Transdermal Delivery of 5-Aminolevulinic Acid by Nanoethosome Gels

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Transdermal Delivery of 5‑Aminolevulinic Acid by Nanoethosome Gels for Photodynamic Therapy of Hypertrophic Scars Zheng Zhang,†,□ Ying Liu,‡,□ Yunsheng Chen,*,†,§,□ Lexiang Li,∥ Ping Lan,⊥ Dannong He,# Jie Song,*,§ and Yixin Zhang*,†,#

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†

Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Road, Shanghai 200011, P.R. China ‡ Cosmetic Laser Center, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Roadd, Shanghai 200011, P.R. China § Institute of Nano Biomedicine and Engineering, Shanghai Engineering Research Center for Intelligent Instrument for Diagnosis and Therapy, 800 Dongchuan Rd, Shanghai Jiao Tong University, Shanghai 200240, P.R. China ∥ Department of Orthopedic, Changzheng Hospital, Second Military Medical University, Shanghai 200240, China ⊥ Institute for Advanced and Applied Chemical Synthesis, Jinan University, Zhuhai, 519070, China # Shanghai National Engineering Research Center for Nanotechnology, 245 Jiachuan Road, Shanghai 200237, PR China S Supporting Information *

ABSTRACT: 5-Aminolevulinic acid (ALA)-loaded nanoethosome (ALA-ES) gels are successfully prepared to realize a transdermal delivery of ALA, and they provide a feasible approach for the photodynamic therapy (PDT) of hypertrophic scars (HS). Herein, the morphological and physicochemical features indicate that ALAES is stable in gel matrix. In vitro transdermal penetration studies suggest ALA-ES gels can overcome the compact dermal barrier and deliver more ALA into human HS tissue. In vivo delivery studies further reveal that ALA-ES gels can penetrate into rabbit HS tissue to facilitate ALA accumulating in hypertrophic scar fibroblast (HSF) and converting into protoporphyrin IX in the cytoplasm. Utilizing transmission electron microscopy, the visual in vivo penetration process indicates ALA-ES penetrate into HS tissue utilizing its deformable membrane, enters HSF by a pinocytotic-like mechanism, and then releases ALA in the cytoplasm. Subsequently, PDT efficacy is assessed using rabbit HS models. The morphological and histological analysis reveal that ALA-ES gels can improve HS by promoting HSF apoptosis, remodelling collagen fibers and increasing MMP3 expression. The results demonstrate that ALA-ES gels are suitable in clinical treatment of HS and make a substantial progress within the field. KEYWORDS: hypertrophic scars, photodynamic therapy, 5-aminolevulinic acid, nanoethosome, transdermal delivery, rabbit models cures HS.4,5 Among various photosensitizers, 5-aminolevulinic acid (ALA) has been most widely used in clinical dermatology since its topical solution (Levulan, Kerastick) was approved by The United States Food and Drug Administration (FDA) in 2000. That ALA accumulates in HSF and converts to protoporphyrin (PpIX) is the crucial premise of ALA-based PDT (ALA-PDT).6 In clinical, ALA can be administered by intravenous and topical administered. Compared to intravenous administration with disadvantage of low bioavailability and long-lasting skin photosensitization, topical administration of ALA through the skin would be preferable. Actually, ALAPDT has been proven to be a local treatment modality in dermatology by topical administration. However, the dysfunc-

1. INTRODUCTION Hypertrophic scar (HS) is caused by persistent dermal fibrosis, irregular collagen deposition, and overproliferative hypertrophic scar fibroblast (HSF).1 Conventional therapy for HS can be divided into noninvasive approaches (i.e., topical silicone sheeting and pressure dressings) and invasive approaches (i.e., surgical resection and laser excision). However, these therapeutic approaches are suffered from various adverse effects, such as low efficacy, high recurrence, and painful processes.2 Recently, photodynamic therapy (PDT) has become an attractive HS treatment approach with much more efficacy but less adverse effects.3 The potential underlying mechanism of PDT in HS divides into three steps: (a) photosensitizer accumulates in HSF at first; (b) after exposure to appropriate laser, the photosensitizers in HSF produce cytotoxic reactive oxygen species (ROS); and (c) ROS causes HSF apoptosis collagen fibers remodelling to © XXXX American Chemical Society

Received: October 10, 2018 Accepted: December 27, 2018 Published: December 27, 2018 A

DOI: 10.1021/acsami.8b17498 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

accumulating in HSF and converting into PpIX in the cytoplasm. By transmission electron microscopy (TEM), the visual penetration process indicated ALA-ES penetrated into HS tissue utilizing its deformable membrane, entered HSF by a pinocytotic-like mechanism, and then released ALA in the cytoplasm. PDT efficacy was assessed in rabbit HS models. The morphological and histological analysis revealed that ALAES gels could improve HS by promoting HSF apoptosis, remodelling collagen fibers and increasing matrix metalloproteinase 3 (MMP3) expression. The results demonstrated that ALA-ES gels were suitable in clinical treatment of HS.

