A photoresponsive transdermal in-situ crosslinking material and a preparation method and use method thereof

Abstract

The application discloses a kind of photoresponsive transdermal in-situ crosslinking materials and its preparation method and use method, belong to biological medical polymer and transdermal skin material technical field.The material includes A component and B component two components separated from each other in storage stage;A component includes short chain, long chain collagen fragment and riboflavin or its derivative, and B component includes aldehyde group modified polysaccharide.When using, A component is delivered to the target area of skin by first stage ultrasonic penetration, and then B component is applied to carry out second stage ultrasonic penetration, so that polysaccharide precursor is co-located with collagen in the target area of intradermal.The first dynamic network of Schiff base is formed by the free amino group of collagen and the aldehyde group of polysaccharide after stopping ultrasonic;then riboflavin is excited by 420-480 nm blue light, and the second enhanced network is formed by mediating photochemical crosslinking of collagen.The delivery and crosslinking time sequence of the application are separated, the delivery efficiency and gel skeleton strength are considered, the double network can be constructed in-situ in the target area of skin, and the effect of firming and anti-wrinkle is realized.

Description

Technical Field

[0001] This invention relates to the fields of biomedical polymer materials, transdermal delivery materials, collagen-based composite materials, visible light responsive materials, and topical skin materials. Background Technology

[0002] Collagen is an important structural component of the skin's extracellular matrix. With age, photoaging, or other factors, the skin's collagen content, fiber continuity, and tissue mechanical state can change. Exogenous collagen, collagen peptides, and polysaccharides are commonly used in topical skin materials, tissue interface materials, and hydrogel systems due to their hydrophilicity and biocompatibility. However, the stratum corneum of the skin forms a significant barrier to most hydrophilic and macromolecular substances; directly applying intact macromolecular collagen or cross-linked polysaccharides to the skin surface does not guarantee their penetration into the target skin layer.

[0003] On the other hand, simply pursuing low molecular weight and high diffusivity in materials weakens their ability to form stable networks locally. Short-chain collagen fragments possess better solubility, spreadability, and migration capabilities, but their chain entanglement and continuous network formation capabilities are relatively limited. Higher molecular weight collagen fragments can provide more amino reaction sites, offering both better chain entanglement and skeletal support, but the system viscosity and transdermal migration resistance will also increase accordingly. Therefore, short-chain collagen can assume the functions of "delivery and replenishment," while long-chain collagen can assume the functions of "interfacial bridging and network support." After delivery, in-situ cross-linking can then be performed, thereby establishing a new balance between transdermal migration capability and structural stability.

[0004] If collagen and cross-linked polysaccharides are thoroughly mixed on the skin surface before transdermal delivery, the pre-formed network increases system viscosity, restricts diffusion, and reduces the efficiency of ultrasound-enhanced permeation. Low-frequency ultrasound (20-100 kHz) primarily disturbs the orderly arrangement of the stratum corneum lipid bilayer through steady-state and transient cavitation effects, increasing intercellular lipid fluidity, constructing transient aqueous channels, and weakening the skin barrier. High-frequency ultrasound (0.5-3 MHz) utilizes mechanical effects and microacoustic flow to propel substances from the epidermis to the superficial dermis. Therefore, a two-stage sequential ultrasound approach—first using low-frequency ultrasound to open the stratum corneum, then using high-frequency ultrasound to promote intradermal transport—is more conducive to efficient co-localization of collagen and polysaccharides in the skin target area compared to single-frequency ultrasound.

[0005] Riboflavin and its derivatives are visible light photosensitizing components. Under blue light excitation, they can undergo both type I and type II photodynamic reactions simultaneously: the type II reaction generates singlet oxygen, which induces the oxidative crosslinking of collagen tyrosine residues in the local microenvironment where riboflavin and collagen tyrosine residues are co-located, thus constructing covalent bonds between collagen molecules; the type I reaction generates free radicals, which, if unsaturated groups such as methacryloyl and acrylamide are present in the system, can initiate free radical polymerization, thereby constructing an additional polymer network.

[0006] Current transdermal collagen crosslinking technologies include ultrasound-enhanced permeation, collagen photocrosslinking, polysaccharide composite hydrogels, and photosensitive crosslinking. However, all have shortcomings, making it difficult to achieve integrated effects of non-invasive collagen delivery, deep penetration, and time-sequential in-situ crosslinking under intact skin. CN100588436C discloses an ultrasound-enhanced permeation scheme, which can adjust parameters to improve the transdermal ability of active ingredients, but it is not optimized for collagen molecular weight and low viscosity characteristics, and lacks "delivery first, crosslinking later" time-sequential control, only able to introduce substances, not to form in situ. CN114874455B uses collagen self-assembly-photocrosslinking to improve gel mechanics and stability, and is mostly used in in vitro tissue engineering. It does not solve the problem of transdermal delivery under intact skin, and the delivery and crosslinking cannot be decoupled in time, making it unsuitable for transdermal scenarios. CN117180494B is a collagen-hyaluronic acid dual-network photocrosslinked hydrogel with good biocompatibility and anti-scarring effects, but it is limited to injection administration, which is an invasive method. Delivery and crosslinking occur simultaneously, and the material solidifies before sufficient penetration, limiting deep repair. CN113769089A discloses riboflavin-blue light collagen crosslinking. When used alone, this technology only achieves photochemical crosslinking of collagen, lacks a matching transdermal permeation enhancement system, cannot overcome the delivery bottleneck caused by the skin barrier, and does not involve temporal coupling with the dynamic covalent network of polysaccharides.

