IR-808 loaded nanoethosomes for aggregation-enhanced synergistic transdermal photodynamic/photothermal treatment of hypertrophic scars

Abstract

Synergistic transdermal photodynamic therapy (PDT)/photothermal therapy (PTT) has emerged as a novel strategy for improving hypertrophic scar (HS) therapeutic outcomes. Herein, a near-infrared heptamethine cyanine dye, named IR-808, has been selected as the desirable photosensitizer owing to its PDT and PTT properties. Benefitting from the transdermal delivery ability of ethosomes (ESs), IR-808 loaded nanoethosomes (IR-808-ES) have been prepared as a novel nanophotosensitizer for the transdermal PDT/PTT of HSs. The special structure of IR-808 aggregate distribution in the ES lipid membrane enhances ROS generation and hyperthermia. The in vitro experiments indicate that the IR-808-ES enhances the PDT/PTT efficacy for inducing the HS fibroblast (HSF) apoptosis via the intrinsic mitochondrial pathway. Furthermore, the in vivo transdermal delivery studies reveal that the IR-808-ES efficiently delivers IR-808 into HSFs in the HS tissue. Systematic assessments in the rabbit ear HS models demonstrate that the enhanced PDT/PTT performance of the IR-808-ES has remarkable therapeutic effects on improving the HS appearance, promoting HSF apoptosis and remodeling collagen fibers. Therefore, the IR-808-ES integrates both the transdermal delivery ability and the aggregation-enhanced PDT/PTT effect, and these features endow the IR-808-ES with significant potential as a novel nanophotosensitizer for the transdermal phototherapy of HSs in the clinical field.

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Introduction

Hypertrophic scar (HS) is a serious skin condition caused by excessive proliferation of fibroblasts and over-deposition of the extracellular matrix, which results in persistent dermal fibrosis.¹ The conventional clinical therapies include surgical resection, laser treatment, corticosteroid injection and other non-invasive methods.^2,3 However, low therapeutic efficacy, high recurrence and painful processes remain the major challenges and hinder their clinical applications. Recently, photodynamic therapy (PDT) has become an attractive non-invasive approach for the HS, owing to its promising therapeutic efficacy and reduced side effects.^4,5

PDT is a non-invasive therapy involving a photosensitizer and an appropriate exciting light, where the excited photosensitizer generates the reactive oxygen species (ROS) to induce apoptosis of the over-proliferating HS fibroblasts (HSFs) and remodeling of the collagen fibers.⁶ The most commonly used topical photosensitizer in dermatology is 5-aminolevulinic acid (ALA), approved by the United States Food and Drug Administration (FDA) in 2000.⁷ However, because of its poor skin penetration ability, 5-ALA for the treatment of HSs is still controversial in its clinical application.⁸ Although the nano-formulations can help the transdermal delivery process, the limited tissue penetration depth of the laser (wavelength = 632.8 nm) and insufficient therapeutic efficacy in the HS are still great challenges.⁹

Visible light (from 380 nm to 700 nm) has a shallow penetration depth (<1 mm) owing to its strong absorption by most tissues. In comparison with visible light, the near-infrared light (NIR, 700–1700 nm) exhibits an excellent penetration depth of more than 1 cm, as the longer wavelength undergoes a lower extent of light scattering and tissue absorption.^10,11 Therefore, the NIR window, known as the biological transparency window, demonstrates an exceptional advantage in HS phototherapy. Meanwhile, a novel synergistic treatment strategy, the combination of PDT with photothermal therapy (PTT), has attracted extensive interest in recent years.^12,13 PTT generally requires a photosensitizer for the conversion of the absorbed light into localized heat energy, thus, leading to the ablation of target cells. In addition, hyperthermia can increase the cellular uptake efficiency of the photosensitizers and enhance ROS generation, resulting in improved PDT.^14,15 Therefore, screening the NIR light activated photosensitizer with both PDT and PTT is critical for improving the treatment of HSs.

Recently, a heptamethine cyanine dye, IR-808, has been reported to exhibit a significant photothermal effect and photodynamic properties.¹⁶ The near-infrared excitation wavelength (≈800 nm) exhibits a penetration depth of 7.0–11.0 mm, which can reach the deep dermis. Previous studies have reported its preferential tumour accumulation without chemical conjugation of the target ligands along with the mitochondrial targeting ability.¹⁷ The synchronous PDT/PTT effects and mitochondrial targeting behaviour provide high PDT/PTT efficacy and exhibit a great potential for HS treatment.¹⁴ However, the hydrophobicity of the dye leads to a marked accumulation in the stratum corneum (SC), and thus how to overcome the compact dermal barrier of HS and realize the transdermal delivery to deep dermis present the current obstacles for the topical application of the dye.

