A Simple Non-Invasive Approach toward Efficient Transdermal Drug

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Biological and Medical Applications of Materials and Interfaces

A simple non-invasive approach towards efficient transdermal drug delivery based on biodegradable particulate system Yulia I. Svenskaya, Elina A. Genina, Bogdan V. Parakhonskiy, Ekaterina Lengert, Ekaterina E. Talnikova, Georgy S. Terentyuk, Sergey R. Utz, Dmitry A. Gorin, Valery V. Tuchin, and Gleb Sukhorukov ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04305 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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ACS Applied Materials & Interfaces

A simple non-invasive approach towards efficient transdermal drug delivery based on biodegradable particulate system Yulia I. Svenskaya1,*, Elina A. Genina1, Bogdan V. Parakhonskiy1, Ekaterina V. Lengert1, Ekaterina E. Talnikova2,3, Georgy S. Terentyuk2, Sergey R. Utz2,3, Dmitry A. Gorin4, Valery V. Tuchin1,5, Gleb B. Sukhorukov6

1 Saratov

State University, Saratov 410012, Russia

2 Saratov

State Medical University, Saratov 410012, Russia

3 Clinic

of Skin and Venereal Diseases, Saratov 410028, Russia

4 Skolkovo

Institute of Science and Technology, Moscow 143026, Russia

5 Tomsk

State University, Tomsk 634050, Russia

6 Queen

Mary University of London, London E1 4NS, United Kingdom

ACS Paragon Plus Environment

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KEYWORDS Depot system; hair follicles; topical, intrafollicular, transfollicular drug delivery; prolonged release; calcium carbonate particles; vaterite

ABSTRACT Transdermal administration via skin appendages enables both localized and systemic drug delivery, as well as minimizes incidental toxicity. However, the design of an appropriate effective method for the clinical use remains challenging. Here, we introduce calcium carbonate-based carriers for the transdermal transportation of bioactive substances. The proposing system represents easily manufacturable biodegradable particles with a large surface area enabling a high payload ability. Topical application of submicron porous СaCO3 particles in rats followed by the therapeutic ultrasound treatment results in their deep penetration through the skin along with the plentiful filling of the hair follicles. Exploiting the loading capacity of porous particles, we demonstrate efficient transportation of a fluorescent marker along the entire depth of the hair follicle down the bulb region. In vivo monitoring of the carrier degradation reveals the active


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dissolution/recrystallization of CaCO3 particles resulting in their total resorption within 12 days. The proposed particulate system serves an intrafollicular depot for drug storage and prolonged in situ release over this period. The urinary excretion profile proves the systemic absorption of the fluorescent marker. Hence, the elaborated transdermal delivery system outlooks promising for medical applications. The drug delivery to different target regions of the hair follicle may contribute to regenerative medicine, immunomodulation and treatment of various skin disorders. In the meantime, the systemic uptake of transported drug opens an avenue for perspective delivery routes beyond the scope of dermatology.

INTRODUCTION Transdermal administration of therapeutic molecules is gaining tremendous scientific interest, especially with regard to the reduction of systemic toxicity.1 Such a delivery route allows the avoiding a first-pass liver effect and the maintaining a continuous drug administration, improving, therefore, a patient compliance.2,3 In addition, transdermal systems are non-invasive and self-administrable. However, this delivery mode has some


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limitations, as the skin provides a protective barrier against external actions.4 The outermost layer of skin, stratum corneum, represents the major limiting component of skin for the drug penetration.5 But there is still an effective alternative route for administration of the topically applied drug, that enables the avoidance of the challenges of delivering through the stratum corneum, calling the appendageal (or transappendageal) pathway.5,6 The delivery through the skin appendages is defined as transportation via hair follicles, sebaceous glands and sweat ducts. Hair follicles represent an efficient reservoir extended deep into the skin tissue and, therefore, play a significant role in both localized and systemic drug delivery.7,8 A dense capillary network associated with the upper dermal vasculature, as well as with the vessels of deep dermis and the subcutaneous tissue, supplies the hair follicles with blood ensuring the systemic uptake of the transported drug.9 In addition to representing a reservoir unit and a penetration pathway, the hair follicle introduces multiple targeted sites for different therapeutic approaches.9 Thus, drugs can be targeted to the bulge region with the stem cell as a site of interest for the gene therapy or can be delivered to the


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sebaceous glands as the targets involving in the aetiology of androgenetic alopecia and acne.10 The therapeutic intervention may also be associated with the intrafollicular delivery of immunomodulators of vaccines and targeted to immune cells.9 Effectiveness of intrafollicular drug delivery depends on the interaction between the drug and the sebum, as well as the choice of drug vehicle.3,6 In general, lipophilic rather than hydrophilic vehicles are used to improve follicular penetration.11 Different vehicles with supramolecular structure (liposomes, microemulsions, dendrimers) have been proposed.2 Thus, various liposomal formulations were shown to enhance the penetration of substances into the hair follicles, as they are able to envelop hydrophilic substances in their inner compartment or to insert lipophilic substances in their membrane.12–14 Various emulsions systems have been proposed as follicle-targeted vehicles as well.15–17 Recent studies have drawn interest on different micro- and nanoparticles as carriers for transfollicular drug delivery.18–20 Such particulate systems have been found to enhance the penetration of the encapsulated substance into the hair follicles.6 Furthermore, the usage of nanoand submicron particles as carriers allows prolongation of the payload release and providing the drug storage in the skin for a longer duration.18 As examples of particulate carriers, biodegradable polymeric polyvinylalcohol18,21, poly-(DL-lactic-co-glycolic acid)22–26, polystyrene27–29, and


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mesoporous silica30–32 particles have been developed. The size of particles was shown as playing an important role in the penetration depth of the follicle.6,19,20,25,33 However, successful transdermal delivery requires a suitable balance between effective transportation and safety for the skin.2,3 In terms of safety, the particles applied as drug carriers should be either biodegradable or able to move out the follicles by the sebum production and excretion.10 Moreover, the delivery system appropriate for the clinical use should be low-cost and easy to apply enhancing the patient compliance. The possibility of self-administration at home can also reveal the promising outlook for transdermal transportation. Storage and sustained-release property of the particulate system may allow the prolongation of therapeutic intervention that provides the transfer of care from the inpatient to the outpatient setting. In this context, elaboration of a carrier system based on biocompatible degradable particles, which provide the effective delivery to the hair follicles and the sustained release of the payload, is an important task. Prolonged drug storage within the follicles can be a beneficial property of this system. Porous micron- and submicron-sized calcium carbonate (CaCO3) particles have been recently intensively elaborated. Previous studies reported on the synthesis of mono-dispersed particles in size range from 2 to 10 µm,34,35 which was sized down to 400-600 nm in our works in order to enhance the cellular uptake.36,37 Vaterite polymorphs appear as porous polycrystals of the spherical shape stand out among the other crystallographic modifications of CaCO3 concerning perspectives of their application as smart containers for various personal care and biomedical applications.38,39 The high porosity of vaterite polycrystalline determines the possibility of different bioactive substance immobilization.34,40 Incorporation of various proteins,40–44 enzymes,45 drugs,46–52 and DNA53 into these matrices was shown revealing the possibility to improve the therapeutic


