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Functional Albumin Nanoformulations to Fight Adrenocortical Carcinoma: a Redox-Responsive Approach
Manuela Curcio1 . Paola Avena1 . Giuseppe Cirillo1 . Ivan Casaburi1 . Umile Gianfranco Spizzirri 1 · Fiore Pasquale Nicoletta 1 . Francesca lemma1 · Vincenzo Pezzi1
Received: 5 July 2019 / Accepted: 27 January 2020 C Springer Science+Business Media, LLC, part of Springer Nature 2020
ABSTRACT
Purpose Solid tumors exhibit an altered redox state in com- parison with normal tissues due to tumor hypoxia, lower pH, and elevated levels of the tripeptide glutathione. This study describes the preparation of functional redox-responsive nanoparticles proposed as delivery vehicle of Doxorubicin in adrenocortical cancer in vitro.
Methods Curcumin and Lipoic acid were conjugated to Human Serum Albumin and nanoparticle systems were pre- pared via a modified desolvation method. Scanning electron microscopy, Fourier transmission IR, dynamic light scattering and differential scanning calorimetry analyses were used to characterize the nanoparticles. Balb3T3 and H295R were used as in vitro models of health and cancer cells, respectively. Results Nanoparticles with a spherical shape and a mean di- ameter of 70 nm were observed, increasing up to ten-folds upon exposure to glutathione 10 mM. Redox responsive Doxorubicin release was recorded, with loaded nanoparticles significantly enhancing the drug cytotoxicity against H295R adrenocortical tumor cells. Cell uptake experiments revealed a rapid and efficient internalization of the nanoparticles.
Conclusions A valuable tools to actively improve the in vitro anticancer activity of Doxorubicin against adrenocortical can- cer was proposed. The effectiveness of the delivery vehicle is related to the presence of both Lipoic acid and Curcumin moieties, enhancing the glutathione responsivity, and the drug cytotoxicity, respectively.
Francesca Iemma and Vincenzo Pezzi are the joint senior authors.
* ☒ Manuela Curcio manuela.curcio@unical.it
1 Department of Pharmacy and Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS), Italy
KEY WORDS adrenocortical carcinoma . curcumin
conjugate · human serum albumin nanoparticles · redox-responsive
INTRODUCTION
Adrenocortical carcinoma (ACC) is a rare and aggressive solid cancer derived from adrenal cortex with a high risk of relapse after radical surgery and 5-year overall survival only between 15 and 44%. (1-3). The great variability in clinical presenta- tion renders the course of disease very difficult to predict; in addition, the protocols of treatment are seriously compro- mised by the low targeting and the severe side effects of the common anticancer drugs used as first-line therapy (4). The adrenolytic drug mitotane, alone or in combination with Etoposide, Cisplatin, and Doxorubicin (DOX), is currently used for ACC treatment in advanced and metastatic stages, although the sum of the adverse effects may represent a limi- tation to its use (5,6).
Nanotechnology represent a suitable and effective tool to enhance the drug efficacy and safety, because of the possibility to design nanoparticle systems with tailor-made features in relation to the site of interest (7). For example, solid tumors exhibit an altered redox state in comparison with normal tis- sues due to tumor hypoxia, lower pH, and elevated levels of the tripeptide glutathione (GSH) (8,9), present at a concentra- tion of approximately 2-10 mM in the intracellular space of cancer cells against 2-20 uM of the normal extracellular ma- trix. Nanoparticles containing disulfide linkages (10), stable in the presence of the low GSH levels of extracellular fluids, but reversibly cleaved at the high GSH levels occurring inside the cancer cells, have been proposed as valuable systems to target the drug release in the tumor cells, limiting the side effects. Among the wide number of natural polymers employed for the preparation of drug carriers, Albumin (from either bovine or human serum) represents an ideal starting material because
of its biocompatibility, biodegradability, and high drug- binding capacity. Moreover, the native protein, possessing several functional groups, can be easily modified with suitable active molecules, carrying out to functional materials with tailor-made properties (11-13).
Albumin nanoparticles can be rapidly prepared under mild conditions by desolvation, emulsion formation or coacerva- tion (14) and were successfully used as drug carrier to sites of malignancy or inflammation (13,15).
