Chemicals and animals

Protoporphyrin IX, coumarin 6, thiazolyl blue tetrazolium bromide (MTT), and dimethyl sulfoxide were acquired from Sigma-Aldrich. Hexadecyltrimethylammonium bromide (CTAB) and Tetraethyl Orthosilicate (TEOS) were acquired from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). Chloral hydrate was obtained from Macklin (Shanghai, China). Neutral balsam and Tween 80 were procured from Solarbio (Beijing, China). Penicillin/Streptomycin Solution and Trypsin 0.25% solution were acquired from HyClone (USA). RPMI Medium 1640 basic and Fetal Bovine Serum (FBS) were purchased from Gibco (Grand Island, USA). B16 cells were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All other chemicals (analytical grade) used in this study were purchased from Sinopharm Group Co. Ltd (Shanghai, China).

Full thickness porcine skin was obtained from YinXiang Group (Xiamen, Fujian, China). Female nude mice (4–5 weeks old) were acquired from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All in vivo experiments conducted in this research adhered to the protocols approved by the Institutional Animal Care and Use Committee at Xiamen University Laboratory Animal Center (XMULAC20210003).

Preparation and characterization of mSHSPreparation of mSHS

The sponge Haliclona sp. spicules (SHS) were derived from cultured sponge (Haliclona sp.) obtained from Dongshan Bay (Fujian, China). Subsequently, they were extracted, prepared, and purified following the methods outlined in our patent (ZL201610267764.6). Specifically, SHS (100 mg) were washed with NaOH (5 mL, 0.1 mol/L) and HCl (5 mL, 0.1 mol/L) successively in an ultrasonic cleaner for 30 min and then cleaned with deionized water. Subsequently, SHS were ultrasonically cleaned for 30 min using 5 mL acetonitrile, and finally lyophilized with a vacuum freeze dryer. To prepare mSHS, SHS (100 mg) were mixed with deionized water (35 mL), CTAB (80 mg), ammonium hydroxide (5 μL, 25 wt%) and ethanol (15 mL) under stirring for 30 min. TEOS (40 μL) was then added dropwise to the above mixture, followed by stirring at 60 ℃ for 24, 48 or 72 h. Finally, the obtained mSHS were purified by repeated centrifugation (2000 g for 10 min) and washing with deionized water to remove the by-products and then aged overnight at 100 ℃. Finally, to remove the CTAB, mSHS were placed into an ethanolic solution of ammonium nitrate and refluxed for 12 h at 80 ℃ [48,49,50].

Visualization of mSHS

The morphologies and structures of SHS and mSHS were first characterized by a scanning electron microscope (SEM, FEI Quanta 650 FEG, USA). The SHS and mSHS underwent platinum deposition using a sputter coater, resulting in the formation of a thin platinum layer. (JEOL JFC1600, Akishima, Japan). SEM micrographs were taken at an accelerating voltage of 10 kV. The SHS and mSHS were then visualized using a transmission electron microscope (TEM, FEI Tecnai G2 Spirit BioTwin, USA). Prior to analysis, the samples were deposited on copper grids. The samples were analyzed using transmission electron microscopy. Nitrogen adsorption and desorption isotherm were acquired using a Micromeritics ASAP 2010 M system. Prior to measurement, the samples were subjected to degassing at 350℃ for 24 h, and the measurements were conducted at 77 K. Pore size distributions and specific surface areas were calculated from the adsorption branches of the N2 adsorption isotherms utilizing the Barrett Joyner Halenda (BJH) and Brunauer Emmett Teller (BET) methods [31, 51].

The drug loading of mSHS

The method of organic solvent immersion [52] was used to load drugs into the mesoporous layer of mSHS. Briefly, Coumarin 6 or PpIX solutions (1, 2, 5, 10, 20, 30 and 40 mg/mL) were dissolved in dichloromethane. Then, 600 μL of Coumarin 6 or PpIX solution at different concentrations was mixed with 10 mg of mSHS. The mixture was shaken for 12 h using a rotating mixer (QB-210, Haimen Kylin-Bell Lab Instruments Co., Ltd., China). Afterwards, the mixture of mSHS and drug solution was centrifuged, and the excess drug solution was removed. Finally, the drug-loaded mSHS was lyophilized using a vacuum freeze dryer to obtain mSHS loaded with the drug (mSHS@drug, including mSHS@coumarin 6 or mSHS@PpIX). For in vitro or in vivo experiments, PpIX was dissolved in N,N-Dimethylformamide to prepare the PpIX solution, which was also mixed with mSHS (10 mg).

