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PLGA based particles as “drug reservoir” for antitumor drug delivery: characterization and cytotoxicity studies
Colloids and Surfaces B: Biointerfaces 180 (2019) 495–502
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PLGA based particles as “drug reservoir” for antitumor drug delivery: characterization and cytotoxicity studies
T
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Laura Chronopouloua,1, Fabio Domenicib,c, ,1, Sabrina Giantullid,1, Francesco Brasilib,c, Chiara D’Erricoa, Georgia Tsaoulid, Elisabetta Tortorellac, Federico Bordib, Stefania Morronee, ⁎⁎ ⁎⁎ Cleofe Paloccia, , Ida Silvestrid, a
Department of Chemistry , Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Department of Physics, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy c Department of Chemical Science and Technology, University of Rome Tor Vergata, Viale della ricerca scientifica 1, 00133, Rome, Italy d Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy e Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Drug delivery Anticancer Doxorubicin PLGA carriers Internalization Burst release
Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy.
1. Introduction Cytotoxic agents remain an important weapon in the challenge against cancer, either alone or combined with other therapies [1–5]. Cytotoxic chemotherapeutics generally interfere with normal cell functions, inhibiting replication or inducing apoptosis. As it is well known, a major issue is represented by the lack of selectivity and associated side effects. Since chemotherapeutics preferentially kill rapidly proliferating cells, they can often induce the shrinkage of solid tumors, but they also affect healthy tissues. These agents, by interfering at different steps with several cellular pathways, may promote multidrug resistance [3]. Moreover, limitations are related to poor pharmacokinetics and inappropriate biodistribution [4]. Low aqueous solubility
and short circulation times contribute to the toxic side effects as well as to decrease the therapeutic effect on tumor cells and metastases [5]. In addition, anthracyclines (e.g. Doxorubicin, DOX) show a characteristic toxicity [6], significantly increasing the risk of heart failure [2]. Within this framework, drug delivery systems, including liposomes, polymeric micelles, polymersomes, nanogels and nanoparticles, have recently emerged as novel therapeutic platforms to improve intravenous administration of anticancer agents [7–12]. Several submicrocarriers have demonstrated promising abilities in enhancing circulation time and selectivity of drugs, with decreased accumulation in healthy tissues and organs. However, in the case of anthracyclines, encapsulated formulations apparently have not been optimized yet, showing significantly reduced drug internalization efficiency [2–4].
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Corresponding author at: Department of Chemical Science and Technology, University of Rome Tor Vergata, Viale della ricerca scientifica 1, 00133, Rome, Italy. Corresponding authors. E-mail addresses: [email protected] (F. Domenici), [email protected] (C. Palocci), [email protected] (I. Silvestri). 1 Equally contributing authors. ⁎⁎
https://doi.org/10.1016/j.colsurfb.2019.05.006 Received 27 February 2019; Received in revised form 5 May 2019; Accepted 6 May 2019 Available online 07 May 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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(20 min) to obtain a mother concentration of DOX c0 = 2 μg/ml. FA-conjugated PLGA (FA-PLGA) particles were prepared according to literature procedures [36]. 3.07 g of PLGA, 0.0583 g of FA and 0.0408 g of EDC were dissolved in 30 ml of acetonitrile and incubated at room temperature for 2 h. The resulting FA-PLGA were further washed with distilled water followed by dichloromethane and freeze dried. FA-PLGA were used for the preparation of fluorescent particles using the methodology described above.
Among different materials, polymers allow to design versatile vectors for the efficient delivery of poorly soluble bioactive molecules [13–21]. The targeting efficacy of polymeric carriers depends on their size and on different surface features, such as charge, chemical derivatization and hydrophobicity, so that the ability to finely control these parameters is pivotal in developing optimized carriers. Poly(lactic-co-glycolic) acid (PLGA), is one of the most employed biodegradable systems for drug encapsulation, since it undergoes hydrolysis in the body to produce biodegradable metabolite monomers, lactic and glycolic acid [21–25]. Therefore, minimal systemic toxicity is associated with the use of PLGA in biomedical applications. The potential of using PLGA based carriers has been recently explored for DOX. Such carriers demonstrated a high encapsulation efficiency together with improved bioavailability and retention time [26–29], moreover their surface can be easily functionalized to enhance the selective accumulation of the capsules onto tumor cell membranes [30,31]. However, the poor internalization of the vectors and the inefficient drug delivery to the cytoplasm remain major issues. In this respect, recent studies showing that PLGA particles are not readily taken up by cells discuss potential strategies to achieve efficient drug delivery. In particular, it has been recently proposed that PLGA vectors may be designed to adhere to the outer cell membrane, thus behaving as drug reservoirs, promoting a delayed release concentrated at the interface with the cell membrane, leading to a more efficient drug uptake [32–34]. In this work we verified such hypothesis, analyzing the ability of PLGA based vectors to modulate DOX effects on MCF-7, a human breast cancer cell line. We demonstrated that our patented one-step methodology [35] yields stable PLGA particles that efficiently entrap DOX. The morphology and size of DOX-loaded PLGA particles (DOXPLGA) were characterized employing Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). DOX release kinetics from PLGA particles were monitored in vitro under physiological conditions. The antitumor activity was evaluated on MCF-7 cells in terms of proliferation activity, clone-forming ability and intracellular distribution by fluorescence microscopy and flow cytometry.
2.3. Size and ζ-potential measurements by dynamic light scattering Particle size and ζ-potential distributions were determined by DLS measurements using a Malvern NanoZetaSizer apparatus equipped with a 5 mW HeNe laser (Malvern Instruments Ltd, UK). Experiments were performed at 25 °C, collecting the intensity autocorrelation functions at a fixed scattering angle θ = 173°, with delay times ranging from 0.8 s to 10 s. Non-negative least-squares (NNLS) or CONTIN algorithms [37] were used to analyze data. The obtained diffusion coefficients D were converted into average hydrodynamic radii R of particles using the Stokes-Einstein relationship R = KBT/6πηD, where KBT is the thermal energy and η is the solvent viscosity. The electrophoretic mobility μ of particles, measured by combined laser Doppler velocimetry (LDV) and phase analysis light scattering (PALS), was converted into their ζ-potential using the Smoluchowski relation ζ=μ η/ε, where ε is the permittivity of the solvent. 2.4. Scanning electron microscopy Particles morphology was investigated by Field Emission Scanning Electron Microscopy (FE-SEM) in both the secondary and the backscattered electron modes using a Zeiss Auriga 405 microscope. A drop of aqueous DOX-PLGA suspension was deposited on an aluminum stab and air-dried. Samples were examined using a low extracting voltage of 1.5–4 kV. 2.5. DOX entrapment efficiency The drug content of DOX-PLGA was measured using a spectrophotometric method. Particles were dissolved in DMF and the total amount of drug was determined by comparing the absorbance of the solution at 483.5 nm with a calibration curve. Validation of the assay demonstrated that this methodology was linear (R2 = 0.99) in the 3–20 μg/ml DOX concentration range. All experiments were performed in triplicate. Results are reported in terms of entrapment efficiency, obtained by the percent ratio between the drug content in the DOXPLGA and the amount of drug employed in their preparation.
2. Materials and methods 2.1. Materials Poly-D,L-lactic-co-glycolic acid (PLGA 50:50, MW 40,000–75,000 Da) Doxorubicin hydrochloride (DOX > 98%), Fluoresceinamine isomer I (FA), 1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium (MTT), crystal violet and all other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and used as received. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, Non-Essential Amino Acids (NEAA), trypsin-EDTA solution, phosphate buffered saline (PBS), Trypan blue (TB) were purchased from Euroclone, Life Science Division, GB (Pero, Italy).
2.6. In vitro release kinetics A fixed amount (5 mg) of DOX-PLGA was incubated in 3 ml of PBS (pH 7.4, 0.1 M) at 37 °C under gentle magnetic stirring (300 RPM). At fixed time intervals, 1 ml of the supernatant was withdrawn and replaced with fresh buffer. The amount of released DOX in the collected samples was determined by measuring the absorbance of each solution at 486 nm and comparing it with a calibration curve. Validation of the assay demonstrated that this methodology was linear (R2 = 0.99) in the 3–20 μg/ml range. All experiments were performed in triplicate. Results are reported in terms of cumulative drug release M(t)/M0, obtained by the percent ratio between the amount M(t) of the drug released by DOX-PLGA and the total drug content in the freshly prepared DOXPLGA M0.