In this work, with the aim to potentiate the efficacy of DOX and improve the therapeutic protocols, we prepared nanoparticle systems based on Human Serum Albumin (HSA) derivatives able to vectorize DOX by virtue of their ability to either accumulate in cancer tissues, exploiting the Enhanced Permeability and Retention (EPR) effect (16,17), or respond to redox gradient (10).
Firstly, the native protein was doubly functionalized with Curcumin (CUR) and Lipoic Acid (LA), in order to obtain a bioconjugate (HSACL) with the biological properties of CUR (a polyphenol employed as adjuvants of the common antineo- plastic agents (18-20) for its antinflammatory and anticancer activities) (21,22), and the enhanced ability to respond to the variation of the redox potential due to the presence of reducible disulfide groups of LA moieties. Such characteristics make the proposed HSACL an ideal starting material to prepare GSH- responsive nanoparticles (CLNPs), which were prepared by a well-established desolvation method reported in literature (23). By this approach it is possible to obtain crosslinked structures only exploiting the disulfide-sulfhydryl interchange reaction, without using any external toxic chemical reagent.
Scanning electron microscopy (SEM) and dynamic light scat- tering (DLS) were employed for the morphological character- ization and evaluation of the size distribution and redox respon- sivity of CLNPs, and DOX release experiments were performed in media mimicking the physiological (phosphate buffer at pH 7.4) and reductive (GSH 10 mM) environments. The bio- compatibility of the carrier was demonstrated by hemocompat- ibility assay and cytocompatibility tests on human fibroblasts. Finally, in vitro cytotoxicity experiments on H295R cells were performed to assess the applicability of these CLNPs for the treatment of ACC, with confocal microscopy used to confirm the effective uptake of the CLNPs by tumor cells. All experimen- tal results were compared to those obtained with the formulation not containing CUR moieties (LNPs), in order to highlight the role of CUR derivatization on carrier performances.
MATERIALS AND METHODS
Synthesis of HSA-Curcumin Conjugate
HSAC was prepared via enzyme catalysis following a proce- dure reported in literature (24). HSA (500 mg) and CUR
(60 mg) were dissolved in 20 mL phosphate buffer solution (10-3 mol L-1, pH 6.8)/DMSO mixture 75/25 v/v and reacted by adding immobilized Laccase (50 mg, 0.23 U), pre- pared as previously reported for 12 h at 37℃ under 70 rpm (25). The conjugate was purified by dialysis (dialysis tubes of 6-27/32” Medicell International LTD, MWCO: 12-14 kDa) and dipped into a glass vessel containing distilled water at room temperature for 72 h. The complete removal of unreacted CUR in the washing media was confirmed by High-Pressure Liquid Chromatography (HPLC) analysis. The resulting solutions were frozen and dried with a freeze drier (Micro Modulyo, Edwards Lifesciences, USA) to afford vaporous solids.
The HPLC analysis conditions were: Jasco PU-2089 Plus liquid chromatography equipped with a Rheodyne 7725i in- jector (fitted with a 20 uL loop), a Jasco UV-2075 HPLC detector operating at 420 nm, Jasco-Borwin integrator (Jasco Europe s.r.l., Milan, Italy) and Tracer Excel 120 ODS-A col- umn particle size 5 um, 15 × 0.4 cm (Barcelona, Spain); mo- bile phase consisting of methanol at a flow rate of 1.0 mL min-1 (26).
The TNBS assay was used to determine the free amino groups in HSA and HSAC, and thus to indirectly calculate the functionalization degree expressed as mg of CUR per g of conjugate (27). Briefly, 0.5 mL 2,4,6-trinitrobenzenesulfonic acid solution (0.01% w/v) were added to 1.0 mg NaHCO3 (0.1 mol L-1, pH 8.5) containing HSA or HSAC (2.0 mg mL-1) and, after mixing in the dark, placed in a water bath at 37℃ for 4 h min under shaking. Then, SDS (50 uL, 0.35 mol L-1) and HCI (25 uL, 1 mol L-1) were added to terminate the reaction. The absorbance was measured at 335 nm on V-530 Jasco UV-Vis spectrophotometer operating with 1.0 cm quartz cells (Jasco Europe, Milan, Italy). CDD(%) was calculated according to the following Eq. 1:
CDD(%)
Aco-Acı Aco × 100 (1)
Aco and Acı the absorbance of HSA and HSAC, respectively.
All chemicals were from Merck/Sigma Aldrich, Germany.
