Enhanced intradermal delivery of Dragon's blood in biocompatible nanosuspensions hydrogel patch for skin photoprotective effect

Dragon's Blood is a member of the Chinese medicinal herb, having anti‐oxygen and anti‐inflammatory activity for the photoprotective effect. However, the poor water solubility of raw Dragon's Blood powder has limited its intradermal delivery process. In this study, we evaluated nanosuspensions to enhance intradermal delivery of Dragon's Blood exerting a photoprotective effect. The prepared nanosuspension was added to a composite hydrogel patch matrix for better skin application. In the present research, we used biocompatible materials hyaluronic acid and amino acid surfactants as nanosuspension stabilizers and agar/gelatin/sodium polyacrylate as hydrogel patch matrix. The prepared Dragon's Blood nanosuspension had a particle size of 447.0 ± 48.6 nm. The micro‐structures morphology and viscoelasticity characteristics by SEM and rheological testing confirmed a sufficient crosslinked hydrogel network. The skin retention amount of Dragon's Blood nanosuspension was 1.48 times of raw Dragon's Blood powder water suspension, and the skin penetration amount of Dragon's Blood nanosuspension was only about 1/3 of Dragon's Blood DMSO solution. In the UVB‐irradiated HaCaT cell phototoxicity model, Dragon's Blood nanosuspension also significantly increased cell viability by about 1 time of the model group and decreased the production of reactive oxygen species about 1/2 times of model group. In vivo safety and efficiency evaluation experiment illustrated that DB‐NS hydrogel patch processes have favorable safety and photoprotective effect with no skin irritancy and phototoxicity. Furthermore, DB‐NS and DB‐NS hydrogel patches could protect skin from UVA and UVB irritating skin reactions. Overall, our study of the combined use of biocompatible and biodegradable materials as excipients of nanosuspension and hydrogel patch could be used as an effective additive of Intradermal delivery and skin photoprotection.


