The effect of a controlled release of Epinephrine hydrochloride from

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Biological and Medical Applications of Materials and Interfaces

The effect of a controlled release of Epinephrine hydrochloride from PLGA microchamber array: in vivo studies Olga A. Sindeeva, Olga I. Gusliakova, Olga Inozemtseva, Arkady S. Abdurashitov, Ekaterina P. Brodovskaya, Meiyu Gai, Valery V. Tuchin, Dmitry A. Gorin, and Gleb B. Sukhorukov ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15109 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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ACS Applied Materials & Interfaces

The Effect of a Controlled Release of Epinephrine hydrochloride from PLGA Microchamber Array: in vivo Studies Olga A. Sindeeva†, #,*, Olga I. Gusliakova†, Olga A. Inozemtseva†, Arkady S. Abdurashitov‡, Ekaterina P. Brodovskaya⊥,#, Meiyu Gai#,~,+, Valery V. Tuchin ‡,∥,§, Dmitry A. Gorin†,| and Gleb B. Sukhorukov†,#,~* †

Remote Controlled Theranostic Systems Lab, Department of Nanotechnology, Educational and

Research Institute of Nanostructures and Biosystems, Saratov State University, 83 Astrakhanskaya str., Saratov 410012, Russia. Email: [email protected] #

School of Engineering and Materials Science, Queen Mary University of London, Mile End,

Eng, 215, London E1 4NS, United Kingdom. Email: [email protected] ‡

Research-Educational Institute of Optics and Biophotonics, Saratov State University, 83

Astrakhanskaya str., Saratov 410012, Russia ⊥ Ogarev

~

Mordovia State University, 68 Bolshevistskaya str., Saransk 430005, Russia

Tomsk Polytechnic University, 30 Lenin Avenue, Tomsk 634050, Russia

+ Max Plank Institute of Polymer research, 10 Ackermannweg, Mainz 55128, Germany

ACS Paragon Plus Environment

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∥ Interdisciplinary

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Laboratory of Biophotonics, Tomsk State University, 36 Lenin Avenue,

Tomsk 634050, Russia §

Laboratory of Laser Diagnostics of Technical and Living Systems, Institute of Precision

Mechanics and Control of RAS, 24 Rabochaya str., 410028 Saratov, Russia |

Laboratory of Biophotonics, Center for Photonics and Quantum Materials, Skolkovo Institute of

Science and Technology, Nobel str, Building 3, Moscow 121205, Russia Keywords: microchamber array, biocompatibility and biodegradability, ultrasound-stimulated cargo release, epinephrine hydrochloride, vascular response in vivo Abstract This paper presents the synthesis of highly biocompatible and biodegradable poly-lactide-coglycolide (PLGA) microchamber arrays sensitive to low-intensity therapeutic ultrasound (1 MHz, 1–2W, 1 min). A reliable method was elaborated that allowed the microchambers to be uniformly filled with epinephrine hydrochloride (EH), with the possibility of varying the cargo amount. The maximum load of EH was 4.5 µg per array of 5 mm x 5 mm (about 24 pg of EH per single microchamber). A gradual, spontaneous drug release was observed to start on the first day, which is especially important in the treatment of acute patients. Ultrasound triggered a sudden substantial release of EH from the films. In vivo real-time studies using a laser speckle contrast imaging system demonstrated changes in the hemodynamic parameters as a consequence of EH release under ultrasound exposure. We recorded a decrease in blood flow as a vascular response to EH release from a PLGA microchamber array implanted subcutaneously in a mouse. This response was immediate and delayed (one and two days after the implantation of the array). The


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PLGA microchamber array is a new, promising drug depot implantable system that is sensitive to external stimuli. 1 Introduction Intensive development of new potent drugs requires the elaboration of new effective ways to reduce their systemic side effects and make the drug used locally and in time-specific manner. To solve these problems, nowadays the intensive research is concentrated on developing and characterizing various drug delivery systems. The key advantages of such systems include increase in drug accumulation at the diseased or pathological area, enhanced cellular uptake, prolonged drug release or circulation time, high stability, and reduction of toxic effects of the encapsulated compounds on healthy tissues1,2. Responsiveness to external stimuli3 also affects the of delivery systems, because in the case of sharp patient deterioration, it is necessary to speed up drug uptake in local areas of acute disease. These smart carriers change their structures in response to an external physical or biochemical stimulus, which culminates in the release of the encapsulated cargo. The effectiveness of external stimuli such as pH, temperature, and laser and microwave radiation for controlled drug release has been proven 4. However, all these stimuli have their limitations when used in therapy5. The main limitations of laser use are strong light scattering in tissue layers and in blood, which entail a low penetration depth for visible and even near-infrared irradiation 6,7. Microwaves offer higher penetration depths than do lasers, but the detailed microwave–drug container interaction is not clear8. The pH and temperature changes of the human torso cause undesirable side effects. By contrast, ultrasound can penetrate deeply into tissues and is used widely in medical practice as a safe diagnostic and therapeutic method9.