Our previous studies have successfully established a nanoethosomal system with a high transdermal delivery efficiency in HS treatment.^18,19 The ethosomal (ES) vesicles can disrupt the barrier function of the SC and squeeze through narrow spaces to reach the dermis. Meanwhile, the small fluorescent organic molecules in their solution state generally suffer from a nonradiative decay due to flexible molecular motions resulting in fluorescence quenching.²⁰ The lipid bilayer can provide an ideal environment to form aggregates and thus achieve an aggregation-induced emission enhancement (AIEE).²¹ Moreover, a few AIE luminogens have been reported to show an enhanced PDT effect in the aggregated state.²² As IR-808 is a liposoluble molecule, the lipid bilayer of the ES increases its solubility, contributes to forming the aggregate state and thus serves as an effective drug carrier theoretically. However, the transdermal delivery efficiency and the membrane stability of ES loaded liposoluble photosensitizers are still unknown.

In this study, the near-infrared photosensitizer, IR-808, was introduced for the synergistic transdermal PDT/PTT of HSs by preparing IR-808 loaded ESs (IR-808-ES). IR-808-ES exhibited the advantages of transdermal delivery of IR-808 and enhanced PDT/PTT with its special structure of IR-808 aggregating distribution in the ES lipid membrane (Scheme 1). The in vitro studies showed that the ES structure could help the accumulation of IR-808 in HSFs. Meanwhile, the intact ES structure played a critical role in improving the PDT/PTT effect and inducing the HSF apoptosis. The in vivo transdermal penetration studies suggest that the ES could enhance IR-808 penetration into the rabbit HS tissue and delivery into the HSFs. Furthermore, the in vivo PDT/PTT evaluation reveal that IR-808-ES showed high PDT/PTT efficacy with decreased scar thickness and collagen remodeling. In conclusion, the results suggest that the ES could facilitate the tissue penetration of IR-808 and enhance the synergistic PDT/PTT effects for HSs. This study selected an ideal photosensitizer and developed a suitable transdermal nanocarrier for further clinical treatment of HSs.

Scheme 1 Schematic illustration of IR-808-ES preparation and its application in the synergistic transdermal PDT/PTT of HSs via aggregation-induced enhancement.

Experimental section

IR-808-ES solution and gel preparation

IR-808 and the carbopol gel matrix were synthesized according to previously reported methods (S1, ESI†).¹⁷ The preparation of IR-808-ES is as follows. Phosphatidylcholine (PC, 6.7% w/v, soybean origin) and IR-808 were dissolved in chloroform in a round-bottom flask. After the solvent was removed using a rotary evaporator, PC with IR-808 was hydrated in a hydroalcoholic solution (ethanol/water, 30 : 70, v/v) overnight. Finally, the mixture was dispersed using an ultrasonic bath for 20 min (300 W), and IR-808-ES was obtained with the final content of IR-808 at 0.33 mM. The fluorescent ES was labeled using 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadi-azol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC, 4 mol%). IR-808-ES gels were prepared by mixing the IR-808-ES solution with the carbopol gel matrix (1 : 1, v/v) in a sealed container at 500 rpm, where the concentration of IR-808 in the IR-808-ES gels was 0.33 mg mL⁻¹. An IR-808 hydroalcoholic solution (IR-808-HA, 30% ethanol, v/v, 0.67 mg mL⁻¹ IR-808) was used to prepare IR-808-HA gels using the same method as with the IR-808-ES gels.

Characterization of IR-808-ES

The morphological features of IR-808-ES were observed using a transmission electron microscope (TEM, JEM-2010 JEOL, Japan). The size distribution was assessed using Nanosight 300 systems. The encapsulation efficiency (EE) of IR-808 was determined by ultracentrifugation and demulsification. IR-808-ES (100 μL) was placed in an ultrafiltration tube and centrifuged (10 000 rpm, 20 min). The free IR-808 (Q_f) was passed through an ultrafiltration tube into a centrifuge tube. Then the total IR-808 (Q_t) was obtained through demulsification by adding ethanol (100 μL) to IR-808-ES (100 μL). Then, the EE was calculated as follows:

EE = (1 − Q_f/Q_t) × 100%.