| INTRODUC TI ON
Dragon's Blood (DB) origins from Dracaena cochinchinensis (Lour.) S.C. Chen (Yunnan, China) is a dark red resinous exudate powder. As a member of the Chinese medicinal herb, DB has been traditionally used for the topical therapy of wounds, fractures, and ulcers. 1 DB is composed of massive bioactive flavonoids (39.98% ~ 49.87%), including 4′-hydroxy-2,4-dimehoxydihydrochalcone (loureirin A), 4′-hydro xy-2,4,6-trimehoxydihydrochalcone (loureirin B). 2 Dihydrochalcone is one of the flavonoids, which contain a 1,3-diphenylacetone skeleton and do not have a-b double bond like most flavonoids. Their antioxidant activity does not reside only in the hydroxyl groups of ring A but is also enhanced by the carbonyl group. 3 The dihydrochalcones are mainly responsible for photoprotection. As a commonly accepted opinion, oxygen free radicals are involved in cutaneous photo-damage. 4 Ultraviolet (UV) irradiation leads to skin photodamage, including erythema of sunburn, premature skin photoaging, and even skin tumors. Especially, UVB irradiation leads to the increment of cellular levels of reactive oxygen species (ROS). The excess ROS destroys epidermal and dermal cells' lipids, proteins, and nucleic acids.
Despite DB possessing a relatively higher potential for skin photoprotection, there remains a challenge that achieving the intradermal delivery of DB exerts the photoprotective effect. 5 As illustrated in the Graphical abstract, the skin could be mainly divided into two layers: epidermis and dermis. The epidermis is characterized by four layers: stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. A highly hydrophobic physical barrier layer created by the stratum corneum binds with the stratum granulosum, which prevents the skin penetration of substances applied to the stratum corneum. Thus, the rate-limiting step in substance skin penetration is commonly thought of as traversing the stratum corneum. 6 The dermis consists of three layers: papillary dermis, reticular dermis, and hypodermis. Skin appendant structures are embedded in the dermis, such as the hair follicles, sweat glands, blood vessels, lymphatic vessels, and nerve fibers. 7 There are three defined routes of drugs or active ingredients for skin penetration, including intercellular lipid route; transcellular route; appendages (hair follicles, sweat duct) route. The primary way for lipophilic compounds is the intercellular lipid route; small hydrophilic molecules generally favor the transcellular route; charged molecules and particulate systems trend to the pilosebaceous unit route. 6 Intradermal delivery refers to the delivery of drugs or active ingredients to possess permeability across the stratum corneum (SC) but impermeability into the hypodermis. The permeability across the SC enables drugs to enter into the skin, exerting the active effect, while the impermeability into the hypodermis limits the possibility of side effects. Over-permeation of active agents, on the one hand, reduces their effectiveness; on the other hand, leads to systemic circulation inducing adverse effects. [8][9][10] In recent years, nano-particulate-based drug carriers have gained increasing attention for effective skin drug delivery. Compared to other approaches, they could enhance the penetration of drugs across the skin barrier without raising obvious safety problems. However, they suffer from a few shortfalls, (1) poor drug loading, (2) instability for long-term storage, and (3) a high-content surfactant, leading to the potential of skin irritation. 7,[11][12][13][14][15] Nanosuspensions (NS) are pure drug particulate systems stabilized by stabilizers (polymeric or surfactant based), possessing a particle size range from 1 to 1000 nm. Generally, drug nanoparticles are apt to aggregate to decrease the Gibb's free energy. While stabilizers absorbed on the surface of drug nanoparticles could reduce the aggregation by electrostatic repulsion (ionic surfactants) or steric repulsion (polymers and nonionic surfactants). 16 With regard to the advantages of NS, NS is not only economical and safe but also practical and efficient. Firstly, NS is easy for manufacturing by topdown and bottom-up methods. Secondly, it has a high drug loading and requires a relatively low quantity of stabilizer as compared to other nano-particulate-based drug delivery systems, which contributes to its economic and safe features. Finally, more importantly, NS could enhance the intradermal delivery of drugs, which depends on the following three aspects: (1) the large surface area facilitates dissolution rate; (2) concentration gradient forming from the stratum corneum surface to stratum corneum interior skin layers enhances the diffusion pressure; and (3) better adhesion property of nanosuspension in covering skin surface. 17,18 Specifically, it was demonstrated that NS possessing a particle size of 300-600 nm exhibited a satisfactory potential for intradermal delivery. With these particle sizes, NS showed more skin deposition amounts of drugs, but few transdermal permeation amounts of drugs into the circulation system. [19][20][21] That might be due to skin appendages (hair follicle, sweat dut) acting a reservoir role that trapping the nanocarriers for a slow controlled release of the drugs. 