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One of the new promising biodegradable and biocompatible controlled drug release system sensitive to the high intensity focused ultrasound (HIFU) is the polylactic acid (PLA) microchamber array (MCA) which have been recently introduced 10–13. An MCA is a thin film with a large number of microcontainers arranged evenly and orderly 14–19. Such hydrophobic PLA MCAs possess efficient barrier properties for small hydrophilic molecules and can easily and quickly be synthesized under laboratory conditions at minimal cost. PLA is biocompatible and completely biodegradable, and because its degradation products are nontoxic, PLA-based materials are suitable for polymer engineering, tissue engineering, and various medical implants20. However, the use of poly(lactide-co-glycolide) (PLGA) in medicine as the main component of carriers is more desirable because of higher biocompatibility of this material21,22. The aim of this work was to fabricate highly biocompatible and biodegradable PLGA MCA with a high degree and uniformity loading by biologically active substance enable to change physiological parameters once released in vivo. We showed the reliability of the chosen method of MCAs synthesis for encapsulating weakly chemically stable biologically active substances. We selected the exposure parameters of standard low-intensity therapeutic ultrasound for the effective controlled drug release from this material. Epinephrine hydrochloride (EH) was chosen as the model drug for this study. Epinephrine, also known as adrenalin or adrenaline, is a hormone, neurotransmitter, and medication23,24. Epinephrine is normally produced by both the adrenal glands and the sympathetic nervous system25. It plays an important role in the fight-or-flight response by increasing blood flow to the muscles, increasing coronary and cerebral perfusion pressure26, pupil dilation27, and increasing blood sugar. As a medication, it is used to treat a number of conditions including anaphylaxis28, cardiac arrest29, and superficial bleeding30. Pharmacological


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doses of epinephrine stimulate α1, α2, β1, β2, and β3-adrenoceptors of sympathetic nervous system, which alters the diameter of the vessels, pressure, blood flow velocity, and other hemodynamic parameters24,31 A number of unique properties (the possibility of quantifying in vitro, the weak chemical stability of this substance, the high rate of response from the cardiovascular system25 as a response to release this substance in vivo, dependence between reaction force and dose, the possibility of evaluating this reaction by various methods) made Epinephrine an ideal drug model for detailed characterization of PLGA MCAs containing this substance in vitro and in vivo. In this work, we demonstrated vascular response to the ultrasound-induced EH release from PLGA MCAs implanted subcutaneously 0, 1, 2, and 3 days before exposure to ultrasound. Vascular response was evaluated in real time with a laser speckle contrast imaging system. 2 Experimental Section 2.1 Materials Poly (D,L-lactide-co-glycolide) (lactide:glycolide (50:50), mol wt 30,000–60,000), Epinephrine hydrochloride, sodium bicarbonate, agarose, Nile red, fluorescein isothiocyanate–dextran, Cy7 and bovine serum albumin were all obtained from Sigma-Aldrich. The Poly(dimethylsiloxane) (PDMS) kit (Sylgard 184) was purchased from Dow-Corning, Midland, USA. The Folin & Ciocalteu reagent was obtained from Panreac, Spain. 2.2 Fabrication and characterization of sealed free-standing PLGA MCAs with encapsulated EH


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For the fabrication of sealed free-standing PLGA MCAs, it is first necessary to produce a patterned PDMS stamp. PDMS stamps with microwells were prepared from a mixture of a prepolymer and a curing agent (10:1), which was poured on a silicon master with 190,000 micropillars, degassed for 30 min in vacuum, and cured at 70 °C for 3 h. After hardening, the PDMS was cut out with a sharp scalpel and was separated from the silicon master. The silicon master had been prepared by traditional photolithography and etching at Shenzhen Semiconductor, Shenzhen, China, and has round micropillars with a diameter of 10 µm and a height of 5 µm. PLGA was dissolved in chloroform at a 1.5% mass concentration. The PDMS stamp was dipcoated for 3 s with the PLGA solution to make a patterned PLGA film. The microwells were filled with EH solution. The patterned PLGA microfilm on the PDMS stamp was printed onto a flat PLGA microfilm on cover glass under pressure (2 kg cm−2) for 60 s. Then, the PDMS stamp was removed and the sealed PLGA MCA was separated from the cover glass. The morphological state of the samples was evaluated by scanning electron microscopy with a Tescan MIRA II LMU setup (Tescan, Brno, Czech Republic) at an acceleration voltage of 5 kV. Before measurements, the samples were sprayed with gold (gold layer of ∼5 nm) by using an Emitech K350 sputter coater (Quorum Technologies Ltd, Ashford, UK). 2.3 Fabrication and characterization of sealed free-standing PLGA MCAs with fluorescent cargo For studying fluorescent cargo release, Nile red (concentration of 1 µg/ml) was added to the PLGA solution to label the MCA shell. The microchambers were filled with a water solution of fluorescein isothiocyanate–dextran (FITC–dextran) and Cy7-labeled BSA (concentration of 10


