASH microcapsules and PMASH capsules loaded with doxorubicin
Thiolated poly (methacrylic acid) (PMASH) and PMASH capsules, 1 μm in diameter, were prepared as previously reported . Doxorubicin (DOX) was covalently linked with the carboxyl group of PMASH capsules using EDC chemistry procedure. The carboxyl groups were activated when 100 μl PMASH capsules were incubated with 0.010 mg of EDC (5.3 x 10-5 mmol) at pH 7 in PBS (phosphate buffer saline) for 30 minutes. Subsequently the capsules were incubated with DOX (5.3 x 10-5 mmol) for 30 minutes at pH 7 in PBS buffer and washed to remove free drug. The concentration of (PMASH) microcapsules was determined by using a counting chamber: 0.5 μl of PMASH capsules were diluted 10 times with PBS buffer at pH 7 and 1 μl of the diluted PMASH capsules was placed on a glass slide. Then, capsules were counted from the micrographs obtained by optical microscopy.
Determination of DOX loaded into PMASH capsules
The amount of DOX loaded into PMASH capsules was determined using UV spectroscopy. At first, the concentration of DOX in the medium (initial concentration) was measured. After loading, the PMASH capsules were centrifuged and the absorbance of DOX present in the supernatant was measured. The amount of doxorubicin loaded into PMASH capsules was calculated using the formula: % uptake=Initial DOX Concentration – Concentration of free DOX in the supernatant (after reaction)/ Initial DOX Concentration. Doxorubicin loaded onto PMASH microcapsules was also quantified after microcapsule disassembly. As DOX is covalently bonded with the carboxylic group of microcapsules by amide linkage and the amide linkage is not hydrolyzed, doxorubicin release was performed in the presence of the reducing agent DTT. Under this condition, dissolution of microcapsules caused PMASH-DOX release. In brief, 50 mM of freshly DTT in PBS buffer (pH 7) was added to DOX loaded capsules. Subsequently the capsules were centrifuged at 4.5 rcf for 10 minutes. The absorbance of the supernatant was measured by UV spectroscopy. From the absorbance value the amount of DOX released was calculated. The loading of drug into the microcapsule suspension was 3.4 × 10-10 mg of DOX per microcapsule.
Briefly, SKBR3 cells were seeded into 96-well microtiter plates (NunclonTM, Nunc, Germany) at a density of 1.5 × 104 cells/well. After 24 h cells were treated with polymer, empty PMASH and PMASH loaded with DOX at concentration of 25, 50, 75 or 100 particles/cell. After an incubation period of 24, 48, 72 h, medium was replaced by fresh medium containing 0.5 mg/ml MTT (Sigma, Deisenhofen, Germany). After 2 h at 37°C, unreacted dye was removed and the purple formazan product was dissolved in 200 μl/well dimethylsulfoxide (Merck, Darmstadt, Germany). The absorbance was read at 570 nm on a scanning microtiter spectrophotometer after agitating the plate for 5 min on a shaker. Data were expressed as absorbance values relative to untreated cells considered as 100%. Assays were performed in triplicate and repeated 2-3 times .
Scanning electron microscopy
The effects of PMASH on cell morphology were visualized by scanning electron microscopy (SEM). Cells were grown on glass coverslips (12 mm-diameter) and treated with particles for 24 and 48 h (1:25). After incubation, samples were washed to remove unbound PMASH and fixed in 2.5% glutaraldehyde in 0.2 M Na-cacodylate buffer (pH 7.4) for 2 h at RT. After 3 washes, cells were post-fixed with 1% (w/v) OsO4 for 1 h, dehydrated in an ethanol gradient, critical point dried in CO2. The coverslips were attached to alluminium stubs, mounted with silver print and coated with gold in a sputter coater. The samples were examined with a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments, Cambridge, UK).