22 However, the nanosuspensions are lack compliance for direct skin application, the fluidity of which resulting deficient skin adhesion and duration. In contrast, hydrogel patches have amounts of advantages as topical preparations: (1) high degree of drug loading; (2) abundant water content keeping the skin moisturized, efficient skin adhesion, and duration; (3) better stabilization and uniform distribution of nanosuspension. 23 Unfortunately, conventional surfactants as stabilizer of NS have raised potential toxicity concerns for long-term use. Conventional surfactants may induce skin irritation. When conventional surfactants are delivered over hypodermis and even into the vessels there is a risk of inducing system absorption. Furthermore, synthetic polymers are commonly used materials for both nanosuspension stabilizers and hydrogel matrices. However, in contrast to natural polymers (e.g. gelatin, agar, hyaluronic acid), synthetic polymers generally lack high intrinsic compatibility and degradability. 24 To the best of our knowledge, no research has yet sought to combine the use of biocompatible amino acid surfactants and hyaluronic acid (HA) as stabilizers in preparing nanosuspensions.
Amino acid surfactants are a kind of surfactant composed of oleophilic groups and one or more amino acid hydrophilic groups.
They are degradable, low irritant, and friendly to the human body and environment. 25,26 They can be decomposed into fatty acids and amino acids by enzymes in the human body. Sodium lauroyl sarcosinate (LS) is one kind of commercially available amino acid surfactant, which is commonly used in cosmetic, pharmaceutical, and food products. Relative research demonstrated that toxicity and sensitization were not observed in the tested models of rabbits, rats, and guinea pigs. In the clinical studies, LS was non-irritating, non-sensitizing, and had practically no reaction to moisture loss from the skin. 26 Hyaluronic acid (HA), also called as hyaluronan, is a vital part of the connective tissues extracellular matrix. Nowadays, HA is extensively applied for its unique biomedical benefits including viscoelasticity, biocompatibility, and biodegradability. The application covers skincare, skin rejuvenating, and tissue regeneration. Few researches have shown the potential of HA as a part of the skin topical delivery system in recent years. [27][28][29][30][31] For better skin application, this study aimed to come up with an effective hydrogel patch system, which is biocompatible, comfortable, and suitable for integrating nanosuspensions of DB. Even though various natural polymers possess biodegradability and biocompatibility, most of them still suffer from poor viscoelasticity to adhesion and duration to the skin surface. Two aspects inspired the hydrogel patch design; on the one aspect, thermo-reversible sol-gel transition biopolymer of agar promises a simple preparation; on the other aspect, we hypothesize that a combined utilization of polymers could exert complementary function in viscoelasticity properties. To the best of our knowledge, no research has yet sought to combine three materials of agar, gelatin, and partially neutralized polyacrylates to prepare the hydrogel patch.
Agar is a widely exited polysaccharide originating from red seaweeds, with a strong gelling property by the thermos-reversible sol to gel transition. Nevertheless, agar hydrogel suffers from poor viscidity and stability. 32 Gelatin is a low-cost natural polymer material with biocompatibility and biodegradability. However, the gelatin hydrogel network formed by hydrogen bonds with better viscidity still suffers from poor elasticity and formability. 33 Partially neutralized polyacrylates are the commonly used hydrogel materials, which are commonly used the aluminum ions to cross-link forming a hydrogel network structure. 23 Rheological characteristics could be useful to reflect the polymer chain interaction degrees in the hydrogel systems and provide some vital information for the adhesion and duration to the skin surface. 23 A variety of parameters are commonly used in the rheological characteristics, including the elastic modulus (G′), the viscous modulus (G″), and the complex modulus (G*).
Herein, three parameters illustrate the material properties from three different angles: G′ represents the "solid-like" character, a higher G′ value means greater cohesion of material, allowing it to be peeled off easily; G″ represents the "liquid-like" character, a high G″ value enables the material sufficiently flow for close contact with the skin. G* is used to determine the degree of viscoelasticity of a material, G* = [(G′) 2 + (G″) 2 ] 1/2 . Figure 1D, all reactants of agar, gelatin, partially neutralized polyacrylates, and aluminum ions cross-linker (aluminum glycinate) were added to a beaker for synthesizing DB nanosuspension hydrogel patch via a heating-cooling process.