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mg/ml). Then, the samples were dried, sealed, and placed in a 2% agarose gel to reduce the diffusion rate of the fluorescent cargo after MCA opening and to detect slow release. Before and after ultrasound exposure, the MCAs were analyzed with a laser scanning confocal microscope (Leica TCS SP8 X). The excitation laser lines for FITC–dextran, Nile red, and Cy7 were 495, 552, and 670 nm, respectively. 2D images were captured by using three fluorescence channels: 505–540 nm, corresponding to the fluorescence of fluorescein; 565–619 nm, corresponding to the fluorescence of Nile red; and 712–822 nm, corresponding to the fluorescence of the dye Cy7. Optical images of the samples were also recorded. Z-stack technology was used for the 3D visualization of the MCAs structures. The step of confocal planes formation was 1 µm. After the samples had been scanned in the specified range, LasX Leica software was used to carry out a 3D reconstruction of the region of interest. 3D images were recorded in the same channels. 2.4 Quantification of EH encapsulation For the estimation of encapsulated EH, the PDMS stamp with the patterned PLGA microfilm containing EH crystals in the microwells was immersed in 1 ml of deionized water before the sealing. Although EH crystals dissolve very rapidly in water, we additionally sonicated them at 37 kHz for 1 min to achieve better dissolution. The concentration of EH was determined by a standard procedure with the Folin (Folin & Ciocalteu) reagent32. The Folin & Ciocalteu’s reagent is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenols33. For this reaction, we took 100 µl of the resulting EH solution, added 400 µl of sodium bicarbonate (1 M) and 50 µl of Folin & Ciocalteu’s reagent. The reaction of EH with Folin & Ciocalteu’s reagent gives the blue color. The solution was centrifuged (2 min, 10,000


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rpm) 10 min after the start of the reaction. The amount was estimated by optical density of the supernatant which was measured with a spectrophotometer (Synergy H1, BioTek). 2.5 In vitro ultrasound induced EH and fluorescent cargo release from the sealed PLGA in MCAs A sealed free-standing PLGA MCA included EH was placed in 1 ml of deionized water in a plastic Petri dish (35 mm). For the controlled opening of the MCA in vitro, we used a therapeutic ultrasound (Dynatronics Corp., Ultrasound Generator Model DRF-100) with a frequency of 1 MHz, a power of 1, 1.5 and 2 W. The exposure time was 1 min. The study of fluorescent cargo release was conducted in the 2% agarose gel (to reduce the diffusion rate) and we used 1 MHzultrasound of 1.5 W with exposure of 1 min. 2.6 Assessment of in vivo blood flow changes caused by a controlled EH release: specklecontrast imaging measurements All animal studies were approved and performed in compliance with the regulations of the Animal Ethics Committee of Saratov State Medical University named after V. I. Razumovsky. Experiments were performed on anesthetized white BALB mice 6-8 week old which weighted 20–25 grams. The release of EH was caused by exposure of the MCA implanted subcutaneously in the mouse hind paw to therapeutic ultrasound (1 MHz, 1.5 W, 1 min). Changes in blood flow parameters in femoral vein and artery as a response to the release of EH were evaluated in real time using a home-made laser speckle-contrast imaging system. Irradiation from a He-Ne laser (Thorlabs HNL210L, 632.8 nm, 21 mW) was launched into a single mode (SM) polarization maintained (PM) fiber (PMC63050BAPC). Power level at the sample’s surface was 2 mW. CMOS camera (Basler acA250014 gm) in combination with a


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photo-lens (Computar M1614MP2) were used to detect a subjective speckle pattern from illuminated femoral vein and artery. The F-number was adjusted to meet the Nyquist spatial criteria (more than 2 pixels per one speckle). Linear polarizer was attached prior lens to minimize specular reflection. Speckle contrast was calculated in a sliding window (5x5 pixels); 25 consecutive speckle contrast frames were averaged to increase a signal to noise (SNR) ratio. Recording rate was set to 40 frames per second. Extraction of flow indexes was performed by applying a histogram analysis algorithm to a region-of-interest (ROI)34. 2.7 Statistics All numerical results were subjected to analysis of variance by using the website http://vassarstats.net (One-Way Analysis of Variance for Independent or Correlated Samples). Also was made pairwise comparisons of sample means via the Tukey HSD test, which compares all possible pairs of means and uses the Studentized range distribution. Two levels of significance were established (p< 0.05 and

The effect of a controlled release of Epinephrine hydrochloride from

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