The in vitro release studies were evaluated using the dialysis method as reported in our previous studies.²³ The fluorescence intensities were obtained using a Synergy H4 hybrid reader (Bio-Tek, USA, λ_ex = 635 nm/λ_em = 730 nm). UV-Vis spectra were obtained using a Varian Cary 50 UV-Vis spectrophotometer (PerkinElmer, USA). The fluorescence spectra were recorded using a Hitachi FL-4100 spectrofluorometer.

Singlet oxygen sensor green (SOSG, ThermoFisher, USA) was employed to evaluate the generation of singlet oxygen (¹O₂). Typically, IR-808-ES or IR-808-HA (1 mL, 10 μM of IR-808 equivalent) and SOSG (final concentration of 2.5 μM) were mixed and irradiated for 120 s (808 nm, 2 W cm⁻²). Then, the ¹O₂ was quantified at 525 nm (λ_ex = 504 nm). Analysis of the heating profiles of IR-808-ES or IR-808-HA (10 μM of IR-808 equivalent) was done immediately after laser irradiation (808 nm, 2 W cm⁻²) using an infrared thermal camera (Thermal Intelligence, Shanghai, China).

In vitro PDT/PTT of HSFs

Cell culture. HSFs were isolated and cultured using a common method: the fresh HS tissue pieces (following the ethical guidelines of the 1975 Declaration of Helsinki approved by the Shanghai Ninth People's Hospital) were digested using collagenase type I (Invitrogen, USA) to achieve a single cell suspension. The HSFs were grown in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) at 37 °C and 5% CO₂. The culture medium was changed every 3 days, and the cells were passaged when 80% confluent. The cells at passage 2 and 3 were used in the following experiments.

Dark cytotoxicity and phototoxicity experiments. For the in vitro cytotoxicity test, HSFs were placed in 96-well plates at a density of 5 × 10³ cells per well. After 24 h of incubation, the cells were treated with IR-808-HA or IR-808-ES diluted in a complete medium (from 0 to 100 μM) for 24 h. For the in vitro phototoxicity test, the cells were treated with IR-808-HA or IR-808-ES (from 2 to 10 μM) for 2 h and then irradiated as per PDT/PTT procedure (808 nm, 120 s, 2 W cm⁻²). The cell viability was then measured using a cell counting kit-8 (CCK-8, Dojindo, Japan) following the manufacturer's instructions.

In vitro cell uptake and localization. HSFs were placed in 12-well plates at a density of 5 × 10⁴ cells per well and incubated for 24 h. The cells were treated with IR-808-HA or IR-808-ES (from 2 to10 μM, from 1 to 6 h). After washing with PBS three times, the cell suspension was run through a CytoFLEX Flow Cytometer (Beckman Coulter, USA). The fluorescence signal of IR-808 was measured at 750 nm (λ_ex = 635 nm) and data analysis was performed using the FlowJo 7.6 software. HSFs were seeded on coverslips in 12-well plates at a density of 5 × 10⁴ cells per well and incubated for 24 h. Then, the HSFs were incubated with NBD-labelled IR-808-ES for 2 and 6 h, or co-incubated with IR-808-ES and MitoTracker™ Green FM (Invitrogen™, USA). The fluorescence signals were detected by CLSM at 539 nm (λ_ex = 466 nm) for NBD, 516 nm (λ_ex = 490 nm) for MitoTracker Green, and 730–800 nm (λ_ex = 630 nm) for IR-808. All data were analyzed using the LAS AF software.

Intracellular ROS generation. HSFs were placed in 12-well plates at a density of 5 × 10⁴ cells per well and incubated for 24 h. After treatment with IR-808-HA or IR-808-ES for 2 h, the cells were incubated with 10 μM of DCFH-DA (AAT Bioquest, USA) for 20 min and then washed three times with PBS. Following irradiation (808 nm, 120 s, 2 W cm⁻²), the cells were washed 2 times with PBS and the signal was measured using a flow cytometer or CLSM at the FITC filter set.

PDT/PTT synergistic effect evaluation. To identify the PDT/PTT synergistic effect, the PDT effect and PTT effect were evaluated separately. Cells were treated with N-acetyl-L-cysteine (5 mM, Sigma, USA) for 24 h to eliminate the ROS, or were irradiated (808 nm, 120 s, 2 W cm⁻²) on ice to eliminate the PTT effect. After irradiation, the cells were washed 2 times with PBS and incubated with 200 μl working solution prepared according to the protocol of the calcein-AM/PI double stain kit (Dojindo, Japan) at 37 °C for 15 min. The fluorescence signals were measured using a fluorescence microscope following the manufacturer's instructions.