| Preparation of Dragon's blood nanosuspensions (DB-NS)
DB-NS were produced using the previously reported Top-down method which is a combined method of high-shear homogenization and high-pressure homogenization. 17 Based on the previous experi-

| Preparation of DB-NS hydrogel patch
DB-NS hydrogel patch was prepared by a three-step method as depicted in Figure 1D Formulation code and composition are listed in Table 2.
Briefly, in step one, agar (0.8 g) and deionized water (40 ml) were added to a beaker and heated in an electromagnetic oven for several minutes to boil. Then the agar powder was dissolved, forming a transparent solution. Additionally, gelatin (1.5 g) was added to the resulting agar solution stirring at 30 rpm heating at 90°C in the water bath for 15 min to dissolve, then the obtained transparent solution was cooled down to 50°C. In step two, sodium polyacrylate (0.25 g) and aluminum amino acetate (0.125 g) was added to 10 ml glycerol stirring at 20 rpm for 5 min, after that the suspension was added to another baker with residual deionized water stirring at 30 rpm heating at 50°C in the water bath forming a transparent solution. In step three, the baker containing agar and gelatin solution was added to the solution forming in step two. The mixture was conducted at 50°C in the water bath stirring at 30 rpm for 30 min. Then the DB-NS was added to the solution stirring at 20 rpm for another 10 min. Finally, the solution was added to a glass mold and cooled down at room temperature, forming the gel patch.
Hydrogel patch matrices of agar, gelatin, NP-800, agar/gelatin, agar/gelatin/NP-800 were also prepared at the middle step for the further characterization of scanning electron microscopy and rheological tests.

| Water equilibrium solubility of Raw DB powder and DB-NS
In brief, 300 mg of raw DB powder and DB-NS (containing 300 mg DB) were added to deionized water in 15 ml volumetric flasks. Then F I G U R E 1 Preparation mechanism of (A) nanosuspension with the stabilizer of HA and LS, (B) nanosuspension without stabilizer. (C) The picture of the prepared hydrogel patch above left is agar, above right is agar/gelatin, below left is Agar/gelatin/NP-800, and below right is DB-NS hydrogel patch. (D) Preparation mechanism of DB-NS hydrogel patch.

| Particle size analysis of DB-NS
Both particle size (PS) and polydispersity index (PDI) detection of DB-NS were operated by dynamic light scattering (DLS) detector (90Plus Zeta, Brookhaven Instruments).

| Scanning electron microscopy (SEM)
The morphology of raw DB powder was observed by scanning electron microscopy (Quanta25, FEI). The morphology of lyophilized hydrogel patch matrix of agar, agar/gelatin, agar/gelatin/NP-800, and DB-NS hydrogel patch were observed by scanning electron microscopy (Quanta25, FEI). The samples were coated with gold for the photography.

| Transmission electron microscopy (TEM)
The morphology of DB-NS was analyzed by transmission electron microscopy (Tecnai G2F20, FEI). The sample was previously diluted with distilled water. Then a drop of the diluted suspension was put on a copper grid. Then the samples were stained using a 2% phosphor-tungstic acid aqueous solution (with appropriate dilution).
After that, the drying process was conducted at room temperature for observation.

| Differential Scanning Calorimetry (DSC)
The thermal analysis was performed by DSC (DSC-60, Shimadzu Co.). Raw DB powder, HA, LS, physical mixture, and lyophilized DB nanocrystal were placed in an aluminum pan with a heating rate of 10°C/min between 30°C and 350°C.

| Powder X-ray Diffraction (PXRD)
PXRD analysis of raw DB powder, HA, LS, the physical mixture, and the lyophilized DB-NS were conducted by a Bruker D8-Advance Xray powder diffractometer following a detect condition of Cu Kα radiation generated at 40 mA 40 kV, a scope of 3-50°(2θ) in the increments of 0.02°, following a step time of 0.3 s/step.

| Fourier Transform Infrared Spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy of DB, HA, LS, the physical mixture, and the lyophilized DB-NS were tested for the confirmation of the molecular interaction. The detections were enforced by the KBr method. The record range was 4000-400 cm −1 .

| Rheological tests
The rheological characteristics of all samples were conducted by

| In vitro skin penetration and retention studies
The skin penetration and retention experiment were conducted with pig ear skin, which is coherent with relative research. 21 Pig ear skin

| Cellular uptake
To compare the cellular uptake of DB-NS, raw DB powder water suspension, and DB dissolved in DMSO. The 2.0 × 10 5 HaCaT cells were seeded every well in 12-well plates for 24 h incubation. 38

| In vivo safety and efficiency study of DB-NS hydrogel patch
In vivo patch tests and photopatch tests were conducted to evaluate the safety and photoprotective effect of DB-NS and DB-NS hydrogel patch. 39 The study was conducted in rabbits by intraindividual, right-left comparison, which is coherent with relevant study. 40,41 The  Tables 3 and 4

according to OECD Test
Guideline No. 406 Skin Sensitisation. 42 The study was approved by the Institutional Review Board (IRB no. DWRL2022032).