[0007] In summary, existing technologies are fragmented and lack an integrated solution that combines non-invasive ultrasound-assisted transdermal penetration enhancement, graded delivery of low-viscosity collagen, and sequential in-situ photocrosslinking after transdermal administration. This makes it difficult to simultaneously address the needs for non-invasive drug delivery, deep penetration, in-situ shaping, and stable repair. Therefore, there is an urgent need to develop novel ultrasound-assisted transdermal collagen transdermal and sequential crosslinking repair technologies. This invention therefore separates "delivery" and "crosslinking" sequentially: first, a low-viscosity component A completes the first stage of ultrasound infusion, followed by the application of a low-viscosity component B to complete the second stage of ultrasound infusion and interfacial mixing; only after both delivery stages are completed and ultrasound is stopped does the two components form a first dynamic network in the target area of ​​the skin tissue, and finally, a second enhancing network is formed through a riboflavin blue light reaction. Summary of the Invention

[0008] A photoresponsive collagen-polysaccharide transdermal in-situ crosslinking material comprises a component A and a component B that are separated from each other during the storage stage; the component A is a delivery-type collagen precursor, comprising at least 0.3-10 kDa short-chain collagen fragments, 10-100 kDa long-chain collagen fragments, and riboflavin or riboflavin derivatives, and may also contain collagen fragments modified with methacryloyl and / or acryloyl groups; the component B is a crosslinking-type polysaccharide precursor, comprising at least aldehyde-modified polysaccharides, and may also contain modified polysaccharides containing polymerizable unsaturated groups.

[0009] The above materials are used as follows: Component A is applied first and ultrasonic permeation is performed in the first stage, followed by the application of Component B and ultrasonic permeation or ultrasonic-mediated local mass transfer and interfacial mixing in the second stage; after the ultrasound is stopped, the free amino groups of collagen react with the aldehyde groups of polysaccharides to construct a dynamic covalent first network; finally, after irradiation with 420-480 nm blue light, a second reinforcing network of collagen photochemical cross-linking is formed mediated by riboflavin or its derivatives.

[0010] Preferably, component A, as a delivery-type collagen precursor, consists of 0.3-10 kDa (preferably 0.3-5 kDa) short-chain collagen fragments, optionally 10-100 kDa (preferably 20-80 kDa) long-chain collagen fragments, partially functionalized collagen, and riboflavin or riboflavin derivatives; wherein, based on the total dry weight of the collagen fragments, the mass ratio of short-chain collagen fragments to long-chain collagen fragments is (20:80)-(80:20); component B, as a cross-linked polysaccharide precursor, contains at least aldehyde-modified polysaccharides, and may further contain modified polysaccharides that have undergone methacrylation, acrylation modification, or have other polymerizable unsaturated groups.

[0011] Preferably, the collagen fragments are selected from type I collagen, type III collagen, or a combination of both; at least a portion of the long-chain collagen fragments are attached with one or two functional groups selected from methacryloyl and acryloyl groups, with a functionalization degree of 5%-40%, and the remaining free amino groups after functionalization account for 30%-90% of the initial free amino groups.

[0012] Preferably, the aldehyde-modified polysaccharide is selected from one or more of aldehyde-modified hyaluronic acid, aldehyde-modified chondroitin sulfate, aldehyde-modified dextran, and aldehyde-modified pullulan, with a degree of aldehyde modification of 5%-45% and a molecular weight preferably of 1-80 kDa, more preferably 5-30 kDa; the modified polysaccharide containing polymerizable unsaturated groups is selected from one or more of methacryloyl hyaluronic acid, methacryloyl chondroitin sulfate, acryloyl hyaluronic acid, and acryloyl chondroitin sulfate, with a degree of functionalization of 5%-50%.

[0013] Preferably, after components A and B are fully contacted, the total solid content of the system is 0.5%-15% (w / w), the mass ratio of collagen fragments to total polysaccharides is 1:(0.1-2.0), the pH value of the system is 6.0-7.8, the final concentration of riboflavin or its derivative is 0.005%-0.20% (w / w), and the viscosity of component A during the delivery stage is lower than the viscosity of the system after components A and B form the first network.