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effectiveness for the loaded substance.52,54,55 Furthermore, vaterite is defined as the least stable phase of CaCO3, which slowly dissolves and recrystallizes upon contact with water-based solutions,56–58 including interstitial fluid,59 forming the stable calcite. A controllable release mechanism based on a crystal phase transition has been demonstrated.36,60–62 At the same time, the carriers show high stability in the dried state enabling a long-term storage at standard conditions. For biomedical applications, it was successfully demonstrated that such type of particles allows for the efficient uptake by mammalian cells.36,37,52,54 Moreover, this process provides concentrating and localization of the drug inside the cell increasing its therapeutic efficiency.52,54 Meanwhile, the carriers themselves came out as biocompatible, biodegradable matrices showing the rapid decomposition under mild conditions (pH below 6.5).42,61 Furthermore, by the virtue of unique mechanical, physical and chemical properties, CaCO3 is applied as a constituent in a broad range of personal care products (e.g. tooth- and skin care, makeup and cosmetic products) acting there as abrasive, absorbent, colourant, buffer, filler or emulsion stabilizer.39 Being biologically friendly, this material is widely used in human daily life. By this means, porous calcium carbonate microparticles are sought to be an attractive carrier for transdermal delivery of therapeutics. The aim of this study was to explore those CaCO3 particles of different sizes for folliclemediated transdermal drug delivery. The carriers were loaded with a fluorescent marker in order to follow its transdermal administration for the purposes of localized and systemic adsorption. The kinetics of carrier degradation together with a payload distribution and elimination rate were investigated in vivo in rats.


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2. EXPERIMENTAL SECTION 2.1 Materials

Calcium chloride (CaCl2), sodium carbonate (Na2CO3), ethylene glycol (EG) and ethylenediaminetetracetic acid (EDTA) were purchased from Sigma-Aldrich and used without further purification. Cyanine 7 (Cy7) dye was purchased from Lumiprobe. Milli-Q water was used in all experiments (Milli-Q Purification System, Millipore, Merck, USA).

2.2. Preparation and characterization of calcium carbonate particles

Particle synthesis Calcium carbonate particles were synthesized by precipitation from the mixture of equimolar solutions of CaCl2 and Na2CO3 using different techniques in order to obtain the particles of different sizes. For this purpose, equal volumes of 0.33 M Na2CO3 and 0.33 M CaCl2 water solutions was quickly poured into a glass vessel at room temperature. CaCO3 particles with a size of 4.5±0.5 μm were obtained at mixing of the reaction solution using the magnetic stirrer Mini MR Standart (IKA, Germany) at 500 rpm during 1 min34,63 (synthesis technique ST1). For the preparation of 1.0±0.1 µm spheres,


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ultrasonic homogenizer Sonopuls (Bandelin, Germany) was used. Agitation of the reaction solution was therefore performed under the sonication at the frequency of 20 kHz and radiation intensity of 1 W/cm2 during 90 seconds63 (synthesis technique ST2). Submicron vaterite particles of 0.6±0.1 μm size were fabricated at the presence of ethylene glycol (EG) in the reaction solution60. Water solutions of Na2CO3 and CaCl2 were added each to EG in 2:10 volume ratio, that corresponds to 83% concentration of EG in the reaction solution, and rapidly stirred at 500 rpm for 3 hours. The synthesized CaCO3 particles were thoroughly washed twice with water and once with ethanol, afterwards they were dried for 30 min at 60°C (synthesis technique ST3).

Scanning electron microscopy imaging The morphology and microstructure of prepared calcium carbonate particles were characterized by scanning electron microscopy (SEM) using MIRA II LMU instrument (Tescan) at an operating voltage of 20 kV. Size distribution of CaCO3 particles was investigated by a set of SEM images in order to obtain a minimum of 100 measurements


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per sample. Image analysis and statistics were performed using Image J. The average size was shown as “mean ± standard deviation”.

2.3 Animals and ethics statement

Male albino rats of 6-8 months of age weighing 200±20 g were obtained from Animal Facility of Saratov State Medical University (SSMU). Rats were housed in individual cages under standard conditions: 12 hours light/dark cycle at 22±2 °C, and were fed with normal diet and water at libitum. The protocols on animal experiments were approved by the Ethics Committee of SSMU wide approval No 8 from 10.04.2018. All the experiments were performed with a proper care minimizing pain and suffering of animals in accordance with the principles of bioethics, rules of Good Laboratory Practice (GLP), and conventions for the protection of animals used in experiments and for other scientific purposes (adopted by the Council of Europe in 1986) in accordance with order of the Ministry of Health of the Russian Federation no. 267 from June 19, 2003 “Approval of the Rules of Good Laboratory Practice.” The study was performed in compliance with the ethical standards set out in the Geneva Convention (1971), “International Recommendations for


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Biomedical Research Using Animals” (1985), “Helsinki Declaration on Medical Ethics,” and “International Recommendations for Biomedical Research Using Laboratory Animals” (1989).

2.4 Protocol for transfollicular delivery of calcium carbonate particles

Delivery of calcium carbonate particles into hair follicles was performed in vivo in rats, sonophoresis was used as a physical enhancer of the transdermal particle penetration. One day prior to particle administration, the lower back of each rat was carefully shaved with a razor without any skin damage remaining short hair stubbles (few millimetres length). The next day, animals were anaesthetized with a combination of Zoletil (Virbac Sante Animale, France) at a dose of 0.1 mL/kg and Xylazine (Interchemie, Netherlands) at a dose of 1 mg/kg. The shaved skin area of each rat was cleaned and degreased with 70% ethanol prior to particle application. Topical application of 70% ethanol does not cause any significant skin irritation in vivo.64 The weighted portion of particle powder was dispersed in ethanol achieving 100 mg/mL concentration. Then 100 μL of such suspension were applied to the shaved skin surface, then the soundhead was disposed


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and used to massage this area. No ultrasound gel was applied here, 70% ethanol was used as a sonocoupler. The sonication was performed using 1-MHz ultrasound at 0.5 W/cm2 of power density during 2 minutes by the means of a Dynatron 125 ultrasonicator (Dynatronics, USA), the diameter of the soundhead was 2 cm. By this means, the particles were homogeneously distributed across the investigating area (corresponded to 3.2 cm2) at the dose of 3 mg/cm2. At the end of the procedure, the rest of the particle suspension was gently removed from the skin surface with a wet cotton pad.