| Statistical analysis
Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA with Tukey's test using GraphPad Prism 8.0 (GraphPad), where p < 0.05 was considered the significance level.

| Particle size and morphology analysis
The DB-NS formulations were produced by the top-down method, which combines high-shear homogenization and high-pressure homogenization. Formulation influencing factors are listed in Table 5.  Figure 2D. The drug nanoparticles' surface was covered by stabilizers shaping a dynamically rough surface to undergo continuous thermal motion, which prevents coalescence and aggregation by repulsive entropic forces. The relative preparation mechanism schematic drawing is shown in Figure 1A Intense erythema and swelling lumpy and a particle size of about 5-10 μm. Figure 2D demonstrates the TEM microphotograph of the DB-NS, which is a regular round shape covered by a coat composed of stabilizers.

| Crystalline of DB-NS
PXRD analysis was conducted as the confirmation of the crystalline state. In Figure 3C, the characteristic diffraction peak was not detected in the physical mixture, DB-NS, and HA, which indicated that three of them were in the amorphous state. LS showed particular characteristic diffraction peaks, which might be coved in the curve of the physical mixture and DB-NS.
The DSC thermograms of raw DB powder, LS, HA, physical mixture, and DB-NS were shown in Figure 3A.

| Intermolecular bond formation
The FTIR test detected the intermolecular bond formation between active ingredients and excipients. As shown in Figure 3B, the spectrum of the physical mixture presented an additive composition of DB, HA, and LS. While the spectrum of DB-NS absorption peak among 3700-3000 cm −1 turned out to be further broad compared to the physical mixture, which illustrated the intermolecular hydrogen bonding formation between DB and stabilizer of HA and LS. 45

| Rheological testing
The  patch could be arranged in the following order: DB-NS hydrogel patch > agar/gelatin/NP-800 > agar/gelatin. It was found that DB-NS containing hydrogel patch behaved with higher G′ values than agar/gelatin/NP-800 which might contribute to the nanoparticles embedded into the network enhancing the cross-link between polymer materials as shown in Figure 2H.
Morphology characteristics of hydrogel patch conducted by SEM disclosed the micro-structures of hydrogel patch systems that occurred among polymer chains from another angle. Agar showed a smooth and compact surface morphology ( Figure 2E), which corresponds to a weak and thinner network. Surface morphology of agar/gelatin hydrogel changes to a distorted and fractured pattern of rough and porous surfaces ( Figure 2F), which is related to weak cross-linked composite polymer networks. The surface morphology of agar/gelatin/NP-800 turned out to be a layered high branched network ( Figure 2G) and spherical nanoparticles with core-shell structure were successfully embedded into the network ( Figure 2H).
That morphology demonstrated that the hydrogel networks of agar/ gelatin/NP-800 were sufficiently cross-linked and DB-NS were appropriately integrated.

| Water equilibrium solubility
The equilibrium solubility of raw DB powder, physical mixture, and DB-NS in water were 263.65 μg/ml, 376.62 μg/ml, and 596.08 μg/ ml, respectively. Water equilibrium solubility of DB-NS was 2.3folds of raw DB powder .and 1.6-folds of raw DB powder and physical mixture, respectively. The results are shown in Figure 2B.
Thus, the rise of equilibrium solubility was not only caused by the stabilizer solubilizing but also contributed to the decrease in particle size. 34