Synthesis of HSA-Lipoic Acid and HSAC-Lipoic Acid Conjugates
In separate vials, HSA or HSAC (250 mg) were dissolved in phosphate buffer solution (10 mL, 0.01 M, pH 7.4). Meanwhile, the carboxylic groups of LA were activated by dissolving LA (23 mg), EDC (43 mg) and N- idrossisuccinimmide (NHS) (25 mg) in DMSO (2 mL) at room temperature under magnetic stirring. After 1 h, the DMSO mixture was added to HSA or HSAC solution and left to react for 24 h at room temperature under magnetic stirring. The
obtained LA conjugates (HSAL and HSACL) were purified by unreacted reagents by dialysis (MWCO: 12-14 kDa) against distilled water for 1 week and freeze-dried, obtaining vaporous solids. The LDD(%), indirectly calculated by TNBS assay, was calculated according to the following Eq. 2:
LDD(%) ALo-ALI AL0 × 100 (2)
AL0 is the absorbance of HSA or HSAC and AL1 the ab- sorbance of HSAL and HSACL.
All chemicals were from Merck/Sigma Aldrich, Germany.
Preparation of Nanoparticles
CLNPs and LNPs were prepared from HSACL and HSAL, respectively, following a procedure reported in literature (23). Briefly, in separate experiments, each albumin conjugate (80 mg) was incubated in GSH water solution (1.5 mL, 50 mM) at 37℃ for 1 h under magnetic stirring. Then, nano- particles were obtained by adding 8 mL ethanol to protein solutions. Nanoparticles suspensions were kept under stirring at 37°℃ for 30 min and dialyzed (MWCO: 12-14 kDa) with deionized water at room temperature for 24 h to remove ethanol and GSH. Finally, LNPs and CLNPs were isolated by 30-min cycle of centrifugation at 20,000 rpm and dried under vacuum overnight.
All chemicals were from Merck/Sigma Aldrich, Germany.
Characterization of LNPs and CLNPs
Fourier-Transmission IR (FT-IR) spectra of native HSA, LNPs, and CLNPs were recorded as pellets in KBr using a FT-IR spectrophotometer (Jasco FT-IR 4200) in the wave- length range of 4000-400 cm . Signal averages were obtained for 100 scans at a resolution of 1 cm-1.
Morphological analysis was carried out using SEM (Leo Stereoscan S420; Leica Microsystems, Wetzlar, Germany). The nanoparticles were sprinkled on a double adhesive tape, stuck on aluminum stubs and coated with thin gold layers (thickness of about 300 Å). Nanoparticles size and distribution were determined by dynamic light scattering (DLS) analysis using a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corporation, New York, NY, USA) at 25.0 ± 0.1℃. The autocorrelation function was measured at 90° and the laser beam was operating at 658 nm. Polydispersion index (PDI) was obtained from the instrumental data fitting procedures using inverse Laplace transformation and Contin methods. PDI values lower than 0.3 indicate homogenous and mono-disperse populations (28). The GSH-induced destabili- zation of nanoparticles was evaluated by the same DLS meth- odology incubating LNPs and CLNPs suspensions (5 mg ml-1) in phosphate buffer (0.01 M, pH 7.4) with or without GSH
10 mM for 24 h under stirring at room temperature and measuring the variation of particle size and PDI.
Drug Loading
DOX was loaded into preformed dry nanoparticles as follows: in separate experiments, CLNPs and LNPs (20 mg) were in- cubated with a concentrated drug water solution (1 mL, 0.5 mg mL-1) for 12 h under slow stirring at 25℃. Then, DOX@CLNPs and DOX@LNPS were centrifuged at 20,000 rpm for 10 min at RT and dried under vacuum.
The LE (%) was determined by fluorescence analysis of the filtered solvent according to the following Eq. (3):
LE(%)
Ci-Co Ci (3)
Here, Ci and C0 were the concentrations of drug in the solution before and after the loading, respectively.
The calorimetric analyses of DOX, DOX@LNPs, and DOX@CLNPs were performed using a Netzsch DSC200 PC. The analyses were performed on the dry samples from 30 to 300℃ under an inert atmosphere with a flow rate of 25 mL min-1 and a heating rate of 5℃ min-1.
All chemicals were from Merck/Sigma Aldrich, Germany.