To determine the drug loading capacity of mSHS, 10 mL of ethanol was added to 10 mg of mSHS@drug, which was placed in a shaker for 12 h (28 ℃, 200 rpm). Subsequently, the concentration of coumarin 6 (excitation/emission wavelength = 466 nm/504 nm) or the concentration of PpIX (excitation/emission wavelength = 409 nm/633 nm) in the ethanol solution was determined using a microplate reader. The accuracy of all standard curves was confirmed through the verification of linearity (R2 = 0.9999). The drug loading capacity of mSHS was determined by employing the subsequent formula:

$$}\left( }/}} \right) \, = }*}/};$$

C represents the concentration of durg in ethanol; V represents the volume of the ethanol (10 mL); W represents the weight of mSHS@drug (10 mg).

To visualize mSHS@drug, an appropriate amount of the powders was distributed on a glass slide in a single layer. Then, mSHS@coumarin 6 (excitation/emission wavelength = 466 nm/504 nm) or mSHS@PpIX (excitation/emission wavelength = 409 nm/633 nm) was observed using a confocal microscope (LSM780NLO, Carl Zeiss, Germany).

The drug release behavior of mSHS@drug

The mSHS@drug (PpIX or Coumarin 6) was selected to study the drug release behavior. In brief, a certain amount of mSHS@drug (10 mg) was dispersed into 1L of PBS (200 mM, pH = 7.4) containing 0.5% Tween80. The system was kept away from light and stirred appropriately (600 rpm). Then, 200 μL of the solution was taken at 0, 1, 2, 4, 6, 10, 12, and 24 h, respectively. Following each sampling, the system was supplemented with 0.2 mL of PBS solution containing 0.5% Tween 80. The concentration or content of the drug (PpIX or coumarin 6) in the solutions was assessed utilizing a microplate reader.

Skin drug delivery using mSHS in vitroSkin penetration study in vitro

Full thickness porcine skin was used for the skin penetration study in vitro. Briefly, the subcutaneous adipose tissue beneath the dermis was meticulously excised using a scalpel, while the porcine hair shaft was cut off with the length shorter than 2 mm employing an electric razor. Subsequently, the treated skin was rinsed with deionized water and subsequently stored at a temperature of − 20 °C. Prior to usage, the porcine skin was retrieved from the refrigerator and allowed to thaw at a temperature of 25 °C. The in vitro skin penetration test was performed using Franz diffusion cells (ZhengTong Co. Ltd., China). The skin was punched out in disk samples (3.6 cm). The skin was placed on top of the vertical Franz diffusion cells with the epidermis facing the donor compartment. The receptor compartment was filled with PBS (12 mL, 0.2 M) containing 1% Tween80. The effective penetration area of the diffusion cells was 1.77 cm2. All bubbles between the receptor solution and the skin are completely removed, and then put it in the water bath of 36.5 ~ 37.5 ℃ to maintain the skin surface temperature at 36.5 ~ 37.5 ℃. The skin's conductivity was assessed to evaluate the integrity of the skin barrier according to the method described in previous studies [36].

The application of SHS or mSHS (100 μL, 100 mg/mL) was achieved by massaging (electric massage). The force applied to the skin surface during the massage was about 0.3 N with the application time of 120 s (300 r/min). After the massage, the residual SHS was removed by washing the skin surface with PBS (200 mM) for 3 times. The drug solution (saturated solution of coumarin 6 or PpIX in 30% ethanol, 200 μL) was then topically applied on the surface of the skin. Each experimental formulation underwent triplicate evaluations. All the groups were performed occlusively for 16 h.

At the conclusion of the skin penetration experiment, 1 mL of the solution was extracted from the receptor compartment. Subsequently, the skin surface underwent three washes using PBS.

The experimental method of tape-stripping to remove SC has been described in our previous studies [36, 37]. The SC layers were subjected to ten successive stripping procedures for their removal: The first strip isolated SC 1, while SC 2 was obtained from the second to fifth strips. SC 3 was obtained from the sixth to tenth strips. The viable epidermis layer was surgically separated from the dermis using a scalpel. Subsequently, the remaining dermis layer was fragmented into diminutive fragments. Methanol was employed for the extraction of coumarin 6 (or PpIX) from individual skin layers. The extraction solution was taken out and centrifuged (8000 r/min, 5 min) to precipitate the skin tissue, and the resulting supernatants were collected. Subsequently, the concentration of coumarin 6 (excitation/emission = 466/504 nm) or PpIX (excitation/emission = 409/633 nm) in the receptor phase and various skin layers was assessed by using microplate reader.