2.5 Investigation of penetration of calcium carbonate particles of different sizes into rat hair follicles in vivo

The penetration of calcium carbonate particles of three different sizes was studied in

vivo in order to optimize the carrier size. For this purpose, particles of 4.5±0.5, 1.0±0.1 and 0.6±0.1 μm were synthesized using synthesis protocols ST1-ST3 described in

paragraph 2.2. Transdermal administration of each particle size was performed for 3 different rats (n=3) dividing the lower back of every rat in 3 regions (one region for one


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particle size). The protocol described in paragraph 2.4 was used for the administration. The efficiency of carrier delivery into hair follicles was judged by the means of optical coherent tomography (OCT) monitoring of rat skin in vivo and by scanning electron microscopy (SEM) imaging of the hairs plucked with root sheaths from the skin.

Optical coherent tomography monitoring of the skin OCT-visualization of the follicle filling with particles was performed in vivo using a commercial OCP930SR system (Thorlabs, USA) working at the central wavelength 930±5 nm with 100±5 nm full width at half maximum spectrum, the axial and lateral resolutions of the light source in the air were 6.2 μm and 9.6 μm, respectively.

Hair pluck and SEM investigation of the hairs For the SEM investigation, hairs of each experimental region and of the control region (where no particle application had been done) were extracted by the “forcible hair pluck” using tweezers65 retaining root sheaths or at least their portion. Every specimen of plucked hairs, contained 10-15 rat hairs, was placed on the conductive tape attached to the sample holder and the root sheaths of these hairs were afterwards destructed


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mechanically with a cutter knife. The morphology of calcium carbonate particles, which filled these follicles, was investigated by SEM as described in paragraph 2.2.

2.6 Study on transdermal transportation of a fluorescent marker by means of calcium carbonate submicron particles in vivo

Particle loading with a fluorescent dye Fluorescent dye Cyanine 7 (Cy7) was loaded into the proposing vaterite submicron (0.6±0.1 μm) matrices as a fluorescent marker. Cy7 was chosen for this study as it has a strong fluorescence peak in near-infrared (NIR) area, where much less background fluorescence is observed on biological tissues that allows one to study the distribution of Cy7 molecules in tissue samples. The adsorption method was used for its loading. Accomplish this, the weighted portion of dried CaCO3 particles was incubated in Cy7 ethanol solution with a concentration of 2 mg/mL for 1 hour under constant shaking at room temperature. Then the carriers were centrifuged at 5000×g for 1 minute and supernatants were collected.

Evaluation of the payload concentration


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The amount of Cy7 loaded into CaCO3 particles was evaluated by fluorescence. As a measure of the Cy7 amount in solution, a fluorescence intensity was recorded at 772 nm by a hybrid multi-mode microplate reader Synergy H1 (BioTek, USA) with excitation at 745 nm. Absolute values were obtained via a calibration curve. The estimation of the carrier loading capacity was performed in two different ways. In the first case, the Cy7 amount was evaluated by subtracting the amount of unloaded dye retained in the supernatant from its initial amount in the solution, which had been added to the system. A calibration curve was obtained from the measurements of known concentrations of Cy7 in ethanol. In the second case, the weighted portion of Cy7-loaded CaCO3 particles was dissolved in 0.2 M aqueous solution of ethylenediaminetetracetic acid (EDTA). Here, the calibration curve was obtained by dissolving the known amounts of Cy7 in EDTA solution. The loading capacity of containers was estimated as a ratio of the weight of Cy7 incorporated into CaCO3 particles to the weight of CaCO3 particles, expressed as a percentage.


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Characterization of dye-loaded particles The morphology of loaded particles was characterized by SEM. The optical study of the loading process was performed using a confocal laser scanning microscope (CLSM) Leica TCS SP8 (Leica, Germany) detecting the emission between 750 and 795 nm with the excitation wavelength 670 nm.

Transdermal delivery of the loaded particles The suspension of Cy7-loaded CaCO3 submicron particles in ethanol was applied topically on the lower back of the rat in vivo in accordance with the protocol described in

paragraph 2.4. After the US-assisted intrafollicular delivery of the carriers and consequent removing of undelivered particle and fluorescent dye from the skin surface, forcible hair pluck and biopsies were taken. The specimens of hair pluck were collected using tweezers (15 hairs were taken), the follicle root sheaths were investigated using SEM, as it has been described in

paragraph 2.5. The skin punch biopsies were taken and investigated using CLSM in order to study the penetration of Cy7 dye into the rat skin.


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Skin punch biopsy and confocal laser scanning microscopy imaging The biopsies were taken using 2-mm punches Harris Uni-Core (Whatman) in order to collect 3 specimens per treated area. The samples were frozen and vertical cryosections with a thickness of 20 μm were obtained using cryostat Leica CM1950 (Leica Microsystems, Germany). Cryoslices were placed on a glass slide, treated with 10 μL of glycerol, covered with another glass slide and imaged with CLSM Leica TCS SP8 (Leica, Germany). Glycerol served as an optical clearing agent for tissue scattering decreasing and, thus, increased the CLSM probing depth.66 Bright field and two fluorescence channels were used in order to demonstrate the penetration of Cy7-loaded particles into hair follicles. Blue fluorescence signal was recorded at 420-490 nm under the excitation of 405 nm showing the autofluorescence of hair shafts. Red fluorescence signal was detected at 750-795 nm under the 670-nm excitation corresponding to fluorescence of the Cy7 dye.

2.7 Study of carrier degradation, distribution of the delivered dye and its elimination rate in vivo


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Carrier degradation investigation Intrafollicular administration of pure and Cy7-loaded submicron calcium carbonate particles (0.6±0.1 μm) was processed in vivo in rats using the protocol specified in

paragraph 2.4. For this purpose, 20 mg of particles were distributed across the investigating area of 6.5 cm2 on two successive sonication procedures keeping the dosage (3 mg/cm2) and all the sonication parameters fixed. The process of particle degradation inside the rat hair follicles was monitored using SEM. Accomplishing this, the specimens of hair plucks were collected before, right after and then 24, 48, 96, 120, 168, 216, 264 and 312 hours after the particle administration. The procedure was performed as described in paragraph 2.5: the hairs were taken using tweezers, their follicle root sheaths were destructed mechanically with a cutter knife and investigated thereafter using SEM. Every specimen contained 15-20 hairs with their root sheaths. In the case of investigation of pure CaCO3 particle degradation, SEM imaging was accomplished by energy dispersive X-ray (EDX) analysis using an Inca Energy 350 EDX


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system (Oxford Instruments) in order to identify the elemental composition of the investigating samples.