Apoptosis and necrosis assay. HSFs were seeded on coverslips or directly in 12-well plates at a density of 8 × 10⁴ cells per well and incubated for 24 h. After 2 h of incubation with IR-808-HA and IR-808-ES, the cells were irradiated (808 nm, 120 s, 2 W cm⁻²). The detection followed the protocol of the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, USA) using a CLSM or flow cytometer at 525 nm (λ_ex = 488 nm) for Annexin-FITC, and at 615 nm (λ_ex = 535 nm) for PI. The western blot procedure was conducted as per the protocol (S2, ESI†).

In vivo PDT/PTT studies

Construction of the rabbit HS model. The rabbit HS models were constructed using Morris's Method (S3, ESI†) under approval from the Animal Experimentation Ethics Committee at the Shanghai Jiao Tong University School of Medicine. The rabbits were raised separately under controlled temperature and humidity. Before the excision of the HS, the rabbits were euthanized by injecting an overdose of pentobarbital sodium.

In vivo transdermal penetration studies. IR-808-HA or IR-808-ES gel was applied on the formed hypertrophic scar for 3 h. HS tissues were harvested and washed using PBS (pH = 7.4). HS tissue pieces were cryostat sectioned (10 μm thickness, perpendicular to the surface), affixed to polylysine-coated glass slides and then incubated with DAPI for nuclear staining. These sections were investigated for the distribution of IR-808 and the visualization of HSFs using a CLSM at 461 nm (λ_ex = 358 nm) for DAPI and at 730–800 nm (λ_ex = 630 nm) for IR-808. IR-808-ES in HS tissue was directly visualized using a TEM (S4, ESI†).

In vivo treatment protocol. Rabbits were randomly divided into three groups: the IR-808-HA group that received topical administration of IR-808-HA gels (n = 8), the IR-808-ES group that received a topical administration of IR-808-ES gels (n = 8), and the control group that received a topical administration of only the carbopol gels (n = 4). After 3 h of administration, HS tissues were irradiated for 120 s (808 nm, 2 W cm⁻²). 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).

In vivo PDT/PTT assessment. After the completion of the four therapeutic sessions, the HS samples were excised from all of the groups and consisted of full thickness scarred skin and cartilage from the rabbit ears. In vivo ROS generation was visualized with a DHE (dihydroethidium) assay kit (Sigma, USA) immediately after the laser irradiation. Three days after the last treatment, Masson's trichrome staining for histopathological analysis, Sirius red staining for collagen and immunohistochemical staining of the TUNEL, MMP-3 and TGF-β with respective antibodies were conducted. The western blot procedure was conducted as per the protocol (S2, ESI†).

Statistical analysis

All of the data were shown as mean ± standard deviations. GraphPad Prism 9.0 (GraphPad Software, Inc.) was used for the statistical analysis. One-way ANOVA followed by Dunnett's test was performed for multiple comparisons with P < 0.05 as the minimum level of significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Results and discussion

Characterization of IR-808-ES

The morphology of the prepared IR-808-ES was first characterized using TEM (Fig. 1A). As observed in the TEM images, IR-808-ES established intact spherical vesicles with a homogeneous size. A high-magnification image with more details exhibited multiple lamellas in even concentric patterns, indicating that IR-808-ES formed the classic multilamellar nanovesicle structure.²⁴ The size distribution analysis provided the precise particle diameter as 107.2 ± 47.3 nm (Fig. 1B). The encapsulation efficiency (EE) of IR-808-ES was measured during preparation and the results show an efficient loading with an EE value of 68.74%.

Fig. 1 (A) TEM image of IR-808-ES with details; (B) diameter distributions of IR-808-ES; (C) release profiles; (D) UV-Vis absorption spectra; (E) fluorescence spectra of IR-808-HA and IR-808-ES; (F) fluorescence signal of SOSG; (G) heating curve of PBS, IR-808-HA and IR-808-ES after laser irradiation; and (H) thermal images of IR-808-ES after laser irradiation.

In IR-808-ES, IR-808 is a hydrophobic molecule loaded between the bilayer membrane. Therefore, the stability of IR-808-ES is an important factor and herein it was evaluated by the extent of the release of IR-808 from IR-808-ES (Fig. 1C). In the release profile of IR-808-HA, over 60% of free IR-808 was observed to be rapidly released to the outside medium within 1 h, while IR-808-ES showed a relatively slow drug release pattern with 45% and 60% release of free IR-808 within 1 h and 2 h, respectively. An equilibrium release was achieved after 2.5 h. The observed results indicate that IR-808 was effectively encapsulated in the IR-808-ES.