Visualization of skin drug penetration

The skin penetration of drug (coumarin 6 or PpIX) was also visualized using confocal microscopy. After the skin penetration experiment in vitro, a small piece of skin measuring 2.5 mm in radius was extracted by punching and promptly preserved in the OCT compound (Sakura Finetek, USA) rapidly. The porcine skin was then cut into a section of 10 μm. Subsequently, the skin section was placed onto an adhesive glass slide, neutral balsam (50 μL) was added to the skin, and a cover slip was then positioned on top. The resulting sample was visualized using a confocal microscope (Carl Zeiss, LSM780NLO, Jena, Germany). The parameters of the confocal microscopy system remained constant for all samples.

Cytotoxicity of PpIX

B16 cells (mouse melanoma cell) were cultivated in RPMI Medium 1640 basic (1 ×) supplemented with 1% 100 × Penicillin/Streptomycin Solution and 10% FBS (fetal bovine serum, Gibco). The cells were incubated at 37 °C in a humidified incubator with 5% CO2. The B16 cells were plated in 96-well plates at a density of 1 × 104 cells/well and cultured for a duration of 24 h. PpIX was dissolved in DMSO at 1 mg/mL and then diluted with water to different concentrations using cell culture media (containing 1% antibiotics and10% FBS). The cells were then incubated with PpIX at different concentration for 6 h away from light. Afterwards, the cell culture medium in 96 well plate was replaced with PBS (0.01 M, pH = 7.4) and B16 cells were exposed to 635 nm radiation at a light dosage of 5 J/cm2 for 10 min. After the irradiation, the PBS solution was replaced with cell culture medium and the cells were incubated in an incubator (37 ℃, 5% CO2) for 24 h in the dark. Thereafter, the cell viability was evaluated utilizing the MTT assay. Finally, the cytotoxicity of different concentrations of PpIX to cells were calculated according to the absorbance.

The internalization of PpIX in cells over time was visualized using a confocal microscope at different time points (2, 4, 6 and 8 h). In brief, B16 cells were inoculated into 96-well plates at a density of 1 × 104 cells/well and subjected to a 24 h incubation period. Following that, the cells were exposed to PpIX at a concentration of 1 μg/mL. Afterward, the culture medium was aspirated, and the cells underwent three washes with PBS. Subsequently, 0.1 mL of PBS was added to each well, and the distribution of fluorescence was examined using a confocal microscope.

Evaluation of mSHS treatment in vivo Evaluation of mSHS efficacy in a melanoma mouse model in vivo

Female nude mice were used to develop the metastatic melanoma mouse model. Briefly, the right forelimb of nude mice underwent sterilization using alcohol, followed by subcutaneous injection of B16 cells into the aforementioned limb of nude mice(4–5 weeks old). The tumor's maximum dimension (L) and minimum dimension (W) were assessed daily using a slide caliper to track tumor progression. Tumor volume can be determined using the formula: V = (tumor width2) × (tumor length) × 0.5. When the tumor volume reached about 500 mm3, a randomly chosen mouse bearing the tumor was selected to evaluate the progression of melanoma.

The mice harboring tumors were subsequently allocated randomly into five distinct experimental groups (4 animals per group), including (1) mSHS group: The tumor of mice was subjected to an even application of mSHS@PpIX (5.6 mg/cm2) for a duration of 3 min, utilizing an applied force of 0.3 N; (2) SHS group: The tumor of mice was subjected to an even application of SHS (5.6 mg/cm2) for a duration of 3 min, utilizing an applied force of 0.3 N. Then a saturated PpIX solution (ethanol) of 200 μL was topically applied to the tumor surface; (3) Injection group: the saturated PpIX ethanol solution (200 μL) was slowly injected into mice by tail intravenous injection; (4) Ethanol group: the saturated PpIX ethanol solution (200 μL) was topically applied to the tumor surface; (5) Control group: the tumor was not treated with PpIX administration and only treated with irradiation.

The treatment period of melanoma in mice was 16 days, and the first day of treatment was recorded as day 0. Before treatment, one tumor-bearing mice was randomly selected from each group to be sacrificed and the tumor was collected. The rest of the mice were treated four times, that is, on day 0, day 3, day 6 and day 9, respectively (Additional file 10: Fig. S10).