Dye distribution and storage within the skin appendages investigation The suspension of 20-mg Cy7-loaded submicron particles was applied on the rat lower back skin as it has been described above. The storage of transdermally penetrated Cy7 dye was studied by the means of CLSM imaging of skin punch biopsies taken from each region of interest. The procedure was performed as described in paragraph 2.6, the biopsies were taken right after, 24, 96, 168, 264 and 384 hours after the particle administration. This 384-hour study was repeated for 3 rats (full independent investigation for each rat). The biopsies from the untreated area were taken as a control: the area was preliminary shaved, but no particles were applied then. The fluorescence intensities and area fractions of the obtained CLSM-images were analysed using Gwyddion 2.51 modular program for scanning probe microscopy data visualization and analysis (http://gwyddion.net). The Mark grains by Threshold and Grain Statistics modules were used. Identical regions of each image were measured for each


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application. Statistics for mean fluorescence intensity and fluorescent area fraction were calculated using the Matplotlib package for Python base version 2.7. Mean values and standard errors were calculated basing on up to 8 measurements for each punch biopsy sample (0-384 hours) of every rat (3 animals).

The study of urinary excretion profiles of the dye In order to study the urinary excretion rate of the transfolliculary delivered dye, a 2-week experiment was carried out in vivo for 5 rats. Two days before the experiments, animals were placed in individual plastic metabolism cages (OpenScience, Russia), food and water were permitted ad libitum. One day prior to particle administration, the lower back of each rat was carefully shaved with a razor without any skin damage and the animals were placed back to the cages. The control urine samples were collected 24 hours after the shaving in order to measure the control values. Application of Cy7-loaded particles was performed in compliance with the elaborated protocol (paragraph 2.4) except that the animals were not anaesthetized in order to avoid any possible effects on their metabolic activity. For the experiment, the area of particle


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application was 5-times expanded with a purpose of increasing the dye concentration introduced to the experimental animal. By this means, 50 mg of particles per each rat were used keeping the dosage at 3 mg/cm2. Urine was collected at 24-hour intervals for 2 weeks (312 hours) following the dye administration. A fluorescence investigation of the urine samples was performed. A hybrid multi-mode microplate reader Synergy H1 (BioTek, USA) was used for the detection of Cy7 in urine. Prior to the measurement, each sample was centrifuged at 3000 rpm for 5 minutes. Fluorescence intensity of the samples was recorded at 772 nm under the excitation at 745 nm keeping the measurement parameters fixed for all the specimens. Four 0.2-mL specimens of each urine sample were examined (n=4 multiplied for 5 rats). Statistical analysis was performed using Microsoft Excel software program. Mean values and standard deviations were calculated basing on 4 repetitions of the measurements for every urine sample (0-312 hours) of every rat (5 animals). The collected urine samples were used also for ascertaining the general health and physiological status of the animals during the experiment. For this purpose, the measurements of urinary glucose, bilirubin, ketones, specific gravity, blood, pH, protein,


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urobilinogen, nitrite, and leukocytes levels were performed with a usage of urine reagent test strips Multistix 10 SG (Siemens, Germany). The measurements were made before the sample centrifugation. In order to specify the impact of intrafollicular drug delivery, the elaborated delivery protocol was compared to the control modes of transdermal transportation: topical application of the Cy7-loaded carriers without ultrasound treatment (control 1) and of the Cy7 solution followed by ultrasound treatment (control 2). The urinary excretion profiles of the delivered dye were studied in vivo during 216 hours for 3 rats per each delivery mode.

3. RESULTS and DISCUSSION 3.1 Transdermal penetration of calcium carbonate particles of different sizes in vivo

Over the last two decades, the transdermal penetration profile of micro- and nanoparticles has gained interest due to the noticeable size-dependence for the depth of follicular penetration.9,33 As known, the usage of particulate substances improves follicular penetration in comparison with non-particulate ones.25,29,67 Polymeric microparticles ranging from 3 to 10 μm in size were demonstrated to penetrate to the


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follicular duct, whilst the larger particles remain on the skin surface.68 The diameter of 5 μm was found to be optimal for the polymeric microcarriers applied topically on a hairless rat or human skin in vitro and in vivo.26 The intrafollicular penetration of topically applied polystyrene particles sized from 0.75 to 6 μm has been demonstrated in human skin in

vitro revealing the microsphere of approximately 1.5-μm size as optimal.29 The study of the penetration depths for polymeric and silica submicron particles ranging from 100 to 1000 nm in size has been performed in vitro on porcine skin demonstrating the deeper penetration for the particles of the size around 600 nm.25 Considering those results, we tested calcium carbonate particles of different sizes for their capability of delivery into hair follicles. For this purpose, we have prepared the microparticles of three different sizes: ~5 μm, ~1 μm and ~600 nm. Wet chemical synthesis of CaCO3 basing on the precipitation of the crystals from the reaction solution, which contains calcium and carbonate ions, have lots of realization ways allowing variation of the particle size.34,60,63,69 The techniques of CaCO3 particles preparation are well elaborated in our research practice.37,40,60,63,69


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SEM-images of the obtained CaCO3 particles are presented in Figure 1. The images demonstrate a spherical shape of the particles with an average size of 4.5±0.5 μm (Figure 1 a), 1.0±0.1 μm (Figure 1 b) and 0.6±0.1 μm (submicron particles in Figure 1 c).

Figure 1. SEM-images of pure calcium carbonate particles of different sizes: (a) 4.5±0.5μm, (b) 1.0±0.1 μm; (c) 0.6±0.1 μm

The synthesized particles were topically applied on the lower back of rat in vivo. For this purpose, we divided the treating area into 3 regions, wherein one region corresponded to one particle size. In addition, we chose the 4th region in order to remain it intact as a control. The investigating suspensions of particles for transdermal delivery were prepared in 70% ethanol as it has been proven to be an effective enhancer for penetration processes.29,31 Moreover, the ethanol solution contributes also to the disinfection and degreasing of the experimental areas.