The influence of the ES on the physico-chemical properties was further studied. Compared with IR-808-HA, IR-808-ES showed a red-shift of the absorption peak (from 705 to 800 nm) with a slight increase in the absorption strength and a significantly increased fluorescence emission intensity at a wavelength of 820 nm (Fig. 1D and E). Being a small fluorescent organic molecule, IR-808 in an aqueous solution suffers from fluorescence quenching. These observed optical enhancements show that the ES structure helps IR-808 to form the aggregate state and to exhibit the aggregation-induced emission enhancement (AIEE) phenomenon.^21,22 The assembly of IR-808 in the ES prevented intramolecular rotation or vibration, and thus realizes AIEE through the restriction of intramolecular motions (RIMs).²⁵

As a photosensitizer, the photodynamic and photothermal characteristics of IR-808 are critical for therapeutic effects. Herein, SOSG was used to selectively detect the ¹O₂ generation under laser irradiation, which is the main therapeutic agent involved in the PDT process (Fig. 1F). Compared with IR-808-HA, the singlet oxygen generation was observed to increase by 1.4-fold. Meanwhile, the photothermal effect was evaluated by detecting the temperature increase of the solution under rated laser intensity and at different times (Fig. 1G and H). The solution temperature of IR-808-ES raised rapidly to over 40 °C within 30 s followed by a decrease to 35 °C in 2 min, while the temperature of the IR-808-HA group slightly increased to 35 °C in 2 min. The fall of the temperature curve was probably attributed to the rupture of the ethosomal structure caused by the rising temperature and molecular motion which further attenuated the hyperthermal effect. These results suggest that the AIEE of the ES could not only decrease the luminescence quenching but also enhance the PDT/PTT effect.

In summary, the ES was confirmed to successfully encapsulate the hydrophobic IR-808 and increase the absorption and emission in the NIR region via the formation of the aggregate state. Moreover, the AIEE enhanced ROS generation and increased the photothermal effect, which was desirable for realizing a satisfactory PDT/PTT effect.

In vitro PDT/PTT of HSFs

In vitro cytotoxicity and photocytotoxicity. HSFs were incubated with different concentrations (0–100 μM) of IR-808-HA or IR-808-ES for 24 h without laser irradiation to investigate the dark cytotoxicity (Fig. 2A). Both IR-808-HA and IR-808-ES showed no significant cytotoxicity up to 10 μM, with the cell viability of 92.74% and 91.70%, respectively. With the concentration increasing from 20 μM to 100 μM, IR-808-ES generated more cytotoxicity than IR-808-HA, which could be attributed to the liposolubility of the ES structure and thus a probable increased cellular uptake and accumulation of IR-808. Therefore, the following in vitro experiments were conducted at concentrations below 10 μM.

Fig. 2 (A) Dark cytotoxicity of IR-808-HA and IR-808-ES; (B) phototoxicity of IR-808-HA and IR-808-ES with laser irradiation; (C) CLSM images of HSFs cultured with NBD-labelled IR-808-ES for 2 h and 6 h; (D) mitochondrial localization of IR-808-ES in HSFs by co-staining with Mito-tracker; (E) flow cytometry and CLSM results of ROS generation with infrared thermal images in different formulation groups after laser irradiation; (F) fluorescent images of PI stained HSFs (dead cells) under different treatments of IR-808-ES; (G) light microscopy images, CLSM images and flow cytometry results of HSF apoptosis; and (H) expressions of cleaved caspase-3, cleaved caspase-9 and Cytochrome c in HSFs in different formulation groups after laser irradiation.

The in vitro photocytotoxicity of IR-808-HA and IR-808-ES (0–10 μM) was investigated after incubation for 2 h (Fig. 2B). The cell viability of the IR-808-ES group significantly decreased after laser irradiation from 6 μM, with over 80% of the decrease detected at 10 μM. While the IR-808-HA group did not exhibit any significant effect on the cell viability. Thanks to the aggregate state of IR-808 in ES, the enhanced PDT/PTT effect induced significant phototoxicity. The effect of single PDT or PTT is investigated in the following section.