Drug Release Experiments
The release experiments were carried out using a dialysis membrane under sink conditions. Aliquots of DOX@LNPs and DOX@CLNPs (5 mg, 0.12 mg mL-1 DOX equivalent concentration) were suspended in phosphate buffer (0.5 mL, pH 7.4), transferred into a dialysis bag (MW cut off 14,000), and dialyzed against phosphate buffer (10 mL, pH 7.4) with GSH of different concentrations (0 mM and 10 mM) under gentle stirring (200 rpm) at 37℃. For comparison, diffusion experiments of plain DOX at the equivalent concentration was subjected to the same dialysis treatment. The drug release was assumed to start as soon as the dialysis bags were placed into the reservoir. At predetermined times (0.5, 1, 1.5, 2, 4, 6, 9, 16, 24 and 48 h), 0.5 mL release medium were withdrawn and replaced with the same volume of fresh medium. Using a standard calibration curve of DOX (0.6-3.0 uM) prepared under the same conditions, drug cumulative release is quanti- fied by fluorescence spectrometer and calculated according to the following Eq. (4):
Release (%) =~ x 100 M tot (4)
Where Mtot is the amount of loaded DOX and Ma is the amount of DOX released at the time t, which is calculated according to the following Eq. (5)
Μ1 = Μ; +0.05 Σ M; j=| i-1
(5)
Mi is the amount of DOX determined at the i-th measure- ment corresponding to the predetermined t.
The drug release data were analyzed by Peppas-Sahlin (Eq. 6) model (29):
MĮ M tot
ML = Kıt1/2 + K2t (6)
K1 and K2 describing the Fickian (first term) and a Case II relaxation (second term) contributions to the drug release, respectively.
All chemicals were from Merck/Sigma Aldrich, Germany.
Hemocompatibility Assay
Fresh blood was taken from human volunteers after informed consent was obtained. Erythrocytes were firstly separated from citrated blood by centrifugation at 3000 rpm for 5 min and washed three times in a phosphate buffer at pH 7.4 con- taining 123.3 mM NaCl, 22.2 mM Na2HPO4 and 5.6 mM KH2PO4. Finally, the cells were suspended at a density of 8 × 109 cells per mL.
Aliquots of erythrocytes were exposed to increasing con- centrations of LNPs and CLNPs (from 0.05 to 0.3 mg mL-1) and incubated for 30 min at room temperature with constant shaking. Cell suspensions were then centrifuged at 3000 rpm for 5 min, and the hemolysis degree was determined by com- paring the absorbance of the samples with the control (totally hemolyzed with distilled water), at 540 nm. The percentage of hemolysis was expressed as the ratio of hemoglobin in the supernatant of the sample solutions related to the hemoglobin concentration after the complete hemolysis of erythrocytes in water, as follows (Eq. 7):
Free Hemoglobin
Hemolysis (%) = Total Hemoglobin x 100 (7)
Data are expressed as the mean + SD from triplicates (30). All chemicals were from Merck/Sigma Aldrich, Germany.
Cell Growth and Cytotoxicity Experiments
Human adrenocortical carcinoma H295R cell line (ATCC® CCL-163™M) was grown in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient mixture (DMEM/F12) (Sigma D-2906; Sigma) supplemented with 5 ml L-1 of ITS and 1% antibiotic solution (10,000 units/ mL penicillin/ and 10,000 µg/mL streptomycin). BALB/ 3 T3 (ATCC® CCL-163TM) were cultured in DMEM, sup- plemented with 10% BCS and 1% antibiotic solution (10,000 units/mL penicillin/ and 10,000 µg/mL
streptomycin). Both cell lines were seeded in 48-well plates at a density of 2 × 104 cells per well and cultured in complete medium overnight at 37℃ with a 5% CO2 atmosphere and 90% RH. Cytotoxicity studies were carried out by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. For BALB/3 T3 experiments, blank LNPs and CLNPs suspensions freshly prepared at different concen- trations (0.05, 0.10, 0.20, 0.30 mg mL-1) in culture medium were added to the wells containing cells.
H295R cells were treated with free DOX, DOX@LNPs and DOX@CLNPs at equivalent DOX concentration of 2.5, 5.0, 10.0, 15.0 uM for 24 and 48 h. At the end of treatments, fresh MTT, resuspended in PBS, was added to each well (final concentration 0.33 mg mL-1). After 120 min incubation, cells were lysed with 200 uL of DMSO. Each experiment was performed in triplicate and the optical density was measured at 570 nm in a spectrophotometer.