tional and persistent extracellular matrix (ECM) of HS is a huge barrier for ALA, so that ALA-PDT for HS is controversial because of the poor penetrability of ALA into both HS tissue and HSF.7 Therefore, the delivery and accumulation of ALA in HSF become desirable goal for improving ALA-PDT toward HS. Recently, transdermal delivery has incorporated nanotechnology, and various nanodelivery systems have attracted considerable attentions.8,9 Nanoethosome (ES), a novel lipid nanodelivery system without cytotoxicity, exhibits a high efficacy in transdermal drug delivery.10,11 The prepared ES in hydroalcoholic solution can act as both drug permeation enhancer and drug carrier by its lipid fluidity. However, this ES solution is limited its clinical applications because it is unsuitable for continuous drug topical administration. Fortunately, gels, a substantially diluted cross-linked system with properties between solid and liquid, can overcome this problem.12 Therefore, the ES gels might have practical significance for clinical transdermal drug delivery.13 In clinically, carbopol gel has been widely applied in topical administrations since it was approved by the FDA. In our previous work, ALA-loaded nanoethosome (ALA-ES) enhanced the penetrability of ALA in vitro.14 Therefore, the combination of ALA-ES and carbopol gels is a feasible approach for PDT treating HS in the clinic, because ALA-ES gels can integrate the advantages of ALA-ES and carbopol gels with higher transdermal delivery efficiency than conventional liposomes and higher feasibility than liquid delivery systems. Although ES loading 5-fluorouracil can transfer into carbopol gels from its solution in our reported paper, whether ES gels play expected role is still unknown.15 In the current study, ALA-ES gels were prepared for transdermal delivery of ALA into HS, and they provided a feasible approach for PDT treating HS for the first time (Scheme 1). Here, the morphological and physicochemical features of ALA-ES gels indicated that ALA-ES were stable in the gel matrix. In vitro transdermal penetration studies suggested ALA-ES gels could enhance ALA penetration into human HS tissue. In vivo delivery studies further revealed that ALA-ES gels can penetrate into rabbit HS tissue facilitate ALA

2. EXPERIMENTAL SECTION 2.1. Materials. Soybean phosphatidylcholine (PC, soybean lecithin with 95.8%), absolute alcohol, propanetriol, and triethanolamine were purchased from Aladdin (Shanghai, China). 1-Palmitoyl2-{6-[(7-nitro-2-1,3-benzoxadi-azol-4-yl)amino]hexanoyl}-sn-glycero3-phosphocholine (NBD-PC) were from Avanti Polar Lipids (Alabaster AL). Carbopol 950, ALA hydrochloride (99% purity), and rhodamine 6G (R6G) were bought from Waldeck GmbH (Muenster, Germany). Hoechst 33342 was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of analytical grade and were obtained from Sinopharm Chemical Reagent, Co., Ltd. (Shanghai, China). 2.2. Preparation of ALA-ES Gels. ALA-ES solution was prepared using the protocol reported in our previous works, and the details was shown in S1, Supporting Information.14 The prepared ALA-ES (4 mg/mL ALA) was characterized as 81 ± 23 nm in size and 53.6 ± 4.2% in encapsulation efficiency. The carbopol gel matrix was prepared as follows: (a) 1.6 g of carbopol and 4 mL of propanetriol were added to 42 mL of double-distilled water; (b) the mixture was stirred for 2 h at room temperature, and then neutralized to Ph 7.4 by using 2.5 mL of triethanolamine; (c) carbopol gel matrix was obtained after hydrating overnight.15,16 Therefore, ALA-ES gels were prepared through mixing ALA-ES solution with the carbopol gel matrix (1:1, v/ v) in a sealed container at 500 rpm, and the concentration of ALA in ALA-ES gels was 2 mg/mL. An ALA hydroalcoholic solution (ALAHA, 30% ethanol, v/v, 4 mg/mL ALA) was used to prepare ALA-HA gels using the same method as with ALA-ES gels. The fluorescent labeled ALA-ES gels was prepared by adding 4 mol % NBD-PC during the preparation of ALA-ES. 2.3. Morphological and Physicochemical Characterization of ALA-ES Gels. ALA-ES gels and their sections were examined using a JEM-2010 TEM (JEOL, Japan) at an accelerating voltage of 120 kV. The diluted ALA-ES gels were placed on the copper grid coated with carbon film and air-dried overnight. Meanwhile, ultrathin ALA-ES gel samples (100 nm thickness) were cryosectioned using a cryoultramicrotome (UC6/FC6, Leica Austria). All TEM samples were negatively stained using 1.5% (w/v) phosphotungstic acid (pH 7.0, Aladdin, Shanghai, China). For scanning electron microscopy (SEM) tests, ALA-ES gels were sputter-coated with gold and then examined using a JSM-6360LA SEM (JEOL, Japan) with an accelerating voltage of 10 kV. Small-angle X-ray scattering (SAXS) studies were carried out using an on a SAXSess mc2 instrument with Cu Kα radiation (0.1542 nm, Anton Paar, Austria). Thin-walled 2 mm glass capillaries were filled with ALA-ES gels and solution for the scattering experiments. All scattering curves, recorded at were reproduced three times, and a representative curve was selected. The working q-range (Å−1) was 0.08 ≤ q ≤ 0.6, where q = 4π sin(θ)λ−1 is the modulus of the scattering wave vector (θ the scattering angle and λ the wavelength.). SAXS patterns were analyzed in terms of a global model using the program GAP (Global Analysis Program) developed by Pabst that permitted to obtain relevant structural parameters on the membrane thickness.17 The membrane thickness was obtained through the definition dB = 2(ΖH + σH), where ΖH and σH derived from SAXS curve fitting with GAP.