2.2. Preparation of DOX-loaded PLGA carriers DOX-PLGA were prepared by using a patented osmosis-based methodology [35]. 25 mg of PLGA and different amounts of DOX in the 50–375 μg range were dissolved in 5 ml of dimethylformamide (DMF). The solution was transferred in a dialysis bag and immersed into 100 ml of a non-solvent of the polymer (H2O). After 72 h the precipitated polymer was recovered by centrifugation (14.000 RPM, 20 min), washed twice with H2O, centrifuged and freeze dried. Unless otherwise stated DOX-PLGA were prepared at a weight ratio of 2.0 μg DOX/mg PLGA, where we observed the maximum loading efficiency (see Section 3.1), and dispersed in PBS under sterile conditions by sonication
2.7. Cell lines MCF-7 cell-line (luminal A: estrogen receptor-positive and progesterone receptor-positive, ErbB2-negative) obtained from Interlab Cell 496
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wavelengths, and the resulting epiluminescent signal was acquired above 590 nm wavelength by combining pass-band and long-pass dichroic filters, respectively. The uptake efficiency was evaluated in terms of relative fluorescence intensity ΔI/I of the treated samples with respect to the controls, where I is the spatially integrated intensity, at a fixed integration time (4000 ms), on a large frame (10x, Obj) by ZEN 2011 software. The fluorescence experiment of FA-PLGA and DOX colocalization onto MCF-7 cells adhered onto Petri dishes (©ibidi GmbH, Martinsried, Germany) was performed by using a Nikon Eclipse (Ti-E) inverted C1 confocal microscope equipped with two lasers and a motorized stage. The lasers used were: an Argon ion (Spectra Physics, Mountain View, California) laser emitting at 488 nm wavelength and a green He-Ne laser (Melles Griot Florence, Italy) emitting at 543 nm wavelength. For scanning along the axial (z) dimension, a Plan Apo 60×, high numerical aperture (NA = 1.40) oil immersion objective (Nikon Florence, Italy) was chosen. The imaging software was NISelement AR 4.3 (Nikon Florence, Italy).
Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) was grown in DMEM supplemented with 10% FBS, 100 U/ ml penicillin/100 μg/ml streptomycin and 1% Non-Essential Amino Acids. Cells were maintained in a tissue culture incubator at 37 °C, 5% CO2. 2.8. Cell proliferation assay 5 × 103 cells were seeded in 96-well flat plate in culture medium and after 24 h free DOX, DOX-PLGA or non-loaded PLGA particles were added in triplicate. Different drug concentrations c0 (0.02, 0.1, 0.2 and 1 μg/ml), free or loaded into PLGA particles were tested at 24, 48 and 72 h of incubation. Cells were then incubated in FBS-free media with MTT for 4 h. Formed formazan crystals were dissolved in 100 μl of DMSO for 15 min. The absorbance was measured in a plate reader spectrophotometer (Labsystem Multiskan MS), using a test wavelength of 540 nm and a reference wavelength of 690 nm [38]. Cells incubated with culture medium alone represented the control, wells containing medium alone represented the blank. Surviving cells were expressed as percentage of the corresponding control. The reported results are the average of at least three experiments performed with independent preparations of entrapped DOX.
2.12. Flow cytometry analysis Flow cytometry was used to analyze DOX-PLGA intracellular uptake compared to free DOX and non-loaded PLGA particles. Exponentially growing MCF-7 cells were seeded into six-well plates at a density of 3 × 105 cells/ml and after 24 h were treated for 2, 5, 8 and 18 h with free DOX or DOX-PLGA at a DOX concentration c0 = 1 μg/ml. Cells incubated with non-loaded PLGA particles and with culture medium alone were used as controls. The cells-associated fluorescence was measured by acquiring 1 × 104 events for each sample using the FACSCalibur flow cytometer (BD Biosciences) equipped with two excitation lasers (argon 488 nm and red diode 635 nm) and analyzed by the CellQuest software. Cell fluorescence distribution is expressed as percentage of fluorescent cells on total cells analyzed.
2.9. Cell viability assay Cells, 1 × 104, were seeded in 24-well microplate. After 24 h, free DOX, DOX-PLGA or non-loaded PLGA particles were added in triplicate and incubated for 24 h, 48 h and 72 h. Final concentration c0 = 1 μg/ml of DOX, free or loaded into PLGA particles, was used. The cell viability was expressed as percentage compared to corresponding control. The cell viability in control samples was always 95–98% of the total number of cells. Reported values result from the average of at least three independent experiments.
2.13. Hemolysis assay 2.10. Clonogenic assay Hemolysis assay was performed using fresh human blood. Erythrocytes were collected by centrifugation at 1500 RPM for 15 min and washed three times with PBS at pH 7.4 and resuspended in 3 ml of PBS. Into 200 μl of this solution we added 100 μl of free DOX, DOXPLGA, non-loaded PLGA particles or PBS. Lysis control was provided by adding distilled water (10-100 μl) instead of PBS. The solutions were mixed and incubated for 3 h at 37 °C on a shaker and centrifuged at 1800 RPM at 10 °C for 5 min, then 100 μl of each supernatant was transferred into a plate and the optical density determined at 414 nm with a plate reader spectrophotometer (Labsystem Multiskan MS). Hemolysis data points are presented as percentages of complete hemolysis. PBS was used as negative control with 0% hemolysis and 100 μl of distilled water was used as positive control with 100% hemolysis [39,40].
Exponentially growing MCF-7 cells, 1 × 103, were seeded into 60 × 15 mm tissue culture dishes, after 24 h treatment with DOX and DOX-PLGA (DOX concentration c0 = 0.2 μg/ml), and then maintained in drug-free complete culture medium for 14 days at 37 °C, 5% CO2. Proliferation was evaluated by 0.5% crystal violet staining. Cells incubated with non-loaded PLGA particles and with culture medium alone represented the control. 2.11. Fluorescence analysis The intracellular distribution of free DOX, DOX-PLGA and FA-PLGA was visualized by fluorescence microscopy. 5 × 105 MCF-7 cells were plated into 60 × 15 mm tissue culture dishes (Falcon). After 24 h, cells were treated with free DOX, DOX-PLGA or FA-PLGA, at a DOX concentration c0 = 1 μg/ml. Cells incubated with culture medium alone represented the control. After 2 and 5 h cells were rinsed with PBS and images were acquired by using an inverted Leica DMIL fluorescence microscope designed for bright-field, phase contrast and integrated modulation contrast, coupled with a high resolution and sensitivity CCD photocam (Zeiss AxioCam ICc3) driven by Zen 2011 software. The fluorescence microscope is equipped with a 100 W mercury vapor lamp and whole range of objectives (4x-100x magnification) and incident light axis integrated in the microscope stand, incorporating a fluorescence slide for three filter cubes and chromatically corrected collectors to brighten up even the weakest fluorescence. Transmitted light techniques can be used simultaneously or in alternation in order to clearly allocate fluorescent and not fluorescent structures. The fluorescence features of DOX were preliminarily checked by UV–vis Absorbance and spectrofluorimetric techniques (data not shown). According to the collected data, the excitation light was selected at 515–560 nm
2.14. Statistical analysis All experiments were performed at least three times and data were analyzed by ANOVA. The significance was evaluated by the Tukey honestly significant difference (HSD) post hoc test. 3. Results and discussion 3.1. DOX-PLGA preparation and characterization DOX-PLGA were prepared using different drug-polymer weight ratios and characterized in terms of size distribution, ζ-potential, morphology, and encapsulation efficiency. DLS characterization showed that the size distribution depends on the drug-polymer weight ratio, ranging from 600 to 1000 nm (Section 1 of ESI, Fig. S1). DOX-PLGA exhibits a negative surface charge, with a ζ497
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koff M (t ) kon (1 − e−ks t ) + (1 − e−koff t ) = M0 kon + koff kon + koff
(1)
where ks > > koff and ks > > kon was assumed. The best fit of the experimental trend according to Eq. (1) (blue dashed line in Fig. 2) yields an equilibrium dissociation constant koff/kon = 0.61, indicating a low binding affinity between DOX and PLGA backbone. The decay times associated to the two release mechanisms are 1/ks = 1.0 ± 0.5 days and 1/koff = 10 ± 2 days. Due to the large difference, of one order of magnitude, in the characteristic kinetics time scale the second exponential can be approximated by a linear contribution (i.e., 1 − e−koff t ≅ koff t ), so that Eq. (1) becomes
koff kon koff M (t ) (1 − e−ks t ) + = t M0 kon + koff kon + koff Fig. 1. DOX entrapment efficiency in PLGA particles, expressed as percent ratio between the amount of loaded drug in PLGA and the total amount of DOX used in the preparation, at different DOX concentrations.