[0014] Preferably, components A and B are packaged independently in a dual-chamber container, dual-bottle, dual-cylinder injector, dual-bag, or multi-chamber packaging with a release liner; the material also contains one or more of the following additives: glycerol, propylene glycol, trehalose, inorganic salts, buffer components, amino acids, and preservatives.

[0015] Preferably, both component A and component B are subjected to ultrasonic permeation in a low-viscosity state without premixing with the other component. The first stage of ultrasound is used to improve stratum corneum permeability and promote the migration of collagen fragments to the target area; the second stage of ultrasound is used to promote the migration of aldehyde-modified polysaccharides and optional photoreactive polysaccharides to the same target area, while enhancing their interfacial contact with collagen fragments. The two stages can use the same or different frequencies, intensities, duty cycles, coupling methods, and action times, and the delivery stage is separated from the network-forming stage by stopping the ultrasound.

[0016] Preferably, after the first stage of ultrasound delivery of component A is completed, excess component A on the skin surface is removed or thinned, and then component B is applied to the same area and the second stage of ultrasound penetration is performed to ensure that the aldehyde polysaccharide comes into full contact with the free amino groups in component A, the collagen fragments that have entered the stratum corneum, and the collagen-related reaction sites that can be accessed at the skin tissue interface. After stopping the ultrasound and letting it stand for 1-10 minutes, the aldehyde polysaccharide and the free amino groups of collagen form a reversible imine bond / Schiff base type dynamic covalent connection, accompanied by hydrogen bonding and chain entanglement effect, forming a first network with local positioning capability.

[0017] Preferably, a second enhancement network is formed through riboflavin-blue light interaction: after the second stage of ultrasound stops and the first network is formed or begins to form, the network is irradiated with blue light at 420-480 nm (preferably 440-470 nm).

[0018] Preferably, the preparation and modification steps of components A and B are as follows: S1. Collagen fractionation: Type I and / or Type III collagen are prepared into an acidic dispersion of 0.5%-2.0% (w / v) and subjected to chain scission by controlled treatment with pepsin, collagenase, or other restricted hydrolysis methods at 4-20℃. The reaction is terminated after the target viscosity or molecular weight distribution is reached. Fractionation is performed using a combination of membrane modules with a retention range of 1 kDa, 3 kDa, 10 kDa, 30 kDa, or 100 kDa. The separation range is then adjusted according to the results of GPC, SDS-PAGE, or LC-MS to finally obtain short-chain collagen components that can be used for transdermal delivery and long-chain collagen components that can be used for structural support.

[0019] S2. Partial Functionalization of Collagen: Dissolve long-chain collagen fragments in a buffer system with a pH of 8.0-9.0, and slowly add methacrylic anhydride or other activating agents that can introduce unsaturated groups at 0-10℃. By controlling the amount of feed and the reaction time, the degree of functionalization is maintained at 5%-40%, while retaining enough free amino groups for the formation of the first network. After the reaction is completed, the product is dialyzed, desalted, and lyophilized for later use.

[0020] S3. Aldehyde-modified polysaccharides: Using hyaluronic acid, chondroitin sulfate, dextran, or pullulan as raw materials, the polysaccharide components with a degree of 1-80 kDa, preferably 5-30 kDa, are first obtained through degradation or fractionation. Then, controlled oxidation is performed using periodate. After the reaction is completed, ethylene glycol or other appropriate terminators are added to terminate the reaction. After purification by dialysis and ultrafiltration, aldehyde-modified polysaccharides with a degree of aldehyde modification of 5%-45% are obtained.

[0021] S4, Photoreactive Polysaccharide: Hyaluronic acid, chondroitin sulfate or other polysaccharides containing hydroxyl / amino groups are modified by methacrylation or acrylation to obtain photoreactive polysaccharides with a functionalization degree of 5%-50%; this component can be prepared separately from the aldehyde-modified polysaccharide and then formulated together into component B.

[0022] S5, Group A allocation method: Short-chain collagen fragments, optional long-chain collagen fragments, optional functionalized collagen fragments, and riboflavin-5-phosphate sodium are dissolved in a buffer solution with a pH of 6.0-7.8 to keep Group A in a low-viscosity state that is spreadable and spreadable during the delivery stage.

[0023] S6 and B component preparation: Aldehyde-modified polysaccharides and optional photoreactive polysaccharides are dissolved in a buffer solution with a pH of 6.0-7.8. Components A and B are treated with sterilization and low bioload processes suitable for polymer materials, and then aliquoted and stored in the dark.

[0024] Preferably, component A is applied to the surface of ex vivo skin or a skin equivalent model, and a first-stage permeation promotion is performed using low-frequency ultrasound of 20-100 kHz, ultrasound of 0.5-3 MHz, or a sequential combination of both. By controlling the frequency, output intensity, duty cycle, treatment