Scheme 1. Overview of Application of ALA-ES Gels for in Vivo PDT

B


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Figure 1. (A) Photo of ALA-ES gels and ALA-ES solution. (B and C) the SEM and TEM images of the prepared ALA-ES gels. Inset: Single ALAES in gels. (D) TEM of the ALA-ES gel cryosection. (E and F) Diameter distributions and SAXS diffraction profiles of ALA-ES gels and ALA-ES solution. Dynamic light scattering (DLS) and polydispersity index were determined by using the NiComp 380ZLS inspection system (Nicomp Instr. Corp., San Diego, CA, USA) at 25 °C. 2.4. In Vitro Transdermal Penetration of ALA-ES Gels. R6G, instead of ALA, was introduced as a fluorescent label to investigate the penetrability of ALA-ES gels. R6G-loaded ES (R6G-ES, 0.01% w/v) gels and R6G hydroalcoholic (R6G-HA, 0.01% w/v) gels were prepare using the same method as ALA-ES gels and ALA-HA gels. The prepared R6G-ES gel and R6G gels were administrated to human HS tissue in 37 °C humid environments. The excised human HS tissues (6 × 2 × 1 cm, without fatty tissue) were obtained from Chinese plastic surgery patients with informed consents at Shanghai Ninth People’s Hospital (the ethical guidelines of the 1975 Declaration of Helsinki). After administrating for 1, 3, and 6 h, the HS tissues were washed with phosphate buffered saline (PBS, 0.1 M, pH 7.4), sectioned into pieces, embedded by O.C.T compound (SAKURA Tissue-Tek, USA) and processed in a cryostat (Leica CM1950, Germany). Ten-micrometer-thick sections, perpendicular to the surface, were cut and then affixed to polylysine-coated glass slides. These sections were investigated for the distribution of R6G using a confocal laser scanning microscopy (CLSM, Leica DMI6000B, Wetzlar, Germany) at 543 nm excitation/560 nm emission. 2.5. Construction of Rabbit HS Models. Rabbit HS models were established using Morris’s Methods under the approval from the Animal Experimentation Ethics Committee of School of Medicine, Shanghai Jiao Tong University.18 Twenty adult New Zealand white rabbits (2.0−2.5 kg, Si-Lai-Ke, Shanghai, China), were single-housed in a regulated environment (22 ± 2 °C), and provided ad libitum access to feed and water. The rabbits were anesthetized using pentobarbital sodium delivered intravenously via ear veins (30 mg/ kg). Four wounds (10 mm diameter) were created down to bare cartilage and the perichondrium was removed on the ventral surface of each ear. The wounds were more than 10 mm apart from each other to prevent potential interference. After surgery, the rabbits were

returned to their cages and the wounds were covered using sterile gauzes for 1 day. After 30 days, the HS models were established with the thickness ratio above 1.5 between HS tissue and healthy skin. 2.6. In Vivo Transdermal Penetration of ALA-ES Gels. R6G also simulated the transdermal penetration of ALA-ES gels in rabbit HS models. After in vivo penetration for 1, 3, and 6 h, HS tissues in the rabbit models were harvested and washed using phosphate buffered saline (PBS, 0.1 M, pH 7.4). Subsequently, HS tissue pieces were cryostat sectioned (10 μm thickness, perpendicular to the surface), affixed to polylysine-coated glass slides and then incubated with Hoechst 33342 (1:1000 dilution in PBS) for nuclear staining. These sections were investigated for the distribution of R6G and the visualization of HSF using a CLSM at 543 nm excitation/560 nm emission and 350 nm excitation/461 nm emission. The penetration amount of ALA-ES gels in the rabbit HS models was also studied. After in vivo penetration of ALA-ES gels (0.4 mL) and ALA-HA gels (0.4 mL) for 1, 3, 6, and 8 h respectively, HS tissues were harvested, washed using PBS, and cut into small pieces. ALA in the HS tissues was extracted through dialysis in PBS for 24 h, and measured using a modified fluorescamine derivatization approach (shown in S2, Supporting Information). 2.7. Visualization of ALA-ES. The actions of ALA-ES gels in both human HS tissue and rabbit HS models were directly visualized by TEM. After administrated with ALA-ES gels for 6 h, HS tissues were harvested and washed using PBS. Subsequently, HS tissue pieces (1 × 1 × 1 mm) were prefixed using 2.5% glutaraldehyde overnight, dehydrated using graded ethanol for 20 min, and infiltrated and post fixed using 2% osmium tetroxide. After they were embedded in epoxy resin, they were cut into ultrasections perpendicular to the epidermis (70 nm thickness), and then, the sections were examined using a JEM-2010 TEM at an accelerating voltage of 120 kV. 2.8. In Vivo PpIX Accumulation. To investigate the formation and distribution of PpIX in rabbit HS models, ALA-ES gels and ALAHA gels were topical administrated in rabbit HS models for 1, 3, 6, C