Cellular uptake and localization. Cell affinity was observed by quantifying the cellular uptake of IR-808 using flow cytometry. After incubation with different concentrations of IR-808-HA or IR-808-ES (2–10 μM) for 6 h, the percentage of the positive HSFs (%) (corresponding to HSFs with a fluorescent signal from IR-808) increased from 80.76% to 95.61% and 84.74% to 97.53%, respectively, indicating that the cell accumulation was dose-dependent (Fig. S1A†). For both groups at 10 μM, the positive HSFs were observed to be over 95%, which indicates a promising cell affinity. Furthermore, the median fluorescence intensity (MFI), corresponding to the amount of accumulated IR-808 in HSFs, was measured at different incubation periods (at 10 μM). The MFI value of IR-808-HA varied with a peak at 2 h of incubation, while the MFI value of IR-808-ES slightly increased with the incubation period (Fig. S1B†). This phenomenon indicates that the ES structure contributed to the accumulation of IR-808-ES in HSFs.

The IR-808-ES accumulation was further investigated using NBD-labelled ES with incubation periods of 2 and 6 h (Fig. 2C). The intense red (IR-808) and green (ES) fluorescence signals at 2 h indicate that IR-808 was delivered in HSFs by IR-808-ES, while the decayed green fluorescence signal at 6 h suggests the degradation of the ES structure within 6 h. Therefore, based on the results of the cellular uptake, the following experiments were conducted with an incubation period of 2 h. As IR-808 is a mitochondria-targeted photosensitizer, the mitochondrial targeting property of IR-808-ES was investigated using a mitochondrial tracker (Mito-tracker Green) (Fig. 2D).¹⁷ The high overlap of the red fluorescence of IR-808 with the green fluorescence of Mito-tracker, indicates that the mitochondrial targeting property was not affected.

Single and synergistic PDT/PTT effects. The PDT/PTT effect depends on both ROS generation for PDT and hyperthermia for PTT. Herein, the intracellular ROS generation was first evaluated by DCFH-DA, which would be converted to the fluorescent DCF (green) by the generated ROS (Fig. 2E). Compared with the control group, the IR-808-HA and IR-808-ES groups both generated ROS, while IR-808-ES showed a higher ROS generation rate as revealed by flow cytometry and CLSM images. Meanwhile, the photothermal efficiency of IR-808 after 2 h of incubation with HSFs was measured. Thanks to the accumulation in HSFs, the solution temperature of IR-808-ES increased to 43 °C after irradiation, which reached the thermal damage threshold of human fibroblasts and exceeded the temperature required for PDT enhancement.^26,27

To further study the single and synergistic PDT/PTT therapeutic effects, laser irradiation was conducted under ice incubation for single PDT and HSFs were pretreated with N-acetylcysteine (NAC, a ROS inhibitor) for single PTT. The phototoxicity was investigated by calcein AM and propidium iodide (PI) co-staining (Fig. S2† and Fig. 2F). The red fluorescence from PI revealed the dead cells, and the higher mortality of the PDT/PTT group than the single PDT or PTT groups suggested that the synergistic PDT/PTT effect contributed to the enhanced phototoxicity of IR-808-ES. In addition, compared with the result obtained after 2 h of incubation, the cell mortality of the 6 h group obviously decreased with the degradation of the ES. These described results show that the accumulated IR-808-ES in HSFs still retained the aggregate form and showed an enhanced synergistic PDT/PTT effect. Moreover, the degradation of the ES along with the attenuated therapeutic effect confirmed its critical role.

HSF apoptosis analysis. The mechanism of the induced phototoxicity was next investigated by CLSM and flow cytometry with Annexin V-FITC/PI co-staining (Fig. 2G). After 2 h of incubation and laser irradiation, the HSFs in the IR-808-ES group presented the plasma membrane retraction and membrane blebs in the bright field images, meanwhile the CLSM images showed the membrane damage (green fluorescence of Annexin V-FITC) and the dead cells (red fluorescence of PI). While HSFs in the control and IR-808-HA groups showed eumorphism. The apoptotic rate was quantified by flow cytometry. The early and late apoptotic rates were 34.4% and 42.9% for the IR-808-ES group, respectively. The western blot analysis revealed that cleaved caspase-3, cleaved caspase-9 and Cytochrome c expression were increased, confirming that the phototoxicity of IR-808-ES induced cell apoptosis via the intrinsic mitochondrial pathway (Fig. 2H).