The tumors were exposed to 635 nm irradiation at a light intensity of 1 W for a duration of 15 min on day 1, day 4, day 7, and day 10, respectively. The spot size is adjusted to match the size of the tumor, and non-tumor areas are protected from light with aluminum foil. The body weight and tumor volume of the mice were assessed on a daily basis throughout the course of the treatment. After treatment for 16 days, the mice were sacrificed and tumor tissues were peeled off. The tumors were collected before and after treatment, and the tumor tissues underwent sectioning and were subjected to H&E staining. Mice received intraperitoneal injections of pentobarbital sodium for anesthesia during treatment.

Predicting the progression of melanoma

A tumor-bearing mouse was randomly selected for euthanasia, the tumor and its adjacent skin tissue were excised to predict the progression of melanoma. Subsequently, organs and tissues, including the liver, heart, spleen, lymph nodes, and kidneys, were collected from mice harboring tumors and normal mice to examine the potential dissemination of melanoma cells to other parts of the body. The tumor and skin specimens were immobilized in a 4% paraformaldehyde solution. Following a 48-h fixation period, all tissue samples were encased in paraffin blocks. Subsequently, sections measuring 5 µm in thickness were obtained from each block using a paraffin cutter (RM2128, Leica, Germany), and stained with immunohistochemical markers (S-100B) for microscope observation (Carl Zeiss, Axio Imager A2, Jena, Germany).

Safety assessment of mSHS in vivoMeasurement of transepidermal water loss (TEWL)

TEWL was measred to study the effect of mSHS on the skin barrier and the recovery time of skin microchannels in guinea pigs in vivo. Briefly, all female guinea pigs (12 weeks old) were subjected to ether inhalation for anesthesia and the back hair of the guinea pigs was clipped. Further, according to the experimental needs, appropriate regions were selected on the back for numbering and grouping, and three parallel data were set for each group. In the mSHS group, mSHS (10 mg/1.77 cm2) was applied with a massage for 2 min by an electric massager. TEWL measurements were performed over 10 days after mSHS treatment and continued for a period of time until measurements were returned to baseline level.

Skin irritation test

The skin irritation induced by mSHS treatment was assessed using guinea pigs. Following a one-week acclimation period, the guinea pigs underwent anesthesia using ether inhalation and the back hair of the guinea pigs was clipped. Further, according to the experimental needs, appropriate regions were selected on the back for numbering and grouping. mSHS (10 mg/1.77 cm2) was applied on back skin of guinea pigs using an electric massager, applying gentle massage for a duration of 2 min. Subsequently, the degree of erythema and edema of each area was observed and recorded at 0 h, 24 h, 48 h, and 72 h after mSHS treatment. The calculation of the Primary Irritation Index (P.I.I.) was based on the following equation:

$$}.}.}.=\frac1}, 24\mathrm72}+\sum \mathrm1}, 24\mathrm72}}\times \mathrm\times \mathrm}$$

The potential for skin irritation caused by the mSHS was assessed using the Draize dermal scoring criteria, which are explained as follows. The criteria employed for Draize dermal scoring.

P.I.I

Classification

0.0–0.4

No irritation

0.5–1.9

Slight irritation

2.0–4.9

Moderate irritation

5.0–8.0

Severe irritation

In addition, skin specimens from both treated and untreated regions were acquired at 0, 1, 2, 3, 5, and 10 days after the mSHS application. Before collecting skin samples, the guinea pigs were killed. The skin samples obtained were cryopreserved in OCT medium and sliced into sections of 10 μm thickness using a freezing microtome (CM1900, Leica). The samples were immobilized using a 10% formaldehyde solution, stained with hematoxylin and eosin, and then photographed with an optical microscope. The quantification of skin cells within the Area of Interest (AOI) were conducted using Image-Pro Plus 6.0 software.

Scanning electron microscopy (SEM) study

Skin samples were obtained from both treated and untreated areas at different time points following the application of mSHS. The collected skin samples were immersed in a 2.5% glutaraldehyde solution for a duration of 2 h. Subsequently, they were subjected to three consecutive washes, each lasting for 10 min, using a 100 μM PBS solution with a pH of 7.4. The samples underwent dehydration at 4 ℃ using various ethanol concentrations for a duration of 15 min and then transferred to 25 ℃, tertiary butanol was used to replace ethanol. After soaking the sample in tert-butanol for 3 times (10 min each time), the sample was further freeze-dried. Finally, skin samples underwent a process of platinum (30 nm) coating utilizing a Sputter Coater, followed by observation through a SEM (FEI Quanta 650 FEG, USA).

Statistical analysis

Statistical significance was assessed using the two-tailed and unpaired Student’s t-test in Microsoft Excel. The data in this study was reported as mean ± standard deviation (SD). A minimum of three independent samples were examined in all experimental analyses. The p values < 0.05 are considered to be significantly different.