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Figure 2. Colocalization images of a cross-section of human HS administrated with R6G-ES gels and R6G-HA gels for 1 (A and D), 3 (B and E), and 6 h (C and F). TEM images of overview of human HS tissue (G), the details in blank frames of melanosomes in epidermis (H), and ALA-ES in dermis (I). The ALA-ES was labeled by black arrows. and 8 h, respectively. The HS tissues were harvested and washed using PBS. After being fixed and dehydrated, the HS tissues were sectioned perpendicular to the surface (10 μm-thickness), incubated with Hoechst 33342, and then examined by CLSM at 450 nm excitation/650 nm emission and 350 nm excitation/461 nm emission. 2.9. In Vivo PDT Protocol. Rabbits were randomly divided into three groups: ALA-ES gel group received topical administration of ALA-ES gels (four rabbits), ALA-HA gel group received a topical administration of ALA-HA gels (four rabbits), and control group received a topical administration of carbopol gels only (two rabbits). After 6 h of administration, HS tissues were irradiated using 635 nm laser (40 mW/cm2, laser spot size of 1.0 cm) generated from a He− Ne laser machine (Shanghai institute of laser technology, China) with a fluence of 5 J/cm2 (10 min). This treatment was administered once a week for 4 weeks and the morphology of the scars in each group was recorded using a digital camera (Canon, Japan). 2.10. Histological Analysis. After the completion of four PDT sessions, the HS samples from all groups containing the rabbit ear fullthickness scar skin and the cartilage were excised. They were fixed in 4% formaldehyde solution, dehydrated using gradient ethanol, embedded in paraffin wax, and sectioned using conventional methods.19 The sections were carried out histological analysis as shown in S3, Supporting Information. 2.11. Statistical Analysis. All data was expressed as mean ± standard deviation (n = 3 independent samples). Statistical significance of obtained results was determined employing t test and analysis of variance (ANOVA) using SPSS 18.0 software (SPSS Inc., Chicago, Illinois, USA) with P < 0.05 as a minimal level of significance.

studied though morphological and physicochemical characterizations. The morphologies of the prepared ALA-ES gels and their cryosections were observed using electron microscopes at first. In SEM image, spherical ALA-ES was clearly visible and densely distributed in the gel matrix according to a topography effect (Figure 1B).20,21 ALA-ES could be observe in the gels as intact spherical or oval vesicles with a homogeneous size of 70−100 nm and characteristic of extended lamellas in TEM image (Figure 1C). Compared with ALA-ES in solution (Figure S1), ALA-ES could keep its structures during the transfer by morphological analysis. Besides, ALA-ES gels were cryosectioned to reveal ALA-ES distribution in the gel matrix in Figure 1D. Abundant intact ALA-ES homogeneously dispersed in the gel matrix. According to the observations of cryosections, ALA-ES in gels had an internal aqueous core, endowing them with excellent entrapment efficiency (77.1% ± 8.2%). Therefore, it did not damage intact ALA-ES during the phase-transfer. Meanwhile, the differences in diameter distribution and lipid bilayer thickness could evaluate the stability of ALA-ES in gels.22 As shown in Figure 1E, the average diameter of ALA-ES in gels had a slight reduction, because some liquid in internal cores transferred into gels. SAXS analysis through GAP showed an almost unchanged dB of ALA-ES gels (40.4 Å) with ALA-ES solution (48.8 Å). Therefore, these slight differences also demonstrated ALA-ES could have its intact and stable structure in gel matrix. Therefore, the similar physicochemical features between ALA-ES gels (size = 83.5 ± 34.5 nm, polydispersity index = 0.22) and ALA-ES solution (size = 82.2 ± 28.4 nm, polydispersity index = 0.17) indicated ALA-ES gels could delivery ALA based on intact and stable ALA-ES. However, whether the intact ALA-ES could penetrate into HS tissue from gel matrix was assessed in transdermal penetration studies.