To quantitative analyze intracellular uptake of doxorubicin-loaded PMASH capsules, SKBR3 cells (5 × 105 cells/well) were seeded in 6-well plates and incubated at 37°C in a 5% CO2 incubator overnight. DOXloaded PMASH capsules were added to the culture medium at the final concentrations of 1:25 cell/DOX-particles at 37°C from 24 h to 48 h. At the end of each treatment, cells were washed with ice-cold Hank’s balanced salt solution (HBSS; Sigma Chemical Company, St. Louis, MO, USA), detached with EDTA and 0.25% trypsin, resuspended in ice-cold PBS and immediately examined for a time course analysis of the uptake by LRSII flow cytometer (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Moreover, to determine cellular glutathione (GSH) content, SKBR3 cells (3×105 cells/well) were seeded in 6-well plates and incubated at 37° C in a 5% CO2 incubator overnight. GSH level was quantified using monochlorobimane (MCB, Molecular Probes, Eugene, OR, USA), a non-fluorescent compound that reacts with GSH to form a highly fluorescent derivative. Cells were treated with DOX-PMASH, at 1:25 cell/capsules, at 37°C from 24 h to 72 h, then incubated with MCB solution (25 μg/ml) for 15 min at 37°C. Samples were washed with HBSS and detached as described above, then analyzed by flow cytometry. Fluorescence emissions from DOXloaded capsules and from MCB were collected through a 570 and 530 nm band-pass filters, respectively, and acquired in log mode. Capsule uptake and GSH level were evaluated as fluorescence intensity values, expressed as mean fluorescence channel and calculated by DIVA software (Becton Dickinson and Company).
Confocal laser scanning microscopy
The analysis of the internalization process of DOX-loaded PMASH capsules was performed by confocal laser scanning microscopy (CLSM) on both cell cultures and multicellular spheroids. SKBR3 cells were grown on coverslips and treated with capsules (1:25) for 24, 48, 72 h. 3D cultures grown for 10 days, as previously described, were treated for 72 h with PMASH at the same concentration. Subsequently, monolayer cultures and spheroids were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature (RT), washed in the same buffer and permeabilized with 0.5% Triton X-100 (Sigma Chemicals Co.) in PBS (5 min for cells, 20 min for spheroids) at RT. For nuclei detection, cells and spheroids were stained with DAPI (150 nM Sigma) at 37°C for 30 min.
For Golgi apparatus detection, cells were incubated with IgG1 monoclonal antibody (Purified Mouse Anti-GM130, diluted 1:100, BD Transduction Laboratories™), 1 h at RT, followed by detection with Alexa Fluor 488-labeled goat anti-mouse IgG for 1 h at RT. After washing with PBS, coverslips were mounted with glycerol-PBS (2:1) and analyzed by intensified charge-coupled device video microscopy (IVM) with a Nikon Microphot fluorescence microscope (Nikon Europe B.V., Amsterdam, The Netherlands) equipped with a Zeiss CCD camera (Carl Zeiss, Oberkochen, Germany). Confocal microscopy experiments were performed on a Olympus IX81 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with Ar laser 488 nm.
One-way ANOVA test was used to perform statistical analysis of the data. Differences between two groups were considered significant at p<0.05.
To evaluate the potential use of PMASH-DOX capsules as in vitro delivery system for breast cancer cells, the effect of thiolated poly- (methacrylic acid) and PMASH capsules on cell growth and viability was analyzed by MTT assay. This test is based on the reduction ability of tetrazolium salts to formazan by metabolically active cells, which correlates with the number of viable cells. As shown in Figure 1A, cell viability was not affected after incubation with different polymer solutions at increasing concentration (0.001, 0.01 and 0.1 mg/ml). Only after 72 h of treatment with 0.1 mg/ml the polymer induced a low cytotoxic effect. Furthermore, treatments with PMASH capsules (25, 50, 75 and 100 PMASH capsules/cell ratio) up to 72 h of incubation did not alter substantially the number of viable cells (Figure 1B). On the basis of this evidence, in the studied concentration range, both polymer and PMASH capsules resulted to be not toxic for SKBR3 cells.
In order to evaluate the potential of PMASH capsules as a carrier to deliver anticancer drugs, doxorubicin (DOX) was covalently linked into the capsules. DOX has an intrinsic fluorescence (excitation at 480 nm, emission at 550-650 nm) that can be exploited to monitor the intracellular accumulation of the drug. SKBR3 cells were treated with different capsules/cell ratios (Figure 1C). The effect induced by these particles was time- and dose-dependent: an evident reduction of viable cell number was observed after incubation for 72 h with all tested capsules/cell ratios (1:50, 1:75, 1:100). These studies indicated 25 capsules/cell as an optimal ratio to study the efficiency and the kinetics of the uptake. Next, SKBR3 cells were incubated with PMASH-DOX at the same capsules/cell ratio, washed and analysed by flow cytometry.