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Topical application of CaCO3 was followed by the ultrasound (US) treatment (sonophoresis). Therapeutic ultrasound has been applied as an influence enhancing the particle penetration. As it is known, sonophoresis induces the delivery of bioactive molecules into the skin.70,71 The US-treatment of skin with a frequency more than 0.7 MHz gives a rise to pressure changes in the medium forming the cavitation bubbles inside the inherent cavities represented by hair follicle shafts and sweat glands.72 Oscillation of the bubbles in the follicles promotes the pushing of a particle suspension down the follicle. It was shown, that the application of 1-MHz ultrasound with a power density of 2 W/cm2 did not induce adverse effects on the rat skin.73 Meanwhile, the low-frequency ultrasound (20–100 kHz) results in significantly increased skin permeation caused by the cavitation-mediated lowering of skin barrier function,74 although possesses the damaging effect on the skin. Its application creates cavitation bubbles near the skin.72 Low-frequency ultrasound increases the cavitation effect resulting in the collapse of bubbles at the bigger size in comparison with high-frequency ultrasound and releases the higher energy by emission of shock waves and jets,75 that causes the structural shifts in surrounding tissues.76 Whereas, the cavitation threshold increases with increasing the US frequency.75


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Here we used the ultrasound for the particle delivery into the skin in a non-damaging manner. The US with a frequency of 1 MHz and a power of 0.5 W has been applied. Relying on a “geared pump” hypothesis,18,77 we expected to achieve the enhanced intrafollicular penetration of the particles. According to this hypothesis, the surface structure of the hair and the hair follicle might act as a pumping system transporting the particles deeply into the hair follicles while the hair is moving. Hence, the penetration of CaCO3 particles of three different sizes was studied in vivo in order to optimize the carrier size. The efficiency of carrier delivery into hair follicles was judged by the means of OCT-monitoring of rat skin in vivo and SEM-imaging of the hairs plucked with root sheaths from the skin. The data are presented in Figure 2.


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Figure 2. In vivo penetration of calcium carbonate particles of different sizes into the hair follicles. OCT-images of rat skin with well-defined hair follicles performed in vivo (a-d), SEM-images of the hairs plucked with a portion of root sheaths from the skin (e-h) and


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SEM-images of the root sheaths of the plucked hairs, which were destructed with a cutter knife, at different magnification (i-p). The bottom line (m-p) presents enlarged images of the red square marked areas in pictures (i-l). The left-most column of images (a, e, i, m) represents “control” hair follicles without particle application, meanwhile the others demonstrate intrafollicular penetration of calcium carbonate particles of different sizes: 4.5±0.5 μm – the second column (b, f, j, n), 1.0±0.1 μm – the third column (c, g, k, o) and 0.6±0.1 μm – the right-hand column (d, h, l, p)

The hair follicles are clearly defined at OCT-images of rat skin (Figure 2 a-d), as they have a refraction index different to the surrounding tissue. Specifically, the empty hair follicles (control image, Figure 2a) are less scattering and therefore seen darker than surrounded tissue. Incorporation of the CaCO3 particles inside the volume of follicles makes them appear as bright white channels since the particles have a higher refraction index compared to surrounding. By that way, the areas of the CaCO3 localization become well-distinguished in OCT-images (Figure 2 b-d). Optical depth of the particle detection in skin and optical length of follicle filling were evaluated and the data are shown in Figure


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S1 (see Supporting Information). Although all the particles have demonstrated deep penetration into the hair follicles, it evidences that 5-μm (Figure 2 b) and 600-nm particles (Figure 2 d) have provided the higher contrast displaying a denser follicle filling, while 1μm particles (Figure 2 c) showed the less efficient penetration. The morphology of the delivered particles should be taken into consideration in order to make a conclusion on the delivery effectiveness. For this purpose, the hairs of each experimental region (Figure 2 f-h) and the control one (Figure 2 e) were extracted by the “forcible hair pluck” using tweezers retaining root sheaths or at least their portion. The root sheaths were afterwards destructed mechanically with a cutter knife (Figure 2 i-p). The “forcible hair pluck” was chosen as a method of extracting hairs, as it allows the study of the root morphology for anagen hairs, which are actively growing and have high metabolic activity (in contrast to catagen and telogen ones representing the phases of hair regression and rest, respectively). The vast majority of hairs on the normal hair covering represent anagen follicles, but they are so firmly rooted that can only be examined either by biopsy or by forcible pluck.65 According to forcible pluck technique, several neighbouring hairs shafts are grasped near the skin surface by tweezers and


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forcible plucked then from the skin in the direction of their growth. By that approach, the investigating hairs retain the root sheaths, or at least their portion, available for the analysis. In order to avoid false judgement on particle penetration, we increased the number of investigating hairs. It provides the better statistics and increasing of the anagen hair numbers in the specimen. The SEM-images (Figure 2 j-l, n-p) have proven out the fact of hair follicle filling with the particles. A deep carrier delivery down to the lower follicle was observed revealing the most plentiful intrafollicular penetration for the submicron particles (0.6±0.1 μm). Furthermore, the particles of this size were delivered to the hair follicles without any integrity loss, as no changes in particle morphology observed (Figures 2 l, p). Meanwhile, there is clear evidence, that particles of the 4.5±0.5 μm size were fragmented during the administration (Figures 2 j, n). It was demonstrated previously, that the particles at a size similar to the thickness of the keratin cells of the hair (530 nm in human hair) penetrate more efficiently into the hair follicles.25,29 The moving hairs in the follicles act as a “geared pump” because of the zigzag structure of the surface of the hairs. Thus, the particles are entrapped underneath


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the cuticular cells and guided further down as the hair moves back and forth. In our study, this movement is enhanced by the therapeutic ultrasound treatment acting as a mechanical force. That provides a fast administration of the topically applied 600-nm particles down the follicles. Whereas, the larger particles (4.5±0.5 μm) are faced with a difficulty of cuticular entrapping. Taking into account the presence of lots of particle fragments, we assume the impeded penetration of such solid carriers. CaCO3 microparticles are fragile and can be broken due to mechanical force.55 The slower penetration of the particles down the follicles causes their longer exposition to the ultrasound that may promote the particle destruction. As a result, the hair follicles have been filled with a plenty of particle fragments as well as with some amount of the integral carriers (Figure 2 n). Basing on the data obtained, we determined the size of 0.6±0.1 μm as an optimal in terms of efficient delivery of CaCO3 particles into the hair follicles. Submicron particles were used in all the following experiments. The experiment on follicular penetration of CaCO3 submicron particles without ultrasound treatment was performed as a control (see Figure S2 in Supporting


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Information). Topical application of the carriers, in this case, led to their delivery into infundibulum, which is the uppermost part of the hair follicle, and could not allow the deeper transportation. The current results show, that the transportation of topically applied CaCO3 particles down the hair follicle here is caused more by the mechanical forces, than by the chemical interactions. 3.2 Transdermal transportation of a fluorescent marker by means of calcium carbonate submicron particles in vivo

As it was mentioned above, high porosity of proposing CaCO3 particles determines the capability of their loading with various bioactive substances. Considering this, we investigated the possibility of fluorescent dye transportation into the hair follicles by the means of these carriers. Accomplish this, the Cy7 fluorescent dye was loaded into the proposed submicron matrices as a model compound. The physical adsorption technique, where the active substance should be adsorbed from the solution onto preformed particles, has been used.40


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The SEM- and CLSM-images of loaded containers, presented in Figure 3, demonstrate the spherical shape of the particles. Furthermore, the fluorescence signal in Figure 3 b shows, that the particles are successfully loaded with Cy7.