In vivo transdermal delivery studies

Transdermal drug delivery is a critical process in PDT/PTT for HSs. Herein, the transdermal penetration and distribution of IR-808 were evaluated by CLSM using a rabbit HS model. In the previous studies, the carbopol gel had been introduced for continuous topical administration without having any influence on drug delivery and release.¹⁹ Herein, the IR-808-HA gel and IR-808-ES gel have been used for in vivo studies. After topical administration for 3 h, in the IR-808-ES group, the red fluorescence of IR-808 was observed to be evenly distributed in the entire dermal layer (Fig. 3A and B). In addition, co-staining with DAPI confirmed the accumulation in the HS tissue. In the IR-808-HA group, although ethanol could disrupt the lipid organization in the SC, IR-808 mainly accumulated in the epidermis due to its liposolubility. The semi-quantitative analysis of the red fluorescence showed that IR-808 accumulation in the dermis of the IR-808-ES group was 12-fold higher than that in the IR-808-HA group (Fig. 3C), which indicates that IR-808-ES possesses a remarkable transdermal delivery ability. Furthermore, the histological characterization of in vivo delivery were explored in detail using TEM (Fig. 3D). IR-808-ES was observed to penetrate through the dense collagen fibers into the deep dermis. It was suggested that the transdermal ability could be attributed to the remarkable deformability and fluidity of the vesicle membrane and confirmed the transdermal penetration via an intercellular pathway.²⁸ The framed and enlarged details of IR-808-ES illustrated the deformed but intact vesicles in both extracellular and intracellular matrices, showing that IR-808-ES has fully entered HSFs, which contributed to reserve the AIEE effect and enhance the following PDT/PTT effect. To sum up, compared to IR-808-HA, IR-808-ES shows the desired transdermal penetration ability and retains the structural integrity during delivery into HSFs.

Fig. 3 (A) Schematic diagram of in vivo transdermal administration to the rabbit HS model; (B) CLSM images and (C) semi-quantification of IR-808 fluorescence signal in the HS in vivo treated with IR-808-HA and IR-808-ES; and (D) TEM images of IR-808-ES in HS tissue and HSFs in detail.

In vivo PDT/PTT efficacy

In vivo PDT/PTT efficacy was assessed using the rabbit HS models (Fig. 4A). After re-epithelialization of the wounds and formation of the HS, the PDT/PTT was conducted once a week. Before treatment, the HS of all of the groups showed a dark-red color and thick texture. After 4 therapeutic sessions, compared to the control group, the HS tissues of IR-808-ES exhibited a faded color and flattened appearance, while the IR-808-HA group revealed a lesser degree of improvement (Fig. 4B). The scar elevation index (SEI), the ratio of the total HS tissue thickness to the normal skin thickness, was calculated to quantify the improvement (Fig. 4C). The SEI value of the IR-808-ES group was observed to significantly decrease from 1.7 to 1.1, indicating a more efficient therapeutic effect.

Fig. 4 (A) Schematic diagram of the in vivo PDT/PTT; (B) appearance changes after PDT/PTT of different groups; (C) SEI values of different groups after PDT/PTT sessions; (D) Masson's trichrome staining; (E) Sirius red staining; and (F) statistical analysis of the ratio of collagen I to collagen III of HSs after PDT/PTT sessions.

The histological analysis based on Masson's trichrome staining as well as Sirius red staining was chosen to observe the collagen deposition in the HS tissue (Fig. 4D and E). The control group showed abundant collagen deposition with an uneven arrangement of the collagen fibers. After treatment, the collagen deposition remarkably decreased in the IR-808-ES group and was remodeled parallel. Although the IR-808-HA group also showed a small extent of the therapeutic effect, the collagen fibers still showed a bulky appearance. The Sirius red staining further showed the distribution of collagen I (bright yellow) and collagen III (green) through polarized light observation. During the process of wound healing, the ratio of type III to type I collagen plays a crucial role, in which the higher ratio results in reduced scar formation.²⁹ In the control group, the major collagen fibers appeared yellow in color, indicating the major deposition of collagen I. The ratio of collagen I to III significantly decreased in the IR-808-ES group, suggesting the therapeutic efficacy of IR-808-ES leading to improve collagen deposition (Fig. 4F).

Although IR-808-ES showed a significant enhancement of PDT and PTT in the in vitro studies, the in vivo ROS generation and photothermal effects were different because of the different transdermal delivery ability and drug accumulation in the HS tissue. The in vivo ROS generation and distribution corresponded to the distribution of IR-808, confirming the ROS generation from IR-808 and the retention of the PDT enhancement after penetration in the HS tissue (Fig. 5A and S3†). A superficial temperature elevation was observed immediately after laser irradiation using an infrared thermal camera (Fig. 5B). The control group showed a minor increase from 29.7 °C to 38.7 °C, while IR-808-HA and IR-808-ES groups were elevated to 49.3 °C and 46.1 °C, respectively. The contrasting results from the in vitro studies were due to the extreme accumulation of IR-808 in the epidermis, as revealed by the in vivo transdermal delivery studies. In summary, IR-808-ES retained both enhanced PDT and PTT effects after the transdermal penetration and distribution, while IR-808-HA showed extremely high temperatures because of the excessive accumulation in the epidermis, which would lead to the risk of epidermal damage.