Figure 1: Cell viability (% respect to untreated cells considered 100%) by MTT test. A) SKBR3 cells were treated with different concentrations of PMASH polymer for 24, 48 and 72 h. B) Cells were incubated with PMASH capsules at different capsule/cell ratio (ranging from 25 to 100) for 24, 48 and 72 h. C) SKBR3 cells were treated with DOX-loaded PMASH capsules at different concentration and incubation times.
The increase of DOX-related fluorescence signal was detected as a function of time. Figure 2 shows that the cellular uptake process of PMASH-DOX capsules was already complete after 48 h incubation as the fluorescent signal did not change at longer incubation times (data not shown). Scanning electron microscopy (SEM) was used to gain an understanding of the mechanism of interaction between cells and capsules during the uptake process.
Figure 2: Flow cytometric study of the uptake process of DOXloaded PMASH capsules by SKBR3 cells. After 24 and 48 hincubation, fluorescence signals arising from capsules that entered the cells were analyzed and represented as mean fluorescence channel. Data show the mean value ± the standard deviation of three independent experiments (k, untreated cells).
SEM micrographs revealed the adhesion of PMASH-DOX capsules on cell surface. Some PMASH-DOX particles could be observed on cell membranes after 24 h incubation (Figure 3A, arrows). After 48 h, numerous membrane protrusions and ruffles could be observed on cell surface (Figure 3B, arrow) suggesting that the internalization process (Figure 3C, arrow) might involve these structures related to cell cytoskeleton. No evidence of cell damage could be detected after 48 h of incubation (Figure 3B and 3C). In Figure 3D (72 h of incubation) arrows indicate the PMASH-DOX capsules inside the cytoplasm underlying the plasma membrane as demonstration of capsules uptake.
Further insight into the internalization and intracellular distribution of PMASH-DOX capsules was gained by confocal laser scanning microscopy (CLSM) observations (Figure 4) after an incubation period of 48 h. Red, green and blue colours indicate PMASH-DOX, actin filaments and nuclei stained with Hoechst 33342, respectively. The arrows in the micrographs indicate the internalization of PMASHDOX capsules and the localization of DOX inside the nucleus after microcapsules disassembling (Figures 4A and 4B, respectively). These observations suggested that PMASH-DOX conjugate was able to migrate into the nucleus crossing the nuclear membranes. In order to investigate if other cellular compartments could represent localization sites for PMASH-DOX capsules, Golgi apparatus was stained with the monoclonal antibody anti-GM130. After 48 h incubation with PMASH-DOX capsules, was observed the presence of intact capsules near Golgi apparatus (Figure 4C, arrows). However, some SKBR3 cells showed evidence of yellow merged fluorescence signal arising from DOX conjugate polymer co-localized near the nucleus, in Golgi area (Figure 4C, inset). Other studies reported that free doxorubicin shows a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus . In particular, Golgi apparatus plays a pivotal role in the intracellular distribution of DOX . These results indicate that other organelles in addition to nuclei are important sites of accumulation of PMASH-DOX polymer. In order to investigate intracellular PMASH-DOX capsule fate in suffering and/or dead SKBR3 cells after 72 h of incubation, detached cells were collected from the supernatant medium and placed on glass coverslips, previously covered with polylysin, then processed for CLSM analysis. The induction of apoptotic death by PMASH--DOX capsules was confirmed by the observation of nuclei with typical areas of condensed chromatin (Figure 4D). The inset shows a 3D reconstruction image of an apoptotic cell.
Figure 4: CLSM images of SKBR3 cells incubated with PMASHDOX capsules. (A) SKBR3 cells at 48 h of incubation with capsules. (B) A three-dimensional CLSM reconstruction of the same samples (CLSM 3D mode image). (C) CLSM images of adherent SKBR3 cells after 48 h of incubation with PMASH-DOX. Golgi areas are green while PMASH-DOX capsules are red. Inset shows open PMASH-DOX capsules in Golgi area at 72 h of incubation (yellow signal from merged image). (D) Apoptotic SKBR3 cells into present in the supernatant after 72 h of PMASH-DOX incubation. The inset shows a CLSM 3D mode image. Red, green and blue indicate PMASH-DOX, actin filaments and nuclei stained with Hoechst 33342, respectively.
As previously described, PMASH capsules are assembled by subsequent cross-linking of the thiol groups . The disulfide-bonded capsules are stable in oxidizing conditions (such as the bloodstream) but disassemble in reducing environments such as the cytoplasm of the cell . The intracellular reducing condition promotes PMASH-DOX capsules dissolution into polymer-DOX soluble conjugate. As DOX is linked to PMASH polymer chains via amide linkages, the DOX intercalation into DNA grooves is likely sterically hindered by the polymer chain. We speculate that PMASH-DOX caused antiproliferative and cytotoxic effect in SKBR3 cells through different mechanisms including generation of free radicals and direct membrane alterations.