3. RESULTS AND DISCUSSIONS 3.1. Morphological and Physicochemical Characterization of ALA-ES Gels. During the ALA-ES gel preparation, ALA-ES had a phase-transfer from liquid to solid-like gels. Compared to ALA-ES solution, ALA-ES gels had enough adhesion to realize a continuous ALA topical administration (Figure 1A). It could apply directly on HS, which was a simple and effective approach. However, the stability of ALA-ES in gel matrix influenced the administration, and it was extensively D


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ACS Applied Materials & Interfaces 3.2. In Vitro Penetration Distribution of Study of ALA-ES Gels. Whether ALA-ES gels enhance the penetration of ALA in disorder and hyperplastic human HS tissue was the primary issue in PDT of HS. Therefore, R6G simulate the penetration of ALA-ES gels by its fluorescence distribution in human HS tissue. Figure 2A−F shows the merging of brightfield and fluorescence images with different penetration time. At the first 1 h, the fluorescence was confined in the epidermis in both R6G-ES gels and R6G gels group (Figure 2A and 2D). After 3 h, the red fluorescence filled in whole dermis for R6GES gels group (Figure 2B). In contrast, much weaker fluorescence disturbed in epidermis/dermis junction area in R6G gels group (Figure 2E). With 6 h penetration, the fluorescence had a significant increase throughout the whole scar in R6G-ES gels group (Figure 2C). The corresponding image of R6G gels group showed a relatively low fluorescence intensity in the deeper dermal area (Figure 2F). All images had the concentrated fluorescence in the epidermis, because the gels could realize continuous transdermal delivery. Therefore, ALA-ES gels could overcome the compact dermal texture barrier and deliver more ALA into dermis area. Furthermore, whether ES could penetrate into the human HS tissue was also verified by TEM. Figure 2G was an overview of the dermalepidermal junction area of HS with characterized of keratinocytes in epidermis and collagen fiber bundles in dermis. The amplification the two frames showed important phenomenon: (a) Few ES could be found in epidermis, because ES could easily pass through the disrupted barrier of stratum corneum and epidermis (Figure 2H).23 (b) plenty of ES were observed among fiber bundles in the dermis, meaning that ALA-ES had reached the target area in HS (Figure 2I). The results indicated that ALA-ES could move freely in gel matrix, which might attribute to carbopol gels with the properties of both solid and liquid. Furthermore, the irregular membrane of ES in HS tissue exhibited the deformable property, which could facilitate ES to squeeze through narrow space during penetration (Figure S2).24 In a word, ALA-ES gels, an efficient carrier, could overcome the compact dermal texture barrier and continuously deliver more ALA into human HS tissue. Furthermore, more in vivo studies of ALA-ES gels treating HS were further studied in rabbit HS models. 3.3. In Vivo Transdermal Penetration Study of ALAES Gels. 3.3.1. In Vivo Penetration Distribution Study. R6GES gel also simulated the penetration behavior of ALA-ES gels and the distribution of ALA in rabbit HS models.14 After topical administration of 1 h, the red fluorescence was confined in the epidermis, and no obvious fluorescence was detected in the dermis (Figure 3A). After 3 h, the red fluorescence had increased and filled in dermis (Figure 3B). At 6 h, the fluorescence significantly increased in the whole dermis, suggesting that a significant amount of R6G penetrated into the HS (Figure 3C). In contrast, in R6G-HA gel group, red fluorescence was much weaker in the dermis after 6 h because much less R6G had penetrated into the HS (Figure 3D). Therefore, in vivo penetration studies indicated, once again, that ALA-ES gels could deliver more ALA into the whole dermis of the HS in rabbit HS models. Interestingly, the red fluorescence formed many intense spots in the dermis (Figure 3B and 3C), which might be attributed to that R6G was released from R6G-ES and accumulated in the dermis. After amplifying of the zone of the white frame in Figure 3C, the colocalization displayed that red and blue fluorescence were in same positions, suggesting

Figure 3. (A−C) Fluorescence images of a cross-section of HS administrated with R6G-ES for 1, 3, and 6 h. (D) Fluorescence images of a cross-section of HS administrated with R6G-HA for 6 h. (E) the colocalization of the zone of the white frame in panel C (red from R6G and blue from Hoechst).