All chemicals were from Merck/Sigma Aldrich, Germany.
Cell Uptake Experiments
H295R cells were seeded in 4-well glass slides (3 x 104 cells/ well, Lab-tek II Chamber Slide) and incubated for 24 h (37°℃, 5% CO2). Then, cells were washed with PBS, and CLNPs suspension in culture medium (0.3 mg mL-1), were added. After 4 h incubation, medium was removed and cells washed with PBS, fixed with paraformaldehyde 3.7% v/v (20 min) and washed again with PBS (three times). Then, cells were permeabilized with Triton X-100 (0.2% in PBS, 5 min) and washed with PBS (three times) before performing nuclear staining with DAPI (0.2 µg mL-1 in PBS, 5 min). Confocal images were recorded using a confocal laser scanning micro- scope (Olympus, FV3000).
All chemicals were from Merck/Sigma Aldrich, Germany.
Statistical Analysis
Experiments were carried out in triplicate. Values were expressed as means ± standard error of the mean. For viability assay, statistical significance was assessed by two-way analysis of variance followed by post-hoc comparison test (Tukey’s test). Significance was set at p< 0.01.
RESULTS AND DISCUSSION
Synthesis and Characterization of HSA-Antioxidant Conjugates
As depicted in Fig. 1, HSACL was synthesized by covalent linkage of CUR and LA to native HSA protein in order to add the peculiar characteristics of each antioxidant molecule to the intrinsic properties of the HSA, such as biocompatibility
Fig. 1 Schematic representation of synthesis of HSAC (a), HSACL (b) and preparation of CLNPs by desolvation process (c).
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37 °C, 12 h
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and non-immunogenicity. In addition, the conjugation of antioxidants to macromolecular systems was found to be a suitable strategy to overcome the main drawbacks hindering the clinical applicability of these molecules, namely the poor aqueous solubility, low stability and unfavorable pharmacoki- netics (20). Specifically, CUR was chosen in order to obtain a vehicle able to improve the anticancer activity of DOX, whereas LA was employed to enhance the number of cleav- able intramolecular disulfide groups susceptible to GSH ac- tivity in the conjugate.
CUR was linked to HSA employing a solid biocatalyst, previously characterized (25), consisting of a Laccase immobi- lized into acrylate hydrogel film (Fig. 1a). As reported else- where (24), the hypothesized reaction mechanism involves
DSC/(mW mg-1)
Exo
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the oxidation of CUR by enzyme to quinone derivative, and the subsequent nucleophilic attack of heteroatoms in the side chain of protein.
This approach allows to easily functionalize the protein obtaining high derivatization degree without the formation of toxic by-products. The shift from 420 to 347 nm, recorded comparing the UV-Vis spectra of HSAC and CUR, proved the covalent conjugation of the polyphenol to the protein backbone, whereas the (2,4,6)-trinitrobenzenesulfonic acid (TNBS) assay allowed to calculate a CUR derivatization de- gree (CDD) of 30%.
In the second step (Fig. 1b), HSAC conjugate was further functionalized with LA by a coupling reaction catalyzed by 1- etil-3-(3-dimetilamminopropil) carbodiimmide (EDC),
DOX Comulative release (%)
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| Sample | Medium | M0 M = K · t/2 + K2 -t | ||
|---|---|---|---|---|
| R2 | K1 X 10-2 | K2 X 10-4 | ||
| DOX@LNPs | PB | 0.9836 | 1.92 | -3.19 |
| GSH | 0.7025 | 9.67 | -19.5 | |
| DOX@CLNPs | PB | 0.9595 | 1.27 | -2.01 |
| GSH | 0.8306 | 4.27 | -8.37 | |
PB, Phosphate buffer 0.01 M, pH 7.4; GSH, Glutathione 10 mM in PB
obtaining the final product HSACL with a LA derivatization degree (LDD) of 43% as per TNBS assay. Following the same procedure, for comparison, HSAL sample was prepared by conjugating LA to native HSA, obtaining a LDD of 41%.