(2)
The best fit according to Eq. (2), represented by the green continuous line in Fig. 2, well conveys the drug release trend, yielding a characteristic release time 1/ks = 1.5 ± 0.3 days, which does not differ significantly from the value obtained using the second order exponential model of Eq. (1). This finding suggests that the initial burst release is mainly driven by diffusion while the slower steady state release is described by the linear contribution [42]. Based on the observed release behavior, it can be reasonably hypothesized that a large amount of DOX is entrapped within the polymer network but does not interact strongly with the PLGA matrix. This part is involved in the burst release during the first few days. A successful comparison between the time scales associated to the diffusion driven burst release and to the cytotoxicity is reported in Section 3.4. In the absence of PLGA degradation (e.g. triggered by cell internalization), the dissociation of remaining DOX strongly interacting with the PLGA network occurs at a low rate. The uptake and degradation of such carriers occur on a higher time scale (i.e. several days) [43,44], and, moreover, the internalized capsules could be expelled by cells in shorter times [45,46]. Therefore, it is important to study DOX cell uptake with respect to the localization of PLGA particles.
potential of ∼ −25 mV. DOX-PLGA morphology was investigated through FE-SEM (Section 1 of ESI, Fig. S2). The DOX-PLGA preparation protocol was optimized with respect to the drug entrapment efficiency by varying DOX concentration while keeping constant PLGA concentration at 5 mg/ml. Results are reported in Fig. 1 as a function of DOX concentration. A maximum loading efficiency of ∼99% is observed for a DOX concentration of 10 μg/ml, corresponding to a drug-polymer weight ratio of 2.0 μg DOX/mg PLGA. Further increases of DOX concentration did not produce any significant increase in the drug loading, pointing out that carrier saturation was already reached.
3.2. DOX release kinetics from PLGA particles The cumulative drug release M(t)/M0 from DOX-PLGA in PBS is reported as a function of time (Fig. 2). A burst release, up to 50% of loaded DOX, occurs in the first three days. Afterwards, a slow, steady state release is observed. According to literature [41], such behavior can be ascribed to two combined mechanisms acting on different time scales: the diffusion of the non-conjugated drug within PLGA matrix and the drug-polymer binding interaction. The observed kinetics can be therefore modeled by a second order exponential law accounting for the diffusion rate ks of the loaded drug, and for the association kon and dissociation koff rate constants of the interaction between DOX and PLGA [42]:
3.3. In vitro uptake Comparative experiments were performed on MCF-7 cells to characterize DOX-PLGA uptake with respect to free DOX. Cells were treated with a DOX-PLGA dispersion corresponding to a concentration of free DOX c0 = 1 μg/ml and monitored by bright phase contrast and fluorescence microscopy (Section 2 of ESI, Figs. S3–S5). As we estimated by fluorescence intensity analysis mediated on three distinct frames, the uptake increases with incubation time roughly from 20% to 75%, while the uptake of the free DOX on MCF-7 up to 5 h of treatment is stable around 20% (a more quantitative evaluation was performed by flow cytometry). Selected fluorescence images obtained after 5 h of treatment are shown in Fig. 3. The fluorescence intensity detected in the cells points out the presence of DOX-PLGA (Fig. 3a and b). The connection between the red fluorescence of MCF-7 cells that internalized the drug, and that of the DOX-PLGA aggregates on the cell surface is evident even after rinsing several times with PBS (Fig. 3c and d). The fluorescence images shown in Fig. 3 confirm that PLGA particles (probed according to Section 2.2) remain adherent to the cell membrane peripheral surface. This was further investigated by confocal laser scanning microscopy (CLSM) through a fluorescence co-localization experiment where the backbone of PLGA particles was covalently labeled with FA green fluorescent dye and the FA-PLGA particles were loaded with DOX (red fluorescence) according to Sections 2.2 and 2.11. The results shown in Fig. 3e and Fig. S6 of ESI indicate that an accumulation of co-localized DOX-loaded FA-PLGA onto MCF-7 cell membranes occurs, promoting the release of DOX (in red) concentrated at the interface, while the FA-PLGA carrier (in green) remains confined at the cell membrane interfaces. No green fluorescence is observed into the cells as no cellular internalization of the vectors occurs. Using FE-
Fig. 2. Cumulative DOX release kinetics of DOX-PLGA in PBS. The best fits according to the two models analyzed of Eq. (1) (blue dashed line) and 2 (green continuous line) are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 498
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Fig. 3. Phase contrast bright-field and fluorescence images of MCF-7 cells after 5 h treatment with DOX-PLGA dispersed in cell culture medium (a and b) and then rinsed with PBS (c and d); co-localization of DOX-loaded PLGA carriers green dyed with FA analyzed by CLSM (e): merge of bright field and green fluorescence of FA-PLGA particles interacting with the plasma membrane surface of MCF-7 cells (left), and red fluorescent DOX embedded within FAPLGA particles and inside the cells (right); cells after incubation with free DOX dissolved in the culture medium at the equivalent concentration (c0 = 1 μg/ml) of DOX embedded in PLGA particles (f). Scale bar is 10 μm (from a to e) and 20 μm in f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
SEM analysis, further details of the presence of PLGA particles onto a MCF-7 cell membrane surface are also shown (Section 4 of ESI, Figs. S7 and S8). On the other hand, Fig. 3f shows that MCF-7 cells treated with DOX are able to internalize the free drug, although the uptake efficiency is comparatively lower due to the low DOX concentration (see also flow cytometry and viability analysis). These results highlight the ability of DOX-PLGA to significantly enhance the in vitro drug uptake by cells, also when the vectors are not internalized. Moreover, the fluorescence intensity of cells treated with free DOX (Fig. 3f, S3B and S4B) appears increased to the plateau value already after 2 h and therefore remains essentially constant. The timescale of the internalization process appears therefore extremely rapid compared to that of DOX release from PLGA capsules (1/ks = 1.5 ± 0.3 days, according to the analysis of Section 3.2). This suggests that the drug internalization is mainly driven by the release kinetics when DOX is entrapped in PLGA carriers. Actually, DOX-PLGA particles exhibit a size distribution of several hundred nanometers and significant negative surface ζ-potential (Fig. S1). In these conditions the particles bind non-specifically to the outer surface of the plasma membrane but cannot be efficiently internalized by cells [47]. Notably, even working with smaller particles in order to favor their cell uptake in spite of a reduced volume-to-surface ratio, may result in a too rapid exocytosis of the PLGA carrier [32–34], so that an internalization-mediated delivery might not guarantee a sufficient availability of the drug. However, Fig. 3a–d clearly shows that the internalization of DOXPLGA is not required to achieve a significant drug uptake. Since the bare drug is able by itself to enter cells (Fig. 3f) the cell-membrane adsorbed DOX-PLGA simply acts as a reservoir for the drug, significantly increasing its concentration close to the cell membrane. The high gradient of drug concentration, released from PLGA particles directly at the surface of the cell membrane, promotes a much more effective trans-membrane diffusion (which could also include a direct PLGA-plasma membrane pathway). This would be equivalent to use a much higher concentration of free DOX [48], with DOX-PLGA particles adsorbed on the cell membrane working as microscopic devices for the delivery of spatially concentrated amounts of drug continuously released over a time interval of several hours. Convincing evidences in support of such hypothesis emerge from the comparison of DOX release rate (Fig. 2) with the long-term cytotoxic analysis reported in Section 3.4. Flow cytometry analysis was performed for further quantitative evaluation of drug accumulation in MCF-7cells. No significant effects were observed in cells treated with 1 μg/ml DOX, free or entrapped, for 2 h (data not shown), while 97% or 38% of fluorescence was detected in MCF-7 incubated for 5 h with DOX- PLGA and free DOX, respectively (Fig. 4). The higher cell retention of drug (about 3-fold) remained fairly
Fig. 4. Cellular fluorescence distribution of DOX, DOX-PLGA and non-loaded PLGA particles evaluated by Flow cytometry analysis. MCF-7 cells were treated with each compound at a DOX concentration c0 = 1 μg/ml for 5, 8 and 18 h.