that R6G-ES had effectively entered and accumulated in HSF (Figure 3E). It could be explained as that the action between the ES and HSF could overcome cytomembrane barriers and delivered more R6G into HSF. In contrast, few fluorescence spots in R6G-HA group suggested R6G could not be effectively accumulated in HSF by free diffusion (Figure 3D). Therefore, besides overcoming the dermal barriers, ALAES gels were also efficient carrier to delivery ALA in HSF with the actions between ALA-ES and HSF, and the actions were studied by TEM in following. 3.3.2. Visualization of ALA-ES in HS in Vivo. When ALA-ES gels were administrated in rabbit HS models, ALA-ES should penetrate into HS, enter HSF as intact entities, and then release ALA in the cytoplasm. Although R6G could visually simulate the in vivo penetration distribution of ALA in rabbit HS models, the process of ALA-ES penetrating into HS and releasing ALA were still unknown. Therefore, the actions of ALA-ES gels in rabbit HS models were directly observed by TEM (Figure 4). When ALA-ES penetrated into the HS tissue from ALA-ES gels and approached HSF, HSF changed its cytomembrane to endocytose ALA-ES (Figure 4A). Furthermore, the details of the frame in Figure 4A displayed ALA-ES with intact entities and irregular appearances in HS tissue, exhibiting that the intact ALA-ES could fluently squeeze through narrow space during penetration into tissue utilizing its deformable membrane (Figure.4E). After being endocytosed, ALA-ES was taken up by a pinocytotic vesicle and E


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Table 1. Penetration Amount of ALA in Different Penetration Time penetration amount (%) penetration time (h) 1 3 6 8

ALA-ES gel group 22.9 50.8 72.8 61.7

± ± ± ±

3.34 5.23 6.32 2.3

ALA-HA gel group 19.5 34.8 42.7 46.7

± ± ± ±

1.2 4.2 3.3 4.6

the ALA-ES gel group, penetration amount of ALA reached a maximum (46.8% ± 6.32% of applied dose) with 6 h of penetration. However, ALA-HA gels produced a significantly lower amount (32.7% ± 3.3% of applied dose) with the same penetration time. With increased time (8 h), the penetrated ALA demonstrated a reduction for ALA-ES gels but an increase for ALA-HA gels. This result could be explained in following: (a) some ALA converted to PpIX in HSF, because the maximum in the in vivo study was much less than the one in the in vitro study (80.79 ± 3.18% in our prior work);14 (b) ALA-HA with poor penetrability need more time to reach its maximum penetration amount in the HS tissue. Furthermore, the optimal administration time was 6 h. 3.3.4. In Vivo PpIX Accumulation. According to the mechanism of ALA-PDT treatments, ALA in HSF converted to PpIX, which could produce ROS under irradiation.29 Therefore, the formulation, distribution and accumulation of PpIX in HS tissue affected the PDT efficacy, and they were visually investigated using the red fluorescence of PpiX.30 As shown in Figure 5A, few PpIX in HS tissue in control group indicated that most of the PpIX would be converted from ALA. A weak red fluorescence was observed in the dermis after 1 h of administration because of a few ALA being penetrated into HS (Figure 5B). The red fluorescence increased in a partial area of the dermis with 3 h of administration (Figure 5C). With administration of 6 h, the significantly increased red fluorescence distributed in all the dermis because ALA-ES had enough time to penetrate into HS, enter HSF, and release ALA (Figure 5D). However, the red fluorescence became obviously weaker after 8 h due to the PpIX metabolism in HSF (Figure 5E).31 ALA-HA gels were also measured with administration of 3 and 6 h, and the red fluorescence was mainly in epidermis due to poor penetrability of ALA (Figure 5F and 5G). Similarly, PpIX increased with time prolonging in ALA-HA gel groups, however its red fluorescence was much weaker than the one in ALA-ES gel group. Furthermore, Hoechst visualized the cells with blue fluorescence in HS. The result merging red and blue fluorescence clearly showed that the red fluorescence was intense in the blue regions in Figure 5H, suggesting that PpIX did form and accumulated in the cytoplasm of HSF. Meanwhile fluorescent phospholipids (NBD-PC, green) labeled ALA-ES was used to further confirm the penetration action in HS tissues. The green fluorescence was clearly shown in the dermis, indicating ALA had penetrated into tissue (Figure S4A). Furthermore, the colocalization displayed that green and red fluorescence were in HSF, suggesting that ALAES had effectively entered in HSF and produced PpIX. Especially, some green fluorescence were out of red area and close to cell nucleus, which might attribute to that ALA-ES was entering HSF (Figure S4B). To sum up, ALA-ES gels could effectively enhance ALA penetration in HS tissue, enter HSF and release ALA in cytoplasm. Therefore, with administration of 6 h, much more

Figure 4. (A−D) TEM images of the action between ALA-ES and HSF. (A) ALA-ES penetrated into HS and approached the HSF. (B) ALA-ES was taken up by a pinocytotic vesicle. (C) ALA-ES was in the HSF cytoplasm. (D) ALA-ES released ALA in the cytoplasm. (E and F) TEM images of ALA-ES in HS. (G) Schematic diagrams of the action between ALA-ES and HSF. ALA-ES was labeled by black arrows.