Preparation of GSH-Responsive Nanoparticles
CLNPs and LNPs were prepared in two-step procedure by employing a modified desolvation process, as reported in lit- erature (23). In the first step, HSACL and HSAL were incu- bated in GSH 50 mM water solution in order to break up the intramolecular disulfide bonds of conjugates, then ethanol, used as desolvating agent, was dropped into the conjugate solutions to obtain the final nanoparticles CLNPs and LNPs (Fig. 1c). GSH and ethanol were finally removed through dialysis. It was assumed that the crosslinking process occurs through a disulphide-sulfhydryl interchange reaction that car- ried out to the formation of intermolecular disulfide bonds.
Both nanoparticle samples were found to have a spherical shape and a narrow size range, with a mean diameter of 72 nm and Polydispersion Index (PDI) of 0.15 for CLNPs and 86 nm (PDI 0.23) for LNPs, as confirmed by combined SEM/DLS studies (Fig. 2).
FT-IR spectra of LNPs (Fig. 3b) and CLNPs (Fig. 3c) showed the typical absorption bands of HSA (amide I and II bands at 1680 and 1557 cm-1, respectively, Fig. 3a), with
CLNPs also showing the appearance of new absorption bands at 1028 and 959 cm-1, ascribable to C-O-C stretching and enolic bending vibrations of CUR residues, respectively. In both nanoparticle spectra, no signals could be assigned to the functional groups of LA moieties due to their overlapping with the HSA absorption bands.
Destabilization Experiments of CLNPs and LNPs
The GSH-responsivity of CLNPs and LNPs was investigated by DLS analyses after 24 h incubation in media mimicking the physiological (phosphate buffer 0.01 M, pH 7.4) or the intra- cellular reductive (GSH 10 mM) environments. It was found that, in the absence of GSH, the size range of both nanopar- ticles remained stable over time (72 and 86 nm for CLNPs and LNPs, respectively) whereas, after the addition of GSH (Fig. 2c and d), a considerable increase of the particle size (to 801 and 640 nm for CLNPs and LNPs, respectively), and larger PDI values (> 0.34) was recorded, due to the breakage of crosslinking points inside the nanoparticle structure and the subsequent destabilization of the polymeric network (31).
Dox Loading and Release Experiments
To evaluate the performances of CLNPs and LNPs as GSH- responsive drug delivery vehicles, the antitumor agent DOX was loaded into the preformed empty nanoparticles by soak- ing procedure, obtaining DOX@CLNPs and DOX@LNPs samples. Loading Efficiency percentages (LE(%) of 96% were obtained for both samples, demonstrating the high affinity of the drug for the nanoparticle structure.
The calorimetric analyses of DOX and drug-loaded nano- particles (Fig. 4) allows the nature (e.g. solid solution, metasta- ble molecular dispersion or crystals) of the drug inside the polymer matrix to be assessed (32). The pure DOX showed an onset melting peak at 220℃ (Fig. 4c), demonstrating the crystalline molecular structure of DOX. However, no charac- teristic peak of DOX was identified in the DSC thermograms of DOX@LNPs and DOX@CLNPs (Fig. 4a and b,
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respectively), suggesting that the drug is molecularly dispersed inside the polymer matrix, losing its crystalline structure due to structural deformations (33,34).
Then, release experiments from DOX@LNPs and DOX@CLNPs were performed in phosphate buffer (0.01 M, pH 7.4) with or without GSH 10 mM (Fig. 5), and Peppas-Sahlin model was applied to analyze the obtained profiles (Table I). Moreover, in order to evaluate the effect of formulations on release pattern, experiments using plain DOX in the same conditions were performed.
At pH 7.4 in absence of GSH, for both DOX@CLNPs and DOX@LNPs samples a slow release occurred, due to the strong drug-matrix interactions and the stability of nanoparticles under non-tumor simulating conditions, as previously demonstrated by DLS analyses. Under these conditions, Fickian diffusion through the nanoparticles structure mainly affect the DOX release (K]>>K2), and, after 24 h, release percentages of only 27% and 20% were achieved for DOX@LNPs and DOX@CLNPs, respectively.
For plain DOX, an amount of 60% was detected in the release medium already after 30 min, reaching 100% in 1.5 h (data not shown).
When GSH was added to the release medium, the drug release percentages significantly increased for both nanopar- ticle samples, because of the reductive breakage of crosslinking points. Moreover, compared to DOX@LNPs, a less marked burst release (20% vs 70%) and a more controlled release profile was recorded for DOX@CLNPs. This different be- havior can be ascribable to the presence of CUR residues which enhanced the strength of drug-matrix interactions, keeping DOX longer and prolonging drug release. In this experimental condition the data did not fit with Peppas- Sahlin model, as demonstrated by lower R2 values recorded for both samples (0.7025 and 0.8306 for DOX@LNPs and DOX@CLNPs, respectively), because the drug release was mainly influenced by destabilization phenomena of the matrix induced by the GSH reductive activity.
