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Biological and Medical Applications of Materials and Interfaces
Soft Nanohydrogels Based On Laponite Nanodiscs: A Versatile Drug Delivery Platform For Theranostics And Drug Cocktails Tiago B. Becher, Monique C. P. Mendonça, Marcelo A. de Farias, Rodrigo Villares Portugal, Marcelo Bispo de Jesus, and Catia Ornelas ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06149 • Publication Date (Web): 11 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018
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ACS Applied Materials & Interfaces
Soft Nanohydrogels Based On Laponite Nanodiscs: A Versatile Drug Delivery Platform For Theranostics And Drug Cocktails Tiago B. Becher,† Monique C. P. Mendonça,‡ Marcelo A. de Farias,§ Rodrigo V. Portugal,§ Marcelo B. de Jesus,‡ Catia Ornelas†* †Institute of Chemistry, University of Campinas -Unicamp, 13083-861 Campinas, SP, Brazil. ‡Institute of Biology, University of Campinas - Unicamp, 13083-861 Campinas, SP, Brazil §Brazilian Nanotechnology National Laboratory (LNNano), Brazilian Center for Research in Energy and Materials (CNPEM), 13083-970, Campinas, Sao Paulo, Brazil. Supporting Information _____________________________________________________________________________________________
ABSTRACT: A new nanohydrogel drug delivery platform based on Laponite nanodiscs, polyacrylate, and sodium phosphate salts is described. The hybrid nanohydrogel is tailored to obtain soft and flexible nanohydrogels with G’ around 3 kPa, which has been proposed as the ideal stiffness for drug delivery applications. In vitro studies demonstrate that the new nanohydrogels are biocompatible, biodegradable, nonswellable, pH-responsive, noncytotoxic, and able to deliver antineoplastic drugs into cancer cells. The IC50 of nanohydrogels containing cisplatin, 4-fluorouracil and cyclophosphamide is significantly lower than the IC50 of the free drugs. In vivo experiments suggest that the new nanomaterials are biocompatible and do not accumulate in crucial organs. The simple formulation procedure enables encapsulation of virtually any water-soluble molecule, without the need for chemical modification of the guests. These nanohydrogels are a versatile platform that enables the simultaneous encapsulation of several cancer drugs, yielding an efficient drug cocktail delivery system, which for instance, presents a positive synergistic effect against MCF-7 cells. KEYWORDS: nanohydrogels, nanomaterials, nanomedicine, drug delivery, drug cocktail, co-delivery, cryo-TEM _____________________________________________________________________________________________
1. INTRODUCTION Nanohydrogels have been considered outstanding candidates for drug delivery applications because it combines the advantages of hydrogel systems and nanoparticles.1-8 The nanohydrogel network contains large amounts of water, and even though they are mainly hydrophilic, in physiological media they remain insoluble protecting their cargo from degradation and elimination.9-12 Nanohydrogels’ hydrophilicity evades recognition by the host immune response, whereas its nanometer-size escape the elimination pathways from the kidneys and liver, together these effects increase the circulation lifetime in vivo.13 The high circulation lifetime improves drugs’ therapeutic efficiency allowing the use of lower doses, and enabling nanomaterials to travel through the bloodstream to accumulate more significantly in the tumor tissues through the enhanced permeation and retention effect (EPR).14-15 Therefore, long-circulating nanomaterials
are highly desirable for drug delivery, diagnosis, and theranostic applications. Zhang et al. demonstrated that the nanoparticles’ stiffness plays an important role in their in vivo behavior. Softer nanohydrogels can deform easily facilitating their passage through crucial physiological barriers, which increases their blood circulation lifetime and consequently improves their therapeutic efficiency.16 Recently, Anselmo et al. used nanohydrogels that differ only in the elastic moduli values (10 kPa vs 3000 kPa) to study the impact of nanohydrogels’ elasticity in processes involved in drug delivery, such as biodistribution, blood circulation lifetime, tissue targeting, and cellular uptake.17 Their results showed that when comparing with hard nanohydrogels, softer nanohydrogels present longer circulation lifetime improving targeting to diseased tissues. This increased permanence in the bloodstream