entered HSF (Figure 4B). Therefore, ALA-ES entering HSF conformed to a pinocytotic-like mechanism for nanoparticles (less 300 nm) uptake.25,26 Subsequently, after released from the broken pinocytotic vesicle, intact ALA-ES was found in the cytoplasm (Figure 4C). It was speculated that the broken pinocytotic vesicle might be resulted from the fusion of phospholipid bilayers between ALA-ES and the pinocytotic vesicle membrane.27 The intact ALA-ES in HSF had its characteristic membrane in Figure S3. Finally, ALA-ES with fractured membrane suggested that ALA was released in the cytoplasm (Figure 4D). As shown in Figure 4F, ALA-ES, broken at one end, might be induced by the osmotic pressure between the internal aqueous core of ALA-ES and the cytoplasm of HSF.28 Therefore, the process of ALA-ES penetrating into HSF and releasing ALA could be expressed as the schematic diagram in Figure 4G. In a word, the visualization of ALA-ES in the HS clearly expressed the action of ALA-ES gels in rabbit HS model as following: (a) ALA-ES fluently squeeze through narrow space during penetration into tissue utilizing its deformable membrane and (b) ALA-ES enter the cytoplasm by a pinocytotic-like mechanism and then released ALA in the cytoplasm. 3.3.3. In Vivo Penetration Amount Study. The penetration amount of ALA in HS was also a key parameter in PDT. Meanwhile, the relation between penetration amounts and time also could confirm a suitable administration time. Therefore, the penetration amounts of ALA in different groups with different administration time were shown in Table 1. For F


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Figure 5. (A) Fluorescence images of PpIX in control. (B−E) Fluorescence images of PpIX in HS models administrated with ALA-ES for 1, 3, 6, and 8 h. (F−G) Fluorescence images of PpIX in HS models administrated with ALA-HA for 3 and 6 h. (H) Colocalization of panle D. Scale bar = 200 μm. (Red from PpIX and blue from Hoechst.)

Figure 6. (A and B) Appearance changes of HS in different groups before and after four PDT sessions. (C) Fluorescence images of PpIX in HS models after ALA-ES gel-PDT. (D) TEM images of HS after ALA-ES gel-PDT. (E and F) TEM images of ALA-ES in HS.

G


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Figure 7. (A) Masson’s trichrome staining of healthy skin. (B) SEI value of different groups after four PDT sessions. (C) Masson’s trichrome staining of HS after four PDT sessions with different magnification factors.

produce ROS. Under the action of ROS, HSF lead to apoptosis or necrosis. The necrotic HSF was found in tissue with the characteristic of an expansion of the cell volume, organelle swelling, plasma membrane rupture and loss of cell organelle contents (Figure 6D).32 Furthermore, the broken ALA-ES existed in dead HSF, but not in health HS, indicating ALA-ES played a key role in the apoptosis or necrosis of HSF(Figure 6E and 6F). 3.4.2. Histological Analysis. Histological analysis based on Masson’s trichrome staining was used to reveal the changes to further evaluate PDT efficacy (Figure 7).33,34 Compared with healthy skin, HS tissues had different histological structures in overproliferative HSF, irregular collagen fibers, thickened epidermis and regenerative cartilage tissue (Figure 7A and 7C). After four PDT sessions, the HS tissues in ALA-ES gel groups had the flattest epidermis, which was in consistence with the morphological analysis. Furthermore, scar elevation index (SEI) was employed to quantify the improvement in the HS (Figure 7B). It was calculated as the ratio of the total dermal area (the hypertrophied dermis plus the underlying dermis area) to the underlying dermis area.35,36 Compared with control groups, the ALA-HA gel group had a slightly decreased SEI value (1.3), and the ALA-ES gel group showed a larger reduction (from 1.65 to 1.03). Additionally, the microstructural features, including collagen fibers and HSF,

ALA could accumulated in HSF, and then converted into PpIX in the cytoplasm of HSF. Whether ALA-ES gels had significant potential for application in PDT treating HS was studied in following section. 3.4. In Vivo PDT Efficacy of ALA-ES Gels. 3.4.1. Morphological Analysis. Morphological changes could be used to evaluate PDT efficacy in rabbit HS models. After reepithelialization of all wounds, the HSs were formed in the rabbit ears. These HS tissues had dark-red color and a firmer and thicker texture than the surrounding tissue, which were the characteristic of HS (Figure 6A). After four PDT sessions, there were different morphological changes in different groups (Figure 6B). In ALA-ES gel groups, the HS tissues were flattened and softened, and their color had obviously faded. Therefore, the ALA-ES gel groups had an obvious PDT efficacy to improve the color and texture of HS tissues as healthy skin. The HS tissues of ALA-HA gel groups, by contrast, were worse in terms of color and texture. Meanwhile, there was almost no difference in the control groups, and the HS tissues remained red in color and firm in texture. Therefore, the morphological analysis indicate that ALA-ES gels provided the best PDT efficacy to improve the color and texture of HS tissues. Furthermore, more details in tissue were also studied after PDT. As shown in Figure 6C, there was few PpIX in tissue, indicating most PpIX had been consumed to H