| In vitro skin penetration and retention studies
The skin permeation and retention study results are shown in The skin permeation and retention amount of 2 mg/ml DB-NS and DB-NS hydrogel patch showed no significant difference.
There is a slight increment of skin permeation amount of DB-NS hydrogel patch that might be influenced by stratum corneum hydration, which can enhance the drug partition into the stratum corneum.
The mechanism of DB-NS enhanced intradermal delivery might be explained in four aspects as illustrated in Figure 4E. (1) The large surface area facilitates dissolution rate thus increasing the concentration gradient between the formulation on the stratum corneum surface and stratum corneum interior skin layers.
(2) The improved adhesion property of nanosuspensions enhances skin surface covering, enlarging the surface for drugs to penetrate the skin. (3) Specifically, it was demonstrated that nanosuspension possessing a particle size of 300-600 nm exhibited a satisfactory potential for intradermal delivery. With these particle sizes, NS showed more deposition amounts within the skin but few transdermal permeation amounts into the circulation system.
That might contribute to skin appendages (hair follicle, sweat dut) creating a reservoir, which assists in trapping the nanocarriers for a slow release of the drug. (4) Furthermore, as the cellular uptake results ( Figure 6D) showed, DB-NS expressed a significantly higher uptake amount than DB dissolved in DMSO and raw DB powder water suspension, which also contributes to skin retention. Compared with raw DB powder water suspension and DB dissolved in DMSO, DB-NS displayed the least skin penetration amount and the most skin retention amount, which realized the improvement of the skin bioavailability and the enhancement of intradermal delivery.

| DPPH radical scavenging activity
As shown in Figure 5, DB scavenged DPPH free radicals at a dosedependent form, from 0.008 mg/ml to 5 mg/ml producing the scavenging percentage of 21.52%-94.26%. Especially, at the concentration of 0.008 mg/ml DB produces a relatively higher scavenging percentage of 21.52% compared with vitamin C of 18.03%. At the concentration of 0.2 mg/ml, DB produces a relatively lower scavenging percentage of 69.38% compared with vitamin C of 91.36%.
Therefore, DB process favorable free radicals scavenging ability to exert the photoprotective effect. As a commonly accepted opinion, oxygen free radicals are involved in cutaneous photo-damage.

| Cell viability and photoprotective activity
Before the photoprotective activity evaluation, the cell viability influence of DB-NS, raw DB powder, and DB dissolved in DMSO was first tested by MTT experiment. 37,38 As the result shown in All groups of DB-NS, raw DB powder, and DB dissolved in DMSO showed a downward ROS compared with the model group.
What's more, compared with raw DB powder, and DB dissolved in DMSO the DB-NS group showed the maximum decline amount of ROS, which was 2/5 times of the model group as presented in Figure 6D,E.

| Cellular uptake
The study assessed the cellular uptake of raw DB powder, DB dissolved in DMSO, and DB-NS. With the treatment of 8 h, DB-NS group cells showed an uptake amount of 13.3 μg/ml. However, the group of raw DB powder and DB in DMSO showed no obvious uptake. As shown in Figure 6C, a significantly higher amount of DB uptake by DB-NS group cells may contribute to the adhesion effect of nanoparticles and the HA molecular also could enhance the contact of nanoparticles with HaCaT cells. Relative research has shown that for topically applied formulation enhancing skin drug retention, CD44 protein could play a potential role in the active-targeting drug delivery system. 49

| In vivo safety and efficiency evaluation of DB-NS hydrogel patch
The skin reaction after application in 24

| CON CLUS IONS
The present study demonstrated that DB-NS stabilized by biocompatible stabilizers HA and LS having a particle size of 447.0 ± 48. Thus, Dragon's Blood nanosuspension hydrogel could be used as an effective additive to enhance intradermal delivery for skin photoprotection. Moreover, this study also provides a new thought to improve the efficacy and safety of cosmetic formulation.

FU N D I N G I N FO R M ATI O N
This work was supported by the Double first-class innovative team (CPU2018GY28).

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PPROVA L
The study was approved by the Institutional Review Board (IRB no. DWRL2022032).