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is explained by the fact that hard nanohydrogels are phagocytized by immune cells much faster than softer nanohydrogels.17 Even though these studies predict nanohydrogels’ behavior in vivo based on their size and storage moduli, it is worth noting that the swelling behavior of nanohydrogels in physiological conditions significantly affect their size and mechanical properties. Therefore, it is important to study nanohydrogels’ properties at its fully swollen state or to develop nonswellable nanohydrogel systems. One of the most important advantages of nanohydrogels when comparing with other nanomaterials (dendrimers, polymeric nanoparticles, and metallic nanoparticles) is their capability of carrying large amounts of payload trapped within the tridimensional network, avoiding the chemical modification of the guest molecules.18-21 However, the method used to load drugs and imaging agents into nanohydrogels can be limiting.22-26 Usually, the guest molecules possess a variety of functional groups that can be sensitive to the reaction conditions used in the hydrogel formation such as high temperature, polymerization initiators, radical polymerization propagation, metal catalysts, or extreme pH.27 The majority of reports in the literature use drug loading methods that involve insertion of the payload into the gel network post-gelification. These methods are mainly controlled by the diffusion of the drug molecules into the hydrogel, which can be very limiting causing low drug loading per nanohydrogel.22-26 To achieve the maximum potential of payload in nanohydrogels, it is important to incorporate the guest molecules prior to gelification and use non-aggressive gelification procedures. The main problem of nanohydrogels studied for drug delivery applications is that most of the systems reported consist of crosslinked polymers that due to their high molecular weight present low biodegradability causing high long-term toxicity.28 Moreover, to minimize harmful side effects, nanohydrogels should degrade into small non-toxic molecules that can be easily excreted by the body.29 Before proposing a new hydrogel system for nanomedicine applications, it is important to evaluate the degradation conditions and metabolites of all hydrogel system components, to avoid toxicity issues from the carrier. Recently, we have developed biocompatible nonswellable hydrogels with tunable mechanical properties, assembled through non-covalent interactions between Laponite nanodiscs, sodium polyacrylate, and sodium phosphate salts.30 Hydrogel’s elastic modulus strongly depend on the relative amounts of each component, ranging from 0.2 kPa to 14 kPa.30 In the present work, our bulk hydrogel system was translated to the nanoscale realm to obtain 100 nm nanohydrogels using a nanoemulsion procedure as template. The
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hydrogel composition was chosen to achieve flexible nanogels with storage moduli G’ around 3 kPa, which is within the range established by Anselmo et al. as ideal for drug delivery applications.17 The new nanohydrogels are biocompatible, biodegradable, nonswellable, pH-responsive, non-cytotoxic, and able to deliver antineoplastic drugs into cancer cells. 2. EXPERIMENTAL SECTION General data. Laponite XLG was obtained from Southern Clay Products. Sodium polyacrylate 5 kDa (average Mw 5,100 by GPC), rhodamine B, fluorescein, SPAN85, cyclohexane, cisplatin, 4-fluorouracil, cyclophosphamide, and WST-8 assay were obtained from Sigma-Aldrich. Phosphate buffer solution (PBS) was prepared using sodium salts only. NIR-dye 1 was synthesized following published protocols. Synthesis of nanohydrogels NHG1-NHG4. Nanohydrogels were prepared by the inverse miniemulsion technique. 