Figure 3. SEM- (a) and CLSM- (b) images of calcium carbonate submicron particles loaded with Cy7 fluorescent dye

The concentration of Cy7 loaded into CaCO3 particles was evaluated by the spectrofluorometric method in two different ways: as a measure of Cy7 amount in supernatants collected after the adsorption procedure and by dissolution of the weighted portion of Cy7-loaded CaCO3 particles in mild acidic conditions to ensure a total dissolution of the carriers followed by a release of the Cy7 loaded into. Basing on the data obtained, the loading capacity of containers was calculated. A good agreement and accuracy of the data were obtained. Thereby, the loading capacity of containers


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corresponds to 0.6 % (w/w), that means 120 μg of Cy7 incorporated to 20 mg of CaCO3 particles. Transdermal delivery of the Cy7-loaded carriers under the therapeutic ultrasound was studied in vivo. The successfulness of dye transportation into hair follicles was estimated basing on SEM-images of the hair plucks (Figure 4 f) and CLSM-images of the skin punch biopsies (Figure 4 a-e).

Figure 4. CLSM-images of frozen skin slices (a-e) and SEM-image of the destructed root sheaths of a plucked hair (f) demonstrating in vivo intrafollicular penetration of Cy7-loaded


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calcium carbonate submicron particles. Blue fluorescence signal corresponds to autofluorescence of the hair shafts recorded at 420-490 nm under the excitation at 405 nm, while the red signal indicates Cy7 distribution detected at 750-795 nm under the excitation at 670 nm. Images (a, b) and (d, e) represent two consequent 20-μm slices of frozen rat skin containing the same group of follicles revealing the delivery of Cy7 to the deepest region of hear follicle – the hair bulb: (a, d) – overlay of transmission and fluorescent images, (b, e) – fluorescent images. Image (c) is a zoomed region of the image (b) enabling a clear determination of the penetration depth for Cy7-loaded calcium carbonate particles. The inset in (f) represents the enlarged white-square marked area of the main image

Red fluorescence signal in CLSM-images of the skin punch biopsies (Figure 4 a-e) corresponds to the Cy7 marker demonstrating its distribution in tissue. The bright fluorescence signal registered from the skin surface here corresponds to the drug penetrated and accumulated into the stratum corneum and hair funnels. Although we are focused mainly on intrafollicular drug accumulation, the delivery to these skin components


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should be also taken into consideration due to its importance in terms of various dermatoses (e.g. contact or atopic dermatitis, dermatomycoses) treatment. Blue fluorescent signal represents the autofluorescence of hair shafts. The overlay of these channels convincingly demonstrates the intrafollicular localization of the fluorescent marker, as the red signal is distributed down along the blue hair shafts. In order to clearly illustrate the delivery of the fluorescent marker, a region of interest of Figure 4 b was zoomed. The enlarged image (Figure 4 c) was rotated to vertical orientation and brightened using the Leica software for a more accurate evaluation of the Cy7 penetration depth. Superposition of these fluorescence channels with the bright field (Figure 4 a, d) allowed us to control a quality of the biopsy slices in order to investigate precision-cut samples avoiding the obliquely-cut ones. Two consequent vertical slices of the rat skin containing the same group of follicles (Figure 4 a, b represents slice 1, Figure 4 d, e – slice 2) illustrated the distribution of the Cy7 dye along the entire depth of the hair follicle from the skin surface (Figure 4 a, b) to the hair bulb (clearly remarkable red-coloured bulb-shaped regions in Figure 4 d, e).


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The SEM-images of the plucked hair root sheath are presented in Figure 4 f, where the white-square marked area of the main image was zoomed and presented as an inset. The images have confirmed, that Cy7-loaded CaCO3 submicron particles plenteously fill the rat hair follicles along their entire depth when applied in vivo.

3.3 Payload release and intratissue distribution during carrier degradation inside the hair follicles in vivo: dye storage and elimination rate

The sustained-release property of the drug-loaded carriers transported into the hair follicles ensures the payload distribution within the different tissue regions targeted for therapeutic action or its uptake by vasculature.20,78,79 Basically, the therapeutic agents immobilized into the carriers are released by gradual diffusion and particle decomposition, if no additional external influence is specified. Here, we investigated in vivo degradation of the submicron calcium carbonate carriers after their intrafollicular administration in rats using SEM and EDX analysis. The specimens of hair plucks were collected at different time points during 2 weeks for this purpose. The SEM-images of pure CaCO3 particles are represented in the left-hand


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column in Figure 5 (a, d, g, j, m, p), the result of their EDX-analysis is shown in Figure S4 (see Supporting Information). Key moments of Cy7-loaded CaCO3 carrier degradation are illustrated with the SEM-images presented in Figure S3 (see Supporting Information).

In vivo storage of transdermally penetrated Cy7 dye within the skin appendages was studied subsequently by the means of CLSM imaging of skin punch biopsies. The images are

presented

in

the

middle

and

the

right-hand

columns

in

Figure 5 (b, c, e, f, h, i, k, l, n, o, q, r), the data on the mean fluorescence intensity and fluorescent area fraction are shown in Figure 6 and Figure S5 (see Supporting Information). The urinary excretion study for the delivered dye was performed as well. For this purpose, urine samples were collected at different time points during 2 weeks after the intrafollicular dye administration. The results of their fluorescence investigation are summarized in the graph represented in Figure 7.