constant until 18 h, highlighting the enhanced drug delivery efficiency provided by PLGA carriers. 3.4. Viability of cells treated with DOX-PLGA The anti-proliferative effect was evaluated by MTT assay on MCF-7 cells treated with DOX and DOX-PLGA. In Fig. 5 the results obtained at varying the concentration c0 of DOX, free or loaded into PLGA particles, for treatments of 24, 48 and 72 h, are reported in terms of cell viability. Every experiment included controls on non-treated cells and on cells treated with an amount of non-loaded PLGA particles equal to that employed to treat samples with DOX-PLGA at the DOX concentration c0 = 0.2 μg/ml. No significant difference was observed between the two controls. Such lack of cytotoxicity of non-loaded PLGA particles confirmed that they could be safely employed as drug carriers. No effect is detected in cells treated with free DOX until 48 h and only low proliferative reduction occurs after 72 h. Noteworthy, DOX-PLGA induced cytotoxicity at lower concentration (IC50 = 8 × 10−2 μg/ml) with respect to free DOX (IC50 = 2.4 μg/ml), already detected in cells treated for 24 h and higher after 48 and 72 h of treatment. We deepened the analysis of the cell viability kinetics by relating it 499
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Fig. 5. Viability of MCF-7 cells treated with different DOX concentrations, free or loaded into PLGA capsules, for 24 (a), 48 (b) and 72 h (c), according to MTT assay. Non-treated (CTRL) and treated cells with non-loaded PLGA particles are also shown. The histograms represent the means from densitometry quantifications of three different experiments. * p < 0.05; ** p < 0.01. Analysis of the viability of cells treated with DOX-PLGA at DOX concentration c0 = 0.2 μg/ml: experimental data as a function of the cumulative DOX release calculated for the corresponding time using the kinetics curve of Eq. (2), green line of Fig. 2 (d), and time evolution together with the best fit according to Eq. (6) (e). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
PLGA capsules and represents the asymptotic value of c(t). The following expression is obtained:
to the DOX release data discussed in Section 3.2, that show a rapid diffusion-driven release from PLGA with a characteristic time 1/ks of 1.5 ± 0.3 days. In Fig. 5d we reported the viability values as a function of the cumulative DOX release and calculated the correlation coefficient between the two quantities. The obtained value of −0.98 points out a strong correlation. To better investigate such correspondence, we derived an expression for the fraction of viable cells NDOX-PLGA(t)/NCONTROL(t) with respect to the population NCONTROL(t) of the control nontreated sample as a function of time. By assuming, for the growth of untreated cells, a first order model with rate constant r, valid in the limit of a population N(t) of viable cells much smaller than the carrying capacity of the habitat [49], and accounting for the effect of the drug by an additional term proportional to both N(t) and drug concentration c (t), the equation for the instantaneous growth rate dN/dt is given by:
dN dN = rN (t ) + dt dt
dN dt
M (t ) M0
(5)
k off
⎡ 1 k t 2+ t − 1 (1 − e−ks t ) ⎤ −αc0 NDOX − PLGA (t ) k on + k off ⎣ 2 on ks ⎦ =e NCONTROL (t )
(6)
Eq. (6) was employed to fit the viability kinetics of cells treated with DOX-PLGA at c0 = 0.2 μg/ml, reported as a function of time in Fig. 5e. The best curve, obtained by fixing ks, kon and koff according to the DOX release analysis and using α as fitting parameter, is represented by the dotted line. The procedure yielded α = 0.08 ± 0.01 h−1(μg/ml)−1 with a reduced χ2 of 0.97, assessing a striking accordance of the model with the viability when employing the kinetics parameters obtained from the release analysis. Such accordance between the two independent experiments suggests that the observed viability kinetics is mainly driven by the diffusive release of DOX from PLGA adsorbed on the cells, rather than by vector internalization and subsequent intracellular degradation. Cell viability was studied also at DOX concentration c0 = 1 μg/ml, also employed for the uptake analyses performed by fluorescence microscopy and flow cytometry. MTT assay (ESI, Fig. S9) was supported by an evaluation of the number of viable and non-viable cells by the TB exclusion test. Results are reported in Fig. 6a. DOX-PLGA induced a
(3)
Here, the constant α can be interpreted as a cell death rate constant normalized to the concentration of drug released from PLGA capsules. It therefore accounts for both the transport of single DOX molecules across the cell membrane as well as the efficacy of internalized drug in inducing a cytotoxic response. Drug concentration c(t) can be expressed in terms of cumulative release M(t)/M0 from PLGA:
c (t ) = c0
M (t ) N (t ) M0
The substitution in this equation of Eq. (2) and a straightforward integration yield:
= rN (t ) − αc (t ) N (t ) drug
= −αc0 drug
(4)
where c0 is the concentration corresponding to the total DOX content in 500
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Fig. 6. (a) Number of viable cells detected as a function of time by TB exclusion test; (b) viability kinetics of cells treated with DOX-PLGA at a drug concentration c0 = 1 μg/ml as a function of time, the best fit according to Eq. (5) is represented by the dotted line.
Acknowledgments
42% reduction in cell viability, while free DOX was unable to induce significant effects after 24 h. 12% and 70% MCF-7 remained alive after 72 h DOX-PLGA and free DOX treatment, respectively. The correspondence between viability and DOX release from PLGA was also analyzed by reporting the viability of cells treated with DOX-PLGA as a function of time in Fig. 6b. The best fit of Eq. (6) to the experimental data yields α = 0.085 ± 0.009 h−1(μg/ml)−1, in accordance with the value obtained from the MTT assay experimental data, with a reduced χ2 of 1.3. The clonogenic ability of MCF-7 cells was also studied, pointing out that both DOX-PLGA and free DOX can impair it (ESI, Fig. S10). Colony forming activity in cells growing with PLGA is similar to the control sample, suggesting that DOX release from PLGA can occur without loss of efficacy compared to the free drug treatment.
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3.5. Hemolytic properties of DOX-PLGA Since carriers used in drug delivery applications should avoid any detrimental interaction with blood constituents, a hemolysis assay was performed. A higher concentration of both non-loaded PLGA particles and DOX-PLGA, as well as free DOX were compared with different volumes of DD H2O in fresh human blood samples. A marked hemolysis was observed by adding 1:20 of DD H2O, while blood samples added with non-loaded PLGA particles, DOX-PLGA or free DOX did not have observational hemolytic properties, supporting the idea of their potential use in medical devices. 4. Conclusions Stable PLGA particles loaded with DOX were prepared and characterized. Such particles adhere to the outer side of cell membranes and act as a drug reservoir. Notably, our study strongly suggests that the vector is able to provide a controlled and persistent release of DOX in the proximity of tumor cells in vitro, enhancing drug retention and concentration within cells without the need of carrier internalization. The analysis of long-term biological effects pointed out a striking correspondence between DOX release from PLGA and the kinetics of treated cells viability, showing an enhanced cytotoxicity with respect to free DOX, starting from a DOX concentration of 0.2 μg/ml. Such formulations could therefore be of potential interest for in vivo therapies. In a forthcoming work we will attempt to optimize the size and targeting of our vector to tumor cells through ligand-receptor biorecognition towards personalized medicine. Conflict of interest The authors state no conflict of interest. 501
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
PLGA based particles as “drug reservoir” for antitumor drug delivery: characterization and cytotoxicity studies
T
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Laura Chronopouloua,1, Fabio Domenicib,c, ,1, Sabrina Giantullid,1, Francesco Brasilib,c, Chiara D’Erricoa, Georgia Tsaoulid, Elisabetta Tortorellac, Federico Bordib, Stefania Morronee, ⁎⁎ ⁎⁎ Cleofe Paloccia, , Ida Silvestrid, a
Department of Chemistry , Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy Department of Physics, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy c Department of Chemical Science and Technology, University of Rome Tor Vergata, Viale della ricerca scientifica 1, 00133, Rome, Italy d Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy e Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Drug delivery Anticancer Doxorubicin PLGA carriers Internalization Burst release
Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy.