1 mL of Laponite aqueous solution (solution prepared with 250 mg of Laponite and 5 mg of sodium polyacrylate 5K in 10 mL of deionized water) was added to the organic phase (75 mg of SPAN85® dissolved in 8 mL of cyclohexane). The mixture was ultrasonicated (Branson Sonifier 450Digital, ½" tip) for 6 min with 50% amplitude, and at a pulse regime of 0.9 s of sonication and 0.3 s of pause. Then, 100 µL of 1.0 M PBS were added followed by ultrasonication during 6 min with amplitude of 50% and at a pulse regime of 0.9 s of sonication and 0.3 s of pause, promoting the formation of nanogel NHG1. An ice bath was used during sonication to avoid a temperature increase during the sonication process. All NHG2, NHG3 and NHG4 were formulated following a similar procedure to the one described for NHG1, but with addition of 100 µL of 0.001 M rhodamine B, 0.001 M fluorescein or 0.001 M NIR-dye 1 to the nanodiscs solution in NHG2, NHG3 and NHG4, respectively, prior to sonication. Synthesis of nanohydrogels NHG5-NHG8. 500 mL of Laponite aqueous solution (solution prepared with 500 mg of Laponite nanodiscs, 5 mg of sodium polyacrylate 5K in 10 mL of deionized water), 100 µL of 0.001 M rhodamine B and 500 µL of cisplatin aqueous solution 5mg/mL were added to the organic phase (75 mg of SPAN85® dissolved in 8 mL of cyclohexane). The mixture was ultrasonicated (Branson Sonifier 450 Digital, ½" tip) for 6 min with amplitude of 50% and at a pulse regime of 0.9 s of sonication and 0.3 s of pause. Then, 100 µL of 1.0 M PBS were added followed by ultrasonication during 6 min with amplitude of 50% and at a pulse regime of 0.9 s of sonication and 0.3 s of pause promoting the formation of NHG5. All NHG6, NHG7 e NHG8 were formulated following a similar procedure to the one described for NHG5, but by using the NRI-dye 1 instead of rhodamine B, and with addition of 500 µL of
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ACS Applied Materials & Interfaces
cisplatin 5 mg/mL in water, cyclophosphamide 50 mg/mL in water, and 4-fluorouacil 50 mg/mL in water, to the nanodiscs solution in NHG6, NHG7 and NHG8, respectively, prior to sonication. Synthesis of nanohydrogel NHG9. 500 mL of the Laponite aqueous solution (solution of containing 500 mg of Laponite nanodiscs, 5 mg of sodium polyacrylate 5K in 10 mL of deionized water), 100 µl of 0.001 M NIR-dye 1, 160 µl of cisplatin 5 mg/mL, 160 µl of cyclophosphamide 50 mg/mL and 160 µl of 4fluorouacil 50 mg/mL were added to the organic phase (75 mg of SPAN85® dissolved in 8 mL of cyclohexane). The mixture was ultrasonicated (Branson Sonifier 450 Digital, ½ "tip) for 6 min with amplitude of 50%, and at a pulse regime of 0.9 s of sonication and 0.3 s of pause. Then the addition of 100 µl of 1.0 M PBS followed by ultrasonication during 6 min with amplitude of 50%, and at a pulse regime of 0.9 s of sonication and 0.3 s of pause promoting the formation of NHG9. Rheology. Experiments were carried out in a Haake RheoStress RS1 rheometer, using geometry PP20 Ti, Gap 2.500 mm (parallel plates 20 mm diameter; gap between the plates: 2.500 mm). All data reported here were collected in triplicata on hydrogels prepared 24h prior to the measurement. SST defines the material’s response to increasing deformation amplitude (strain: γ) at a constant frequency (1 Hz) and temperature (25 ºC), and gives materials’ linear viscoelastic region (LVR), storage modulus G’ (material’s ability to store energy; measure of elasticity), loss modulus G’’ (material’s ability to dissipate energy), and crossover point (G’=G’’). FST measures the materials’ response to increasing frequency (rate of deformation) at a constant strain (γ = 1%) and temperature (25 ºC). Step-strain measures the response of the material under the application of an oscillatory force of large (100%) and low (1%) amplitude. SST, FST and Step-Strain were carried out for HN2.5PBS (2.5% w/w nanodiscs, [polyacrylate] = 0.05 mM, and [PBS] = 0.005M). Size by dynamic light scattering (DLS) and zeta potential. The mean particle size and zeta potential of nanohydrogels were determined at 25 ºC by dynamic light scattering using a Malvern Zetasizer Nano ZSZen3600 equipped with a 4 mW He-Ne laser with light wavelength of 632.8 nm, and detection at scattering angle of 173°. These measurements were done with disposable capillary cells (DTS1070). All samples were properly diluted 1000 times and 10,000 times in PBS (pH = 7.4) and three measurements were taken of the resulting dispersions. The results are presented in % by number and are representative of the triplicata. Size by nanoparticle tracking analysis (NTA). Size, concentration of particles, and approximated molecular weight of the NHGs were measured by NTA using a Malvern Nanosight LM10. The samples were prepared by diluting 10,000 times the nanohydrogels dispersions.