To demonstrate the intracellular redox-induced rupture of PMASHDOX capsules, we evaluated GSH cytoplasmatic concentration during PMASH capsules trafficking in SKBR3 cells. The modulation of GSH level during PMASH-DOX particles uptake was evaluated by flow cytometry analysis after labelling of GSH with MCB (Figure 5). The lowest fluorescent signal, corresponding to a lowest concentration of GSH, was observed after 48 h incubation at the ratio 25 capsules/cell. This result suggested that the decrease of reduced GSH could be associated with the dissolution of PMASH particles and consequently with the release of PMASH-DOX in the intracellular space. Evidence of PMASH-DOX capsules dissolution was also provided by the homogeneously distributed red emission observed by CLSM analysis (Figure 4A, inset Figure 4D). From these observations we could hypothesize that PMASH-DOX capsules exerted their apoptotic activity in SKBR3 cells within 48 h by disassembling into cytotoxic PMASHDOX polymer.
Figure 5: Flow cytometric analysis of GSH levels by using MCB. SKBR3 cells were treated with PMASH-DOX capsules for 24, 48 and 72 h. Fluorescence signals were represented as mean fluorescence channel. Data show the mean value ± the standard deviation of three independent experiments (k, untreated cells).
To evaluate micro-nanocarriers biological efficacy, monolayer (2D) cell cultures are typically used as in vitro testing models. However, these studies often are unable to predict in vivo results. 2D-cultures do not reproduce the complex cell-cell and cell-matrix interactions, which significantly limit their ability to recapitulate the appropriate level of in vivo cellular response. In vitro 3D-culture models have been introduced to reduce the gap between in vitro 2D-cultures and in vivo models . Multicellular spheroid is considered the most effective 3D cell culture model ; it is characterized by many features of in vivo condition such as the interaction of cells with the extracellular matrix (ECM) components . To assess if 3D organization and ECM presence could affect the entry and interaction of PMASH-DOX capsules with individual cells, SKBR3 cells were grown following a 3Ddimensional cell culture protocol that employs Matrigel . SKBR3 spheroids appeared as multicellular aggregates characterized by poor cell-cell adhesion (Figures 6A and 6B, optical and scanning electron microscopy, respectively), as previously observed by other Authors . To better analyse internal organization, spheroids were labelled with nuclear stain DAPI and observed by confocal microscopy (Figure 6C). To study the interaction with capsules, SKBR3 spheroids after 10 day-growth were treated for 72 h with PMASH-DOX capsules (1:25) and observed by confocal microscopy (Figure 6D). PMASH-DOX capsules and polymer were observed inside the 3D cultures (Figure 6D, arrow). In addition, some nuclei positive for DOX signal could be detected suggesting the presence of drug released from the opened capsules (Figure 6D, arrowhead). These results suggested that the presence of Matrigel (which mimics the ECM structure in in vivo growth condition) allowed the entry and diffusion of capsules inside the 3D organized structures.
Figure 6: SKBR3 spheroid (10 day-growth) observation by phasecontrast optical (A) and scanning electron microscopy (B). CLSM image of control spheroids labelled with nuclear dye DAPI (C). CLSM micrograph of spheroids after 72h treatment with PMASHDOX capsules (D) showing both the presence of capsules near the nuclei (arrow) and some drug-positive nuclei (arrowhead).
We showed that PMASH-DOX capsules exhibited an efficient intracellular controlled release of PMASH-DOX conjugate in breast cancer cells and spheroids. In vitro cell viability assay indicated that PMASH-DOX capsules could efficiently inhibit SKBR3 cell viability at long incubation times, suggesting that the antitumoral drug maintained its activity even if conjugated to a polymer by a nonhydrolyzable linkage. Cellular uptake studies showed that PMASHDOX conjugate was able to access the nuclei of SKBR3 cells, following PMASH-DOX capsules dissolution induced by intracellular reducing condition. Results obtained in SKBR3 cells grown as spheroids indicated that PMASH capsules were also able to enter and move into these 3D organized cellular structures suggesting that PMASH capsules could represent a promising vehicle for intracellular controlled drug delivery in nanomedicine.