1. Introduction Cytotoxic agents remain an important weapon in the challenge against cancer, either alone or combined with other therapies [1–5]. Cytotoxic chemotherapeutics generally interfere with normal cell functions, inhibiting replication or inducing apoptosis. As it is well known, a major issue is represented by the lack of selectivity and associated side effects. Since chemotherapeutics preferentially kill rapidly proliferating cells, they can often induce the shrinkage of solid tumors, but they also affect healthy tissues. These agents, by interfering at different steps with several cellular pathways, may promote multidrug resistance [3]. Moreover, limitations are related to poor pharmacokinetics and inappropriate biodistribution [4]. Low aqueous solubility
and short circulation times contribute to the toxic side effects as well as to decrease the therapeutic effect on tumor cells and metastases [5]. In addition, anthracyclines (e.g. Doxorubicin, DOX) show a characteristic toxicity [6], significantly increasing the risk of heart failure [2]. Within this framework, drug delivery systems, including liposomes, polymeric micelles, polymersomes, nanogels and nanoparticles, have recently emerged as novel therapeutic platforms to improve intravenous administration of anticancer agents [7–12]. Several submicrocarriers have demonstrated promising abilities in enhancing circulation time and selectivity of drugs, with decreased accumulation in healthy tissues and organs. However, in the case of anthracyclines, encapsulated formulations apparently have not been optimized yet, showing significantly reduced drug internalization efficiency [2–4].
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Corresponding author at: Department of Chemical Science and Technology, University of Rome Tor Vergata, Viale della ricerca scientifica 1, 00133, Rome, Italy. Corresponding authors. E-mail addresses: [email protected] (F. Domenici), [email protected] (C. Palocci), [email protected] (I. Silvestri). 1 Equally contributing authors. ⁎⁎
https://doi.org/10.1016/j.colsurfb.2019.05.006 Received 27 February 2019; Received in revised form 5 May 2019; Accepted 6 May 2019 Available online 07 May 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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(20 min) to obtain a mother concentration of DOX c0 = 2 μg/ml. FA-conjugated PLGA (FA-PLGA) particles were prepared according to literature procedures [36]. 3.07 g of PLGA, 0.0583 g of FA and 0.0408 g of EDC were dissolved in 30 ml of acetonitrile and incubated at room temperature for 2 h. The resulting FA-PLGA were further washed with distilled water followed by dichloromethane and freeze dried. FA-PLGA were used for the preparation of fluorescent particles using the methodology described above.
Among different materials, polymers allow to design versatile vectors for the efficient delivery of poorly soluble bioactive molecules [13–21]. The targeting efficacy of polymeric carriers depends on their size and on different surface features, such as charge, chemical derivatization and hydrophobicity, so that the ability to finely control these parameters is pivotal in developing optimized carriers. Poly(lactic-co-glycolic) acid (PLGA), is one of the most employed biodegradable systems for drug encapsulation, since it undergoes hydrolysis in the body to produce biodegradable metabolite monomers, lactic and glycolic acid [21–25]. Therefore, minimal systemic toxicity is associated with the use of PLGA in biomedical applications. The potential of using PLGA based carriers has been recently explored for DOX. Such carriers demonstrated a high encapsulation efficiency together with improved bioavailability and retention time [26–29], moreover their surface can be easily functionalized to enhance the selective accumulation of the capsules onto tumor cell membranes [30,31]. However, the poor internalization of the vectors and the inefficient drug delivery to the cytoplasm remain major issues. In this respect, recent studies showing that PLGA particles are not readily taken up by cells discuss potential strategies to achieve efficient drug delivery. In particular, it has been recently proposed that PLGA vectors may be designed to adhere to the outer cell membrane, thus behaving as drug reservoirs, promoting a delayed release concentrated at the interface with the cell membrane, leading to a more efficient drug uptake [32–34]. In this work we verified such hypothesis, analyzing the ability of PLGA based vectors to modulate DOX effects on MCF-7, a human breast cancer cell line. We demonstrated that our patented one-step methodology [35] yields stable PLGA particles that efficiently entrap DOX. The morphology and size of DOX-loaded PLGA particles (DOXPLGA) were characterized employing Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). DOX release kinetics from PLGA particles were monitored in vitro under physiological conditions. The antitumor activity was evaluated on MCF-7 cells in terms of proliferation activity, clone-forming ability and intracellular distribution by fluorescence microscopy and flow cytometry.
2.3. Size and ζ-potential measurements by dynamic light scattering Particle size and ζ-potential distributions were determined by DLS measurements using a Malvern NanoZetaSizer apparatus equipped with a 5 mW HeNe laser (Malvern Instruments Ltd, UK). Experiments were performed at 25 °C, collecting the intensity autocorrelation functions at a fixed scattering angle θ = 173°, with delay times ranging from 0.8 s to 10 s. Non-negative least-squares (NNLS) or CONTIN algorithms [37] were used to analyze data. The obtained diffusion coefficients D were converted into average hydrodynamic radii R of particles using the Stokes-Einstein relationship R = KBT/6πηD, where KBT is the thermal energy and η is the solvent viscosity. The electrophoretic mobility μ of particles, measured by combined laser Doppler velocimetry (LDV) and phase analysis light scattering (PALS), was converted into their ζ-potential using the Smoluchowski relation ζ=μ η/ε, where ε is the permittivity of the solvent. 2.4. Scanning electron microscopy Particles morphology was investigated by Field Emission Scanning Electron Microscopy (FE-SEM) in both the secondary and the backscattered electron modes using a Zeiss Auriga 405 microscope. A drop of aqueous DOX-PLGA suspension was deposited on an aluminum stab and air-dried. Samples were examined using a low extracting voltage of 1.5–4 kV. 2.5. DOX entrapment efficiency The drug content of DOX-PLGA was measured using a spectrophotometric method. Particles were dissolved in DMF and the total amount of drug was determined by comparing the absorbance of the solution at 483.5 nm with a calibration curve. Validation of the assay demonstrated that this methodology was linear (R2 = 0.99) in the 3–20 μg/ml DOX concentration range. All experiments were performed in triplicate. Results are reported in terms of entrapment efficiency, obtained by the percent ratio between the drug content in the DOXPLGA and the amount of drug employed in their preparation.
2. Materials and methods 2.1. Materials Poly-D,L-lactic-co-glycolic acid (PLGA 50:50, MW 40,000–75,000 Da) Doxorubicin hydrochloride (DOX > 98%), Fluoresceinamine isomer I (FA), 1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium (MTT), crystal violet and all other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and used as received. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, Non-Essential Amino Acids (NEAA), trypsin-EDTA solution, phosphate buffered saline (PBS), Trypan blue (TB) were purchased from Euroclone, Life Science Division, GB (Pero, Italy).
2.6. In vitro release kinetics A fixed amount (5 mg) of DOX-PLGA was incubated in 3 ml of PBS (pH 7.4, 0.1 M) at 37 °C under gentle magnetic stirring (300 RPM). At fixed time intervals, 1 ml of the supernatant was withdrawn and replaced with fresh buffer. The amount of released DOX in the collected samples was determined by measuring the absorbance of each solution at 486 nm and comparing it with a calibration curve. Validation of the assay demonstrated that this methodology was linear (R2 = 0.99) in the 3–20 μg/ml range. All experiments were performed in triplicate. Results are reported in terms of cumulative drug release M(t)/M0, obtained by the percent ratio between the amount M(t) of the drug released by DOX-PLGA and the total drug content in the freshly prepared DOXPLGA M0.