Fig. 5 (A) In vivo ROS generation in HS tissue; (B) infrared thermal images after laser irradiation of different groups; (C) TUNEL staining and statistical analysis of HS tissue; (D) immunohistochemical and statistical analyses of TGF-β; (E) MMP-3 in HS tissue; and (F) expression of HSP70, HSP90, cleaved caspase-3 and cleaved caspase-9 in HS tissue.

The TUNEL analysis was conducted to further investigate the in vivo apoptosis of HSFs (Fig. 5C). Few TUNEL-positive apoptotic HSFs were observed in the control group. In the IR-808-HA group the apoptotic activities were limited mainly in the epidermis and superficial dermis, while the apoptotic HSFs in the IR-808-ES group were distributed throughout the entire dermis. The described results corresponding to ROS generation and temperature elevation confirmed their therapeutic effects. Meanwhile, the expression of TGF-β and MMP-3 in the HS tissue, the indicators of HSF proliferation and collagenolytic activity, was also evaluated (Fig. 5D and E).³⁰ The lowest TGF-β level in the IR-808-ES group indicated the remarkable inhibition of HSF proliferation via generated ROS and hyperthermia. The significantly increased MMP-3 expression in the IR-808-ES group confirmed the influence of the enhanced PDT/PTT effects on collagen metabolism, which was consistent with the finding from Masson's staining. The western blot analysis showed that cleaved caspase-3 and cleaved caspase-9 were increased after PDT/PTT of IR-808-ES, confirming that the HSF apoptosis in vivo was still mediated by the intrinsic mitochondrial pathway (Fig. 5F). Heat shock protein 90 (HSP90) played an important role in the cell response to hyperthermia, hence the increased expression in the IR-808-ES group and no modification in the IR-808-HA group indicating that the limited hyperthermia in the epidermis provided no therapeutic effects. Meanwhile, some studies reported that HSP90 reduced hypothermic efficacy, suggesting that HSP90 could be a potential therapeutic target to overcome the phototherapy resistance.³¹

Overall, IR-808-ES was confirmed to exhibit an enhanced PDT/PTT therapeutic effect for the HS in in vivo studies by promoting the HSF apoptosis and remodeling the collagen fibers.

Conclusions

In this study, IR-808-ES was prepared as a novel nanophotosensitizer for an enhanced synergistic transdermal PDT/PTT for HSs. The morphological analysis and release profile confirmed the successful encapsulation of IR-808 in the ES. The physicochemical features indicated that the ES induced the AIEE effect of IR-808 and enhanced the PDT/PTT effect. The in vitro studies revealed the synergistic PDT/PTT effect via inducing the apoptosis of HSFs and confirmed the critical role of the ES structure in the PDT/PTT. As revealed by the in vivo transdermal penetration studies, IR-808-ES could penetrate evenly to the deep dermis via the deformable membrane and enter HSFs with an intact vesical structure. The in vivo PDT/PTT assessment in the rabbit ear HS model showed the enhanced therapeutic effects in improving the HS appearance, promoting HSF apoptosis and remodeling collagen fibers. Therefore, this work suggests IR-808-ES as an efficient photosensitizer for the transdermal PDT/PTT of HSs. Next, IR-808-ES will be further investigated for its potential in clinical applications.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (81772098, 81801917 and 81801918), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20152227), the Clinical Multi-Disciplinary Team Research Program of ninth People's Hospital, the Shanghai Jiao Tong University School of Medicine (20171007), the Cross Research Project of Ninth People's Hospital, the Shanghai Jiao Tong University School of Medicine (JYJC202009), the Shanghai Health Industry Clinical Research Special Project (20204Y0443) and the Shanghai Municipal Key Clinical Specialty (shslczdzk00901). The authors would like to express their gratitude to EditSprings for the expert linguistic services provided.

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Footnotes

† Electronic supplementary information (ESI) available: Additional experimental details, materials, and methods; flow cytometry results of cellular uptake; fluorescent images of calcein AM/PI co-staining; fluorescent images of in vivo ROS generation. See DOI: 10.1039/d1bm01555a

‡ These authors contributed equally to this work.

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