A 97% of plain DOX was recorded in the GSH-containing release medium after 30 min incubation, as a consequence of
1A
2A
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O
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®
o
o
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the drug protonation at the acidic pH of the GSH-containing medium (pH 4.0), enhancing DOX diffusion (data not shown).
Biological Experiments
The biocompatibility of empty CLNPs and LNPs was assessed by hemocompatibility test and cell viability experiments on Balb3T3. Hemolytic experiments allow to establish the safety of the nanoparticles for intravenous administration by evalu- ating a possible erythrocyte damage (30). The samples were found to be highly hemocompatible, with hemolysis percen- tages less than 5% (acceptable limit) at all the tested concen- trations. Cell viability experiments were performed by direct contact of CLNPs and LNPs, in the 0.05-0.30 mg mL-1 con- centration range, with Balb3T3 cells for 24 and 48 h. The results of MTT test, reported in Fig. 6, show that cell viability was not compromised at any tested concentration, proving that the functionalization processes did not determine any detrimental effect on cytocompatibility of HSA (35).
H295R adrenocortical cells were adopted as tumor cell model to evaluate the anticancer activity of the proposed nanoparticle formulations. As showed in Fig. 6, no toxic effect was observed at 24 and 48 h for empty nanoparticles at any tested concentration (0.05-0.30 mg mL-1); otherwise, DOX- loaded nanoparticles (DOX equivalent concentrations of 2.5- 15.0 µM) determined a significant and dose-dependent cell death (from 27 to 36% and from 13 to 28% for DOX@CLNPs and DOX@LNPs, respectively) already after 24 h incubation, when only few effects (from 3 to 9%) were recorded for free drug at the same concentrations (Fig. 7a).
These results clearly show that nanoparticles are able to efficiently carry DOX into the cells, where high GSH level determines their destabilization through the reductive break of the disulfide bonds, enhancing drug release and DOX availability. At 48 h incubation (Fig. 7b), DOX exerts strong cytotoxic effects, allowing to calculate an IC40 of 8.5 uM, a value that underwent a considerable reduction to 7.8 mM and 3.8 mM after treatment with DOX@LNPs and DOX@CLNPs, respectively. For both systems, the enhanced anticancer activity can be ascribable to a more effective drug vectorization inside the cells. In addition, for DOX@CLNPs, the most robust reduction of IC40 can be referred to an adju- vant effect due to the presence of the CUR moieties.
Finally, the uptake of CLNPs by H295R cells was evaluat- ed by confocal microscopy images, proving that the proposed nanoparticles are rapidly internalized inside the cells and lo- cated in the cytoplasm (Fig. 8).
CONCLUSIONS
In this work, nanoparticles systems based on CUR- and LA- HSA conjugates with enhanced redox responsivity were
proposed as valuable tools to actively improve the in vitro an- ticancer activity of Doxorubicin against ACC, one of the most aggressive and rare solid tumor. DLS analyses and release experiments were performed to demonstrate the redox sensi- tivity of the nanoparticles, obtaining a ten-folds enhancement of the nanoparticles size and a remarkable increase of the release profile, respectively, after incubation in GSH 10 mM, as a result of the more extensive disruption of the nanoparticles crosslinking points. Then, the enhanced anti- cancer activity of the Doxorubicin-loaded nanoparticles was verified by cytotoxicity experiments on H295R tumor adre- nocortical cell line, obtaining a significant enhancement of drug toxicity already after 24 h incubation. By contrast cells treated with free drug showed no relevant toxic effects. Finally, cell uptake experiments revealed a rapid and efficient internalization of the nanoparticles within the cytoplasmic fraction of tumor cells, supporting the suitability of the pro- posed carrier to limit the DOX-related side effects. The ac- quired know-how will be exploited for the development of a mitotane carrier to be used in association with the proposed DOX@CLNPs, opening new perspectives for ACC treatment.
ACKNOWLEDGMENTS AND DISCLOSURES
Supports by University of Calabria funds and MIUR Excellence Department Project funds, awarded to the Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, L.232/2016 are acknowledged.
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