2.2. Preparation of DOX-loaded PLGA carriers DOX-PLGA were prepared by using a patented osmosis-based methodology [35]. 25 mg of PLGA and different amounts of DOX in the 50–375 μg range were dissolved in 5 ml of dimethylformamide (DMF). The solution was transferred in a dialysis bag and immersed into 100 ml of a non-solvent of the polymer (H2O). After 72 h the precipitated polymer was recovered by centrifugation (14.000 RPM, 20 min), washed twice with H2O, centrifuged and freeze dried. Unless otherwise stated DOX-PLGA were prepared at a weight ratio of 2.0 μg DOX/mg PLGA, where we observed the maximum loading efficiency (see Section 3.1), and dispersed in PBS under sterile conditions by sonication
2.7. Cell lines MCF-7 cell-line (luminal A: estrogen receptor-positive and progesterone receptor-positive, ErbB2-negative) obtained from Interlab Cell 496
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wavelengths, and the resulting epiluminescent signal was acquired above 590 nm wavelength by combining pass-band and long-pass dichroic filters, respectively. The uptake efficiency was evaluated in terms of relative fluorescence intensity ΔI/I of the treated samples with respect to the controls, where I is the spatially integrated intensity, at a fixed integration time (4000 ms), on a large frame (10x, Obj) by ZEN 2011 software. The fluorescence experiment of FA-PLGA and DOX colocalization onto MCF-7 cells adhered onto Petri dishes (©ibidi GmbH, Martinsried, Germany) was performed by using a Nikon Eclipse (Ti-E) inverted C1 confocal microscope equipped with two lasers and a motorized stage. The lasers used were: an Argon ion (Spectra Physics, Mountain View, California) laser emitting at 488 nm wavelength and a green He-Ne laser (Melles Griot Florence, Italy) emitting at 543 nm wavelength. For scanning along the axial (z) dimension, a Plan Apo 60×, high numerical aperture (NA = 1.40) oil immersion objective (Nikon Florence, Italy) was chosen. The imaging software was NISelement AR 4.3 (Nikon Florence, Italy).
Line Collection (ICLC) (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) was grown in DMEM supplemented with 10% FBS, 100 U/ ml penicillin/100 μg/ml streptomycin and 1% Non-Essential Amino Acids. Cells were maintained in a tissue culture incubator at 37 °C, 5% CO2. 2.8. Cell proliferation assay 5 × 103 cells were seeded in 96-well flat plate in culture medium and after 24 h free DOX, DOX-PLGA or non-loaded PLGA particles were added in triplicate. Different drug concentrations c0 (0.02, 0.1, 0.2 and 1 μg/ml), free or loaded into PLGA particles were tested at 24, 48 and 72 h of incubation. Cells were then incubated in FBS-free media with MTT for 4 h. Formed formazan crystals were dissolved in 100 μl of DMSO for 15 min. The absorbance was measured in a plate reader spectrophotometer (Labsystem Multiskan MS), using a test wavelength of 540 nm and a reference wavelength of 690 nm [38]. Cells incubated with culture medium alone represented the control, wells containing medium alone represented the blank. Surviving cells were expressed as percentage of the corresponding control. The reported results are the average of at least three experiments performed with independent preparations of entrapped DOX.
2.12. Flow cytometry analysis Flow cytometry was used to analyze DOX-PLGA intracellular uptake compared to free DOX and non-loaded PLGA particles. Exponentially growing MCF-7 cells were seeded into six-well plates at a density of 3 × 105 cells/ml and after 24 h were treated for 2, 5, 8 and 18 h with free DOX or DOX-PLGA at a DOX concentration c0 = 1 μg/ml. Cells incubated with non-loaded PLGA particles and with culture medium alone were used as controls. The cells-associated fluorescence was measured by acquiring 1 × 104 events for each sample using the FACSCalibur flow cytometer (BD Biosciences) equipped with two excitation lasers (argon 488 nm and red diode 635 nm) and analyzed by the CellQuest software. Cell fluorescence distribution is expressed as percentage of fluorescent cells on total cells analyzed.
2.9. Cell viability assay Cells, 1 × 104, were seeded in 24-well microplate. After 24 h, free DOX, DOX-PLGA or non-loaded PLGA particles were added in triplicate and incubated for 24 h, 48 h and 72 h. Final concentration c0 = 1 μg/ml of DOX, free or loaded into PLGA particles, was used. The cell viability was expressed as percentage compared to corresponding control. The cell viability in control samples was always 95–98% of the total number of cells. Reported values result from the average of at least three independent experiments.
2.13. Hemolysis assay 2.10. Clonogenic assay Hemolysis assay was performed using fresh human blood. Erythrocytes were collected by centrifugation at 1500 RPM for 15 min and washed three times with PBS at pH 7.4 and resuspended in 3 ml of PBS. Into 200 μl of this solution we added 100 μl of free DOX, DOXPLGA, non-loaded PLGA particles or PBS. Lysis control was provided by adding distilled water (10-100 μl) instead of PBS. The solutions were mixed and incubated for 3 h at 37 °C on a shaker and centrifuged at 1800 RPM at 10 °C for 5 min, then 100 μl of each supernatant was transferred into a plate and the optical density determined at 414 nm with a plate reader spectrophotometer (Labsystem Multiskan MS). Hemolysis data points are presented as percentages of complete hemolysis. PBS was used as negative control with 0% hemolysis and 100 μl of distilled water was used as positive control with 100% hemolysis [39,40].
Exponentially growing MCF-7 cells, 1 × 103, were seeded into 60 × 15 mm tissue culture dishes, after 24 h treatment with DOX and DOX-PLGA (DOX concentration c0 = 0.2 μg/ml), and then maintained in drug-free complete culture medium for 14 days at 37 °C, 5% CO2. Proliferation was evaluated by 0.5% crystal violet staining. Cells incubated with non-loaded PLGA particles and with culture medium alone represented the control. 2.11. Fluorescence analysis The intracellular distribution of free DOX, DOX-PLGA and FA-PLGA was visualized by fluorescence microscopy. 5 × 105 MCF-7 cells were plated into 60 × 15 mm tissue culture dishes (Falcon). After 24 h, cells were treated with free DOX, DOX-PLGA or FA-PLGA, at a DOX concentration c0 = 1 μg/ml. Cells incubated with culture medium alone represented the control. After 2 and 5 h cells were rinsed with PBS and images were acquired by using an inverted Leica DMIL fluorescence microscope designed for bright-field, phase contrast and integrated modulation contrast, coupled with a high resolution and sensitivity CCD photocam (Zeiss AxioCam ICc3) driven by Zen 2011 software. The fluorescence microscope is equipped with a 100 W mercury vapor lamp and whole range of objectives (4x-100x magnification) and incident light axis integrated in the microscope stand, incorporating a fluorescence slide for three filter cubes and chromatically corrected collectors to brighten up even the weakest fluorescence. Transmitted light techniques can be used simultaneously or in alternation in order to clearly allocate fluorescent and not fluorescent structures. The fluorescence features of DOX were preliminarily checked by UV–vis Absorbance and spectrofluorimetric techniques (data not shown). According to the collected data, the excitation light was selected at 515–560 nm
2.14. Statistical analysis All experiments were performed at least three times and data were analyzed by ANOVA. The significance was evaluated by the Tukey honestly significant difference (HSD) post hoc test. 3. Results and discussion 3.1. DOX-PLGA preparation and characterization DOX-PLGA were prepared using different drug-polymer weight ratios and characterized in terms of size distribution, ζ-potential, morphology, and encapsulation efficiency. DLS characterization showed that the size distribution depends on the drug-polymer weight ratio, ranging from 600 to 1000 nm (Section 1 of ESI, Fig. S1). DOX-PLGA exhibits a negative surface charge, with a ζ497
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koff M (t ) kon (1 − e−ks t ) + (1 − e−koff t ) = M0 kon + koff kon + koff
(1)
where ks > > koff and ks > > kon was assumed. The best fit of the experimental trend according to Eq. (1) (blue dashed line in Fig. 2) yields an equilibrium dissociation constant koff/kon = 0.61, indicating a low binding affinity between DOX and PLGA backbone. The decay times associated to the two release mechanisms are 1/ks = 1.0 ± 0.5 days and 1/koff = 10 ± 2 days. Due to the large difference, of one order of magnitude, in the characteristic kinetics time scale the second exponential can be approximated by a linear contribution (i.e., 1 − e−koff t ≅ koff t ), so that Eq. (1) becomes
koff kon koff M (t ) (1 − e−ks t ) + = t M0 kon + koff kon + koff Fig. 1. DOX entrapment efficiency in PLGA particles, expressed as percent ratio between the amount of loaded drug in PLGA and the total amount of DOX used in the preparation, at different DOX concentrations.