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Figure 5. SEM-images of the root sheaths (the left column – images a, d, g, j, m, p), demonstrating degradation of delivered calcium carbonate submicron particles inside the rat hair follicles during 264 hours in vivo, and CLSM-images of frozen skin slices (the middle and the right-hand columns) showing Cy7 distribution within the follicle and surrounding tissues in vivo. The middle column of images (b, e, h, k, n, q) represents overlay of transmission and fluorescent images, the right-hand column (c, f, i, l, o, r) – fluorescent images. Blue fluorescence signal corresponds to autofluorescence of the hair shafts recorded at 420-490 nm with 405-nm excitation, the red signal indicates Cy7 distribution detected at 750-795 nm with 670-nm excitation. Images of the upper line (ac) illustrate the “control” samples of rat skin and follicles without particle application. Active dissolution/recrystallization of calcium carbonate particles is processing during 1 week (168 hours, images d, g, j, m) and ends up with their total degradation within 264 hours (image p). Intrafollicular storage of Cy7 dye lasts during this period (Figure 5 e, f, h, i, k, l, n, o, q, r)


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Figure 6. Storage and elimination rate of Cy7 dye within the rat skin during 17 days (384 hours) after in vivo delivery of calcium carbonate carriers. The data are represented as average fluorescence intensities and fluorescent area fractions calculated from CLSMimages of skin punch biopsies and expressed as mean±SE based on up to 8 measurements for each time point of 3 independent experiments. The standard errors of the mean for fluorescence intensity measurements are less than 0.5%. The control skin fluorescence intensity, when no particles had been applied, amounted to 8.08±0.16

As mentioned above, the release of the payload from the porous CaCO3, particles is an interplay of drug desorption and carrier degradation.61,80 Furthermore, such porous


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spherical particles represent the vaterite polymorphs connoting the thermodynamically metastable phase of CaCO3. Their immersing in different solutions leads to slow dissolution and recrystallization of the carriers forming a stable calcite phase. In our previous works36,52,61,62 we have demonstrated the controllable release mechanism based on a crystal phase transition, where the external layer of the particles ionized seeding the formation of cubic-like calcite monocrystals from the ions. We should notice here, that these studies were carried out in vitro and did not consider the outflow of Ca2+ and CO32ions from the surface. It is well-known, that the hair follicle together with the sebaceous glands is a complex dynamic structure representing the site of unique biochemical, metabolic and immunological events.81 The interactions between the epithelium and the mesenchyme play pivotal roles in the morphogenesis of hair follicles. During the hair cycle, these interactions are regulated by a distinct set of molecular signals: a large variety of growth stimulatory pathways are activated for proper hair formation.82 These active processes influence the particle degradation. The secretion within the hair follicle forms the release medium for the particles and drugs delivered via the transfollicular route.83


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Current in vivo study on degradation of vaterite submicron carriers reveals, that an active dissolution/recrystallization of calcium carbonate particles is processing during 168 hours (Figure 5 d, g, j, m) and ends up with their total degradation within 264 hours (Figure 5 p). Being delivered into the hair follicles of rats, particles started to cover with proteins, enzymes and other components of follicle secretion. It is evidently seen when we compare SEM-images for just-delivered particles (Figure 5 d and Figure 4 f) with images performed 24 hours after the intrafollicular storage of the carriers (Figure 5 g and Figure 7 a). This follicle secretion represents the surrounding medium for the vaterite carriers providing their decomposition. Two main trends in timewise behaviour of the particles inside the hair follicles should be highlighted (see Figure S3 in Supporting Information). Transdermal transportation of the CaCO3 carriers applying the elaborated protocol leads to abundant filling of the inner volume of hair follicles and to their sticking and incorporation into the walls of follicle root sheaths. The particles incorporated into the walls were then captured by follicle tissue and their occlusion was obviously shown (Figure 5 j and S3). At the same time, the groups of closely-spaced particles (which filled the inner follicle volume) demonstrated a clear


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tendency towards aggregate formation during the first week caused by vaterite dissolution/recrystallization process. By this means, the inner volume of the follicles remains to be quite compactly filled with calcium carbonate after 24 (Figure 5 g) and 96 hours (Figure 5 j) of the investigation. The process of active dissolution/recrystallization continued further and after 168 hours (Figure 5 m) we have found the big fragments of CaCO3 aggregates inside the follicles reflecting in the sparse follicle filling. The total intrafollicular degradation of the carriers was observed within 264 hours (Figure 5 p), when no CaCO3 particles were seen in a specimen. The root sheaths fragments were smooth, thin and similar to control ones (Figure 5 a). The EDX-analysis of the elemental composition in the investigating samples (see Figure S4 in Supporting Information) has proved out the fact of particle degradation inside the hair follicles in vivo showing the decreasing of Ca-content inside the follicle root sheath with time. Intrafollicular storage of Cy7 fluorescent dye was observed during the particle degradation (the middle and right-hand columns of images in Figure 5 and the graph in Figure 6). After the transdermal transportation of Cy7-loaded particles, the bright


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fluorescence signal was registered from the rat skin surface and from the tissues surrounding the hair follicles during 96 hours (Figure 5 e, f, h, i, k, l). That fact is an agreement with particle degradation process, illustrating the compact filling of the inner follicle volume with calcium carbonate after 96 hours (Figure 5 j) of the investigation. Quantification by compiling the data from CLSM-images indicated that the mean Cy7 fluorescence intensity had been lowered by 25 % during this period (from 33.98±0.03 to 25.25±0.04). Meanwhile, the fluorescing area fraction was decreased by 72% (from 37±3 % to 10±2 %) (Figure 6). The payload release was processed as a result of carrier dissolution and recrystallization and the drug was eliminated thereafter, that appeared in gradual decreasing of the fluorescent signal with time. A marked fluorescent lowering in perifollicular tissue took place at 168 hours after the particle administration (Figure 5 n, o), when the recrystallization process had been finished for the majority of the particles and the process of aggregate dissolution was in a progress rarefying the follicle filling (Figure 5 m). The fluorescence area fraction was drastically decreased amounting to 0.8% (Figure 6). But the dye nevertheless remained in tissue showing a weak fluorescence of 15.46±0.07 arbitrary units, which is noticeable in the fluorescent


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image (Figure 5 o). The elimination of Cy7 from the tissue was observed at 264 hours (Figure 5 q, r), that corresponded to the total degradation of the carriers inside the hair follicles (Figure 5 p). However, the fluorescent dye was detected still on the surface of the hair shaft as covered the cuticular cells even after 384 hours of investigation (Figure S5). Such cuticular storage depends on the nature of the applying dye, we assume not to take it into consideration. A fluorescence investigation of the urine samples has proved out the possibility of systemic drug absorption via hair follicles by the means of the proposing particulate system and the elaborated protocol (Figure 7).