Particle sizes are given as the average of three measurements. Cryogenic Transmission Electron Microscopy (Cryo-TEM). Samples were prepared in a controlled environment vitrification system (Vitrobot Mark IV, Thermo Fischer Scientific – formerly FEI, USA) with controlled temperature (22 °C) and humidity (100%). Before the application of the sample, the grids were subjected to a glow discharge treatment using an Pelco easiGlow discharge system (Ted Pella, USA) with 15 mA current, for 25 s in air atmosphere. A 3 µL sample droplet was deposited on a 300 mesh lacey carboncoated cooper grid (Ted Pella, USA) and prepared with a 3 s blot time, -5 blot force and 20 s of waiting time before blotting. Samples were analyzed in low dose condition, using a TALOS F200C (Thermo Fischer Scientific – formerly FEI, USA) electron microscope operating at 200 kV. Images were acquired using a CMOS camera Ceta 16M 4k x 4k pixels (Thermo Fischer Scientific - formerly FEI, USA). Cell culture. Human breast adenocarcinoma cells (MCF-7) and human cervix adenocarcinoma cells (HeLa) were grown and maintained in 12 mL of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 100 µL penicillin, and 0.1 mg/mL streptomycin in T-75 cm2 cell culture flasks at 37 °C, in a humidified atmosphere of 5% CO2. Culture medium was refreshed every two to three days, and cells were not allowed to exceed 80% confluency. At 80% confluency, the cells were counted, diluted, and plated into treated 96 well cell culture plates at a cell density of 15 x 103 cells per well in 200 µL of respective growth medium, and incubated for 24 h to allow cells to adhere to the plates before treatment. Cytotoxicity assays. The cytotoxic activity of the nanohydrogels and drugs were tested in triplicata in serial dilutions of five concentrations ranging from 0.25 mg/mL to 5.00 mg/mL per well of nanohydrogel and from 1.25 µM to 125 µM per well of drugs. The cells were incubated in presence of the compounds at 37 °C, in a humidified atmosphere of 5% CO2 for 24 h or 48 h. The cells viability was evaluated using CCK-8 (Cell Counting Kit-8), which is a colorimetric assay based on the reduction of tetrazolium salt WST-8 ([2-(2methoxy4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium)]) in water. After exposing cells to the nanohydrogels, 10 µL of the CCK8 solution containing the WST-8 were added to each well of the plates. The cells were further incubated for approximately 2 hours at 37 °C under humidified atmosphere with 5% CO2. The number of viable cells was determined by the amount of WST-8 (colorless compound) that was converted to formazan WST-8 (orange compound, soluble without culture medium) by mitochondrial dehydrogenases. To minimize the possible contribution from NHGs turbidity (at high
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concentrations), the solutions present in the tested 96well plates were transferred to another clean 96-well plate for reading. The absorbance of the solution in each well was measured at 450 nm in a plate reader (FlashScan 530 Analitic Jena). The negative control was designated as 100% of dehydrogenase activity and cell viability was expressed as a percentage of the untreated cells. The blank values, corresponding to the absorbance of the CCK-8 solution in growth medium, were subtracted from all samples. In vivo experiments. Experiments were carried out in accordance with the Brazilian Society of Laboratory Animal Science guidelines and approved by the Institutional Committee for Ethics in Animal Use (CEUA/IB/UNICAMP, protocol n. 4491-1). Male Wistar rats (Rattus norvegicus, 6-week-old, 180-220g) received a single tail vein injection of NHG-1 or NHG3 (10 mg/kg), while the control group was given the same volume of physiological saline solution 0.9%. The animals were anesthetized (2:1 v/v ketamine chloride (Dopalen®, 100 mg/kg), and xylazine chloride (Anasedan®, 10 mg/kg) (Fortvale, Valinhos, SP, Brazil) and euthanized 3 and 24 h after treatment (n=34/group). Blood samples were obtained by cardiac puncture prior to perfusion and analyzed using a Coulter T540 hematology system (Fullerton, CA, USA) and Cobas® 6000 C501 Clinical Chemistry Analyzer (Roche Diagnostics, Mannheim, Germany). All data were expressed as mean ±SEM. Statistical significance was determined by two-way ANOVA followed by the Bonferroni post-hoc test with a value of p