(2)
The best fit according to Eq. (2), represented by the green continuous line in Fig. 2, well conveys the drug release trend, yielding a characteristic release time 1/ks = 1.5 ± 0.3 days, which does not differ significantly from the value obtained using the second order exponential model of Eq. (1). This finding suggests that the initial burst release is mainly driven by diffusion while the slower steady state release is described by the linear contribution [42]. Based on the observed release behavior, it can be reasonably hypothesized that a large amount of DOX is entrapped within the polymer network but does not interact strongly with the PLGA matrix. This part is involved in the burst release during the first few days. A successful comparison between the time scales associated to the diffusion driven burst release and to the cytotoxicity is reported in Section 3.4. In the absence of PLGA degradation (e.g. triggered by cell internalization), the dissociation of remaining DOX strongly interacting with the PLGA network occurs at a low rate. The uptake and degradation of such carriers occur on a higher time scale (i.e. several days) [43,44], and, moreover, the internalized capsules could be expelled by cells in shorter times [45,46]. Therefore, it is important to study DOX cell uptake with respect to the localization of PLGA particles.
potential of ∼ −25 mV. DOX-PLGA morphology was investigated through FE-SEM (Section 1 of ESI, Fig. S2). The DOX-PLGA preparation protocol was optimized with respect to the drug entrapment efficiency by varying DOX concentration while keeping constant PLGA concentration at 5 mg/ml. Results are reported in Fig. 1 as a function of DOX concentration. A maximum loading efficiency of ∼99% is observed for a DOX concentration of 10 μg/ml, corresponding to a drug-polymer weight ratio of 2.0 μg DOX/mg PLGA. Further increases of DOX concentration did not produce any significant increase in the drug loading, pointing out that carrier saturation was already reached.
3.2. DOX release kinetics from PLGA particles The cumulative drug release M(t)/M0 from DOX-PLGA in PBS is reported as a function of time (Fig. 2). A burst release, up to 50% of loaded DOX, occurs in the first three days. Afterwards, a slow, steady state release is observed. According to literature [41], such behavior can be ascribed to two combined mechanisms acting on different time scales: the diffusion of the non-conjugated drug within PLGA matrix and the drug-polymer binding interaction. The observed kinetics can be therefore modeled by a second order exponential law accounting for the diffusion rate ks of the loaded drug, and for the association kon and dissociation koff rate constants of the interaction between DOX and PLGA [42]:
3.3. In vitro uptake Comparative experiments were performed on MCF-7 cells to characterize DOX-PLGA uptake with respect to free DOX. Cells were treated with a DOX-PLGA dispersion corresponding to a concentration of free DOX c0 = 1 μg/ml and monitored by bright phase contrast and fluorescence microscopy (Section 2 of ESI, Figs. S3–S5). As we estimated by fluorescence intensity analysis mediated on three distinct frames, the uptake increases with incubation time roughly from 20% to 75%, while the uptake of the free DOX on MCF-7 up to 5 h of treatment is stable around 20% (a more quantitative evaluation was performed by flow cytometry). Selected fluorescence images obtained after 5 h of treatment are shown in Fig. 3. The fluorescence intensity detected in the cells points out the presence of DOX-PLGA (Fig. 3a and b). The connection between the red fluorescence of MCF-7 cells that internalized the drug, and that of the DOX-PLGA aggregates on the cell surface is evident even after rinsing several times with PBS (Fig. 3c and d). The fluorescence images shown in Fig. 3 confirm that PLGA particles (probed according to Section 2.2) remain adherent to the cell membrane peripheral surface. This was further investigated by confocal laser scanning microscopy (CLSM) through a fluorescence co-localization experiment where the backbone of PLGA particles was covalently labeled with FA green fluorescent dye and the FA-PLGA particles were loaded with DOX (red fluorescence) according to Sections 2.2 and 2.11. The results shown in Fig. 3e and Fig. S6 of ESI indicate that an accumulation of co-localized DOX-loaded FA-PLGA onto MCF-7 cell membranes occurs, promoting the release of DOX (in red) concentrated at the interface, while the FA-PLGA carrier (in green) remains confined at the cell membrane interfaces. No green fluorescence is observed into the cells as no cellular internalization of the vectors occurs. Using FE-
Fig. 2. Cumulative DOX release kinetics of DOX-PLGA in PBS. The best fits according to the two models analyzed of Eq. (1) (blue dashed line) and 2 (green continuous line) are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 498
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Fig. 3. Phase contrast bright-field and fluorescence images of MCF-7 cells after 5 h treatment with DOX-PLGA dispersed in cell culture medium (a and b) and then rinsed with PBS (c and d); co-localization of DOX-loaded PLGA carriers green dyed with FA analyzed by CLSM (e): merge of bright field and green fluorescence of FA-PLGA particles interacting with the plasma membrane surface of MCF-7 cells (left), and red fluorescent DOX embedded within FAPLGA particles and inside the cells (right); cells after incubation with free DOX dissolved in the culture medium at the equivalent concentration (c0 = 1 μg/ml) of DOX embedded in PLGA particles (f). Scale bar is 10 μm (from a to e) and 20 μm in f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
SEM analysis, further details of the presence of PLGA particles onto a MCF-7 cell membrane surface are also shown (Section 4 of ESI, Figs. S7 and S8). On the other hand, Fig. 3f shows that MCF-7 cells treated with DOX are able to internalize the free drug, although the uptake efficiency is comparatively lower due to the low DOX concentration (see also flow cytometry and viability analysis). These results highlight the ability of DOX-PLGA to significantly enhance the in vitro drug uptake by cells, also when the vectors are not internalized. Moreover, the fluorescence intensity of cells treated with free DOX (Fig. 3f, S3B and S4B) appears increased to the plateau value already after 2 h and therefore remains essentially constant. The timescale of the internalization process appears therefore extremely rapid compared to that of DOX release from PLGA capsules (1/ks = 1.5 ± 0.3 days, according to the analysis of Section 3.2). This suggests that the drug internalization is mainly driven by the release kinetics when DOX is entrapped in PLGA carriers. Actually, DOX-PLGA particles exhibit a size distribution of several hundred nanometers and significant negative surface ζ-potential (Fig. S1). In these conditions the particles bind non-specifically to the outer surface of the plasma membrane but cannot be efficiently internalized by cells [47]. Notably, even working with smaller particles in order to favor their cell uptake in spite of a reduced volume-to-surface ratio, may result in a too rapid exocytosis of the PLGA carrier [32–34], so that an internalization-mediated delivery might not guarantee a sufficient availability of the drug. However, Fig. 3a–d clearly shows that the internalization of DOXPLGA is not required to achieve a significant drug uptake. Since the bare drug is able by itself to enter cells (Fig. 3f) the cell-membrane adsorbed DOX-PLGA simply acts as a reservoir for the drug, significantly increasing its concentration close to the cell membrane. The high gradient of drug concentration, released from PLGA particles directly at the surface of the cell membrane, promotes a much more effective trans-membrane diffusion (which could also include a direct PLGA-plasma membrane pathway). This would be equivalent to use a much higher concentration of free DOX [48], with DOX-PLGA particles adsorbed on the cell membrane working as microscopic devices for the delivery of spatially concentrated amounts of drug continuously released over a time interval of several hours. Convincing evidences in support of such hypothesis emerge from the comparison of DOX release rate (Fig. 2) with the long-term cytotoxic analysis reported in Section 3.4. Flow cytometry analysis was performed for further quantitative evaluation of drug accumulation in MCF-7cells. No significant effects were observed in cells treated with 1 μg/ml DOX, free or entrapped, for 2 h (data not shown), while 97% or 38% of fluorescence was detected in MCF-7 incubated for 5 h with DOX- PLGA and free DOX, respectively (Fig. 4). The higher cell retention of drug (about 3-fold) remained fairly
Fig. 4. Cellular fluorescence distribution of DOX, DOX-PLGA and non-loaded PLGA particles evaluated by Flow cytometry analysis. MCF-7 cells were treated with each compound at a DOX concentration c0 = 1 μg/ml for 5, 8 and 18 h.