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Figure 8. (A) Immunohistochemical TUNEL staining of HS with different magnification factors. (B) TUNEL staining positive cells in dermis. (C) Fluorescence distribution of MMP3 immunoreactivity (green) in HS. (D) Statistics of mean MMP-3 immuno-fluorescence by using ImageJ. * represents P < 0.05.

indicting ALA-ES gel had much better ability in remodeling collagen fibers. Therefore, the studies of histological analysis, including TUNEL and MMP3 expression assay, were consistent with the result of Masson staining analysis. Furthermore, He−Ne Laser (632 nm) has been proven to be ineffective in HSF apoptosis or necrosis in report works14,39,40 In sum up, the results suggested that ALA-ES gels provided the best PDT efficacy to promote HSF apoptosis and remodel the collagen fibers.

were also assessed by magnifying observations. HSF in control groups had notably proliferation and infiltration in the entire dermis. In ALA-HA gel groups, HSF still proliferated and infiltrated in the dermis, suggesting the poor PDT efficacy. In ALA-ES gel groups, HSF had a remarkable reduction, because ALA-ES had excellent efficacy in promoting the apoptosis or necrosis of HSF. Furthermore, the collagen fiber arrangement was also an important indicator in evaluating PDT efficacy. In control group, there was of a large number of dense collagen fibers with a disordered manner arrangement and a bulky appearance. After PDT, the pathological morphology seen in ALA-HA gel group was similar to that in control group, although some blue-stained collagen fibers had decreased. ALA-ES gel group had the best improvement, and the lighter color collagen fibers were slender with a regular and parallel arrangement. Although ALA-ES had excellent PDT efficacy to promote HSF apoptosis in the in vitro study, the apoptotic activity in rabbit HS models was much difference.14,37 Therefore, to confirm the apoptotic activity in HS tissue after PDT sessions, the apoptotic HSF in each group was examined by TUNEL assay (green fluorescence, Figure 8A). There was poor apoptotic activity in the HS tissue of control groups. After PDT treatment, more TUNEL-positive apoptotic HSF were observed in the HS tissues of the ALA-ES gel group and ALAHA gel group. Furthermore, the apoptotic activities were quantified in the same dermal region. Apoptotic HSF in ALAES gel group was much more than the one in ALA-HA gel group (83 ± 9.0% vs 41 ± 9.8%, Figure 8B). Meanwhile, MMP3 expression was a response in remodeling collagen fibers.38 Therefore, the differences of MMP3 expression in different group were also used to evaluate the PDT efficacy, and it also quantified by green fluorescence intensity (Figure 8C and 8D). The MMP3 expression in ALA-ES gel group was approximately 3-fold to the one in ALA-HA gel groups;

5. CONCLUSION In the current study, ALA-ES gels were prepared for highly efficient transdermal delivery of ALA into HS and HSF, and they provided a feasible approach to PDT treating HS. The morphological and physicochemical features indicated ALA-ES was stable in gel matrix. According to in vitro and in vivo transdermal penetration studies. ALA-ES gels could overcome the compact dermal texture and cytomembrane barriers to continuously deliver more ALA into HSF and produce abundant PpIX in the cytoplasm. Meanwhile, according to the visual penetration process in HS models, ALA-ES penetrated into HS tissue utilizing its deformable membrane, entered HSF by a pinocytotic-like mechanism, and then released ALA in the cytoplasm. PDT efficacy was assessed using rabbit HS models. The morphological and histological analysis revealed that ALA-ES gels could improve HS by promoting HSF apoptosis, remodelling collagen fibers and increasing MMP3 expression. The results demonstrated that ALA-ES gels were suitable in clinical treatment of HS. ALA-ES gels would be further investigated for their potential in clinical treating HS to make a substantial progress within the field. I


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b17498.

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Preparation of ALA-ES, quantitative analysis of ALA, and the details of histological analysis (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected] *E-mail: [email protected]. ORCID

Ping Lan: 0000-0002-9285-3259 Jie Song: 0000-0002-4711-6014 Author Contributions □

Z.Z., Y.L., and Y.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by National Natural Science Foundation of China (NSFC) (Grant Number: 81772098, 81801917, 81822024, 21605102, 11761141006), China Postdoctor Science Foundation (Grant Number: 2017M620159), Clinical Research Program of ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (Grant Number: JYLJ027), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (Grant Number: 20152227). Clinical Multi-Disciplinary Team Research Program of ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (Grant Number: 2017-1007). We thank Dr. Conn Hastings, from Liwen Bianji, Edanz Editing China, for editing the English text of a draft of this manuscript.

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Transdermal Delivery of 5-Aminolevulinic Acid by Nanoethosome Gels

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