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Figure 7. Urinary excretion kinetics of intrafolliculary delivered Cy7 dye in vivo represented as fluorescence intensity of Cy7 in rat urine (excitation at 745 nm, emission at 772 nm) during two weeks (312 hours). The data are expressed as mean±SD basing on 4 repetitions for each time point of 5 independent experiments. SEM-images (a-c) illustrate the key moments of recrystallization/dissolution process for calcium carbonate submicron particles loaded with Cy7 inside the hair follicle: morphology of carriers inside the root sheath 24 hours after the intrafollicular delivery (a) referring to start of recrystallization process (cubic particles appeared); 120 hours after (b) demonstrating the ending of a recrystallization process (the majority of particles either had a cubic-like morphology or formed aggregates); 168 hours after (c) illustrating the process of dissolution of calcite particle and aggregates, which took place up to 288 hours after particle application. The scale bar at SEM-images corresponds to 1 μm

The Cy7 dye excretion profile in urine of rats demonstrates its full agreement with the particle degradation kinetics and intrafollicular dye storage duration. Thus, the fluorescent marker was detected in urine within 264 hours after the application of carriers, but at


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168 hours after the transportation, the signal intensity had been decreased more than 10times and continued lowering up to the moment of total dye elimination. The control experiments on Cy7 urinary excretion performed for the topical application of the dye-loaded CaCO3 carriers without ultrasound treatment and for the application of Cy7 solution followed by ultrasound treatment demonstrated a fast lowering of the fluorescence intensity (see Figure S6 in Supporting Information). In such a manner, the dye was mostly excreted within 24 hours. Moreover, the fluorescence values were at least 5-times lower than for US-assisted delivery of the Cy7-loaded carriers, that highlighted the importance of intrafollicular drug delivery compared to other transdermal transportation modes. The collected urine samples were used also for ascertaining the general health and physiological status of the animals during the experiment. The measurements of urinary glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes levels demonstrated no abnormal levels. The results represented the absence of significant pathological processes regarding the status of carbohydrate metabolism, kidney and liver function, acid-base balance, and urinary tract infection.


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Apart to metabolites monitoring, we did not observe any acute reaction during the hair cover regeneration as seen from the pictures of the rat back performed during 3 weeks of the experiment (Figure S6 in Supporting Information). No skin damaging, inflammatory state or erythema has been registered. The hair cover regenerated completely within 21 days after the shaving and application of the particles. Based on this observation, one can tentatively deduce that no significant toxic effect is relevant for the proposed carrier systems in terms of transdermal drug transportation. By this means, the proposed particulate delivery system provides effective transdermal transportation and intrafollicular storage of bioactive molecules allowing the prolongation of therapeutic intervention over the two-week period. This approach reveals the promising outlook for the treatment of skin disorders as well as for systemic drug delivery via skin appendages. The impactful effect of the prolonged release has been previously reported for particulate transdermal delivery systems loaded with various drugs.20 Immunomodulators and vaccines,84,85 antibacterial drugs,86,87 photosensitizers,52,88 insulin,16,89 siRNA90,91 and hair growth therapeutics20 stand out among the other bioactive compounds here, as their


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successful loading at therapeutic dose range has been demonstrated using CaCO3 particles. Thus, such therapeutics can be considered for future biomedical experiments and their clinical application. 4. CONCLUSIONS

The current study proposed porous calcium carbonate submicron carriers to provide efficient transdermal drug transportation via skin appendages. The optimal particle size was found laying the groundwork for the standardized protocol elaboration. Thus, the topical application of 600-nm CaCO3 carriers in rats in vivo followed by the therapeutic ultrasound treatment during 2 minutes led to their deep penetration through the skin by the means of plentiful filling of hair follicles. The proposing carriers represent biocompatible biodegradable particles with a large surface area enabling a high drug payload ability. Intrafollicular delivery of the fluorescent marker loaded into these carriers as a model drug was successfully demonstrated. The rat hair follicles were filled with the dye-loaded particles along the entire follicle depth down to the bulb region after their topical application in vivo. Our study revealed the active dissolution/recrystallization of CaCO3 particles, that resulted in their total degradation inside the hair follicles within 12


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days. The release of fluorescent payload followed the carrier degradation ensuring the payload distribution in the rat skin and its’ intrafollicular storage during this period. The urinary study evidentiated a systemic absorption of the fluorescent marker via hair follicles. The results on payload excretion kinetics in urine correlated strongly with intrafollicular storage kinetics and the particle degradation data. By this study, such a particulate system presents a promising carrier for the drug transportation and storage while penetrating into the hair follicle. At the same time, degradation-driven in situ release from CaCO3 particles provides systemic adsorption of the delivered drug. Hence, such carriers play a significant role in both localized and systemic drug delivery. Wherein, the elaborated system provides the prolongation of therapeutic intervention over the two-week period, what makes it promising for envisaged medical applications.

ASSOCIATED CONTENT

Supporting Information. Supporting Information (SI) contains the data on optical depth of calcium carbonate particle detection in skin and optical length of follicle filling. The data


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on follicular penetration of CaCO3 submicron particles into the rat skin without ultrasound treatment are also shown in SI. The elemental composition of pure submicron calcium carbonate carriers obtained by EDX-analysis during the investigation of their in vivo degradation after the intrafollicular administration in rats is described in Supporting Information. The SI contains also additional SEM-images illustrating the key moments of Cy7-loaded carrier degradation in vivo inside the follicles of rat. The description of quantification of the mean fluorescence intensity and fluorescent area fraction from CLSM-images is contained in SI. The data on urinary excretion kinetics of Cy7 dye after topical in vivo application of the Cy7-loaded CaCO3 carriers without ultrasound treatment and of Cy7 solution under the sonication are presented in SI as well. Supporting information contains results of the observation of hair cover regeneration after shaving and application of the particles. The photography of the rat back performed during 3 weeks of the experiment are provided there. Supporting Information is available free of charge.

AUTHOR INFORMATION


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Corresponding Author *Dr. Yulia Svenskaya, e-mail: [email protected]

Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Russian Science Foundation, project № 17-73-20172; Government of the Russian Federation, grant № 14.Z50.31.0004. ACKNOWLEDGMENT

The work was supported by the Russian Science Foundation (project № 17-73-20172). The studies related optimization of the protocol for transdermal transportation of the carriers

were

supported

by

Government

of

the

Russian

Federation

(grant

№14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists at Russian institutions and Russian institutions of higher education). We would like to acknowledge Vsevolod Atkin for SEM measurements. In


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vivo experiments were carried out at the laboratories of Saratov State Medical University with the help of Dr Alla Bucharskaya and Dr Olga Sindeeva.

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GRAPHICAL ABSTRACT


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A Simple Non-Invasive Approach toward Efficient Transdermal Drug

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