constant until 18 h, highlighting the enhanced drug delivery efficiency provided by PLGA carriers. 3.4. Viability of cells treated with DOX-PLGA The anti-proliferative effect was evaluated by MTT assay on MCF-7 cells treated with DOX and DOX-PLGA. In Fig. 5 the results obtained at varying the concentration c0 of DOX, free or loaded into PLGA particles, for treatments of 24, 48 and 72 h, are reported in terms of cell viability. Every experiment included controls on non-treated cells and on cells treated with an amount of non-loaded PLGA particles equal to that employed to treat samples with DOX-PLGA at the DOX concentration c0 = 0.2 μg/ml. No significant difference was observed between the two controls. Such lack of cytotoxicity of non-loaded PLGA particles confirmed that they could be safely employed as drug carriers. No effect is detected in cells treated with free DOX until 48 h and only low proliferative reduction occurs after 72 h. Noteworthy, DOX-PLGA induced cytotoxicity at lower concentration (IC50 = 8 × 10−2 μg/ml) with respect to free DOX (IC50 = 2.4 μg/ml), already detected in cells treated for 24 h and higher after 48 and 72 h of treatment. We deepened the analysis of the cell viability kinetics by relating it 499
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Fig. 5. Viability of MCF-7 cells treated with different DOX concentrations, free or loaded into PLGA capsules, for 24 (a), 48 (b) and 72 h (c), according to MTT assay. Non-treated (CTRL) and treated cells with non-loaded PLGA particles are also shown. The histograms represent the means from densitometry quantifications of three different experiments. * p < 0.05; ** p < 0.01. Analysis of the viability of cells treated with DOX-PLGA at DOX concentration c0 = 0.2 μg/ml: experimental data as a function of the cumulative DOX release calculated for the corresponding time using the kinetics curve of Eq. (2), green line of Fig. 2 (d), and time evolution together with the best fit according to Eq. (6) (e). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
PLGA capsules and represents the asymptotic value of c(t). The following expression is obtained:
to the DOX release data discussed in Section 3.2, that show a rapid diffusion-driven release from PLGA with a characteristic time 1/ks of 1.5 ± 0.3 days. In Fig. 5d we reported the viability values as a function of the cumulative DOX release and calculated the correlation coefficient between the two quantities. The obtained value of −0.98 points out a strong correlation. To better investigate such correspondence, we derived an expression for the fraction of viable cells NDOX-PLGA(t)/NCONTROL(t) with respect to the population NCONTROL(t) of the control nontreated sample as a function of time. By assuming, for the growth of untreated cells, a first order model with rate constant r, valid in the limit of a population N(t) of viable cells much smaller than the carrying capacity of the habitat [49], and accounting for the effect of the drug by an additional term proportional to both N(t) and drug concentration c (t), the equation for the instantaneous growth rate dN/dt is given by:
dN dN = rN (t ) + dt dt
dN dt
M (t ) M0
(5)
k off
⎡ 1 k t 2+ t − 1 (1 − e−ks t ) ⎤ −αc0 NDOX − PLGA (t ) k on + k off ⎣ 2 on ks ⎦ =e NCONTROL (t )
(6)
Eq. (6) was employed to fit the viability kinetics of cells treated with DOX-PLGA at c0 = 0.2 μg/ml, reported as a function of time in Fig. 5e. The best curve, obtained by fixing ks, kon and koff according to the DOX release analysis and using α as fitting parameter, is represented by the dotted line. The procedure yielded α = 0.08 ± 0.01 h−1(μg/ml)−1 with a reduced χ2 of 0.97, assessing a striking accordance of the model with the viability when employing the kinetics parameters obtained from the release analysis. Such accordance between the two independent experiments suggests that the observed viability kinetics is mainly driven by the diffusive release of DOX from PLGA adsorbed on the cells, rather than by vector internalization and subsequent intracellular degradation. Cell viability was studied also at DOX concentration c0 = 1 μg/ml, also employed for the uptake analyses performed by fluorescence microscopy and flow cytometry. MTT assay (ESI, Fig. S9) was supported by an evaluation of the number of viable and non-viable cells by the TB exclusion test. Results are reported in Fig. 6a. DOX-PLGA induced a
(3)
Here, the constant α can be interpreted as a cell death rate constant normalized to the concentration of drug released from PLGA capsules. It therefore accounts for both the transport of single DOX molecules across the cell membrane as well as the efficacy of internalized drug in inducing a cytotoxic response. Drug concentration c(t) can be expressed in terms of cumulative release M(t)/M0 from PLGA:
c (t ) = c0
M (t ) N (t ) M0
The substitution in this equation of Eq. (2) and a straightforward integration yield:
= rN (t ) − αc (t ) N (t ) drug
= −αc0 drug
(4)
where c0 is the concentration corresponding to the total DOX content in 500
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Fig. 6. (a) Number of viable cells detected as a function of time by TB exclusion test; (b) viability kinetics of cells treated with DOX-PLGA at a drug concentration c0 = 1 μg/ml as a function of time, the best fit according to Eq. (5) is represented by the dotted line.
Acknowledgments
42% reduction in cell viability, while free DOX was unable to induce significant effects after 24 h. 12% and 70% MCF-7 remained alive after 72 h DOX-PLGA and free DOX treatment, respectively. The correspondence between viability and DOX release from PLGA was also analyzed by reporting the viability of cells treated with DOX-PLGA as a function of time in Fig. 6b. The best fit of Eq. (6) to the experimental data yields α = 0.085 ± 0.009 h−1(μg/ml)−1, in accordance with the value obtained from the MTT assay experimental data, with a reduced χ2 of 1.3. The clonogenic ability of MCF-7 cells was also studied, pointing out that both DOX-PLGA and free DOX can impair it (ESI, Fig. S10). Colony forming activity in cells growing with PLGA is similar to the control sample, suggesting that DOX release from PLGA can occur without loss of efficacy compared to the free drug treatment.
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3.5. Hemolytic properties of DOX-PLGA Since carriers used in drug delivery applications should avoid any detrimental interaction with blood constituents, a hemolysis assay was performed. A higher concentration of both non-loaded PLGA particles and DOX-PLGA, as well as free DOX were compared with different volumes of DD H2O in fresh human blood samples. A marked hemolysis was observed by adding 1:20 of DD H2O, while blood samples added with non-loaded PLGA particles, DOX-PLGA or free DOX did not have observational hemolytic properties, supporting the idea of their potential use in medical devices. 4. Conclusions Stable PLGA particles loaded with DOX were prepared and characterized. Such particles adhere to the outer side of cell membranes and act as a drug reservoir. Notably, our study strongly suggests that the vector is able to provide a controlled and persistent release of DOX in the proximity of tumor cells in vitro, enhancing drug retention and concentration within cells without the need of carrier internalization. The analysis of long-term biological effects pointed out a striking correspondence between DOX release from PLGA and the kinetics of treated cells viability, showing an enhanced cytotoxicity with respect to free DOX, starting from a DOX concentration of 0.2 μg/ml. Such formulations could therefore be of potential interest for in vivo therapies. In a forthcoming work we will attempt to optimize the size and targeting of our vector to tumor cells through ligand-receptor biorecognition towards personalized medicine. Conflict of interest The authors state no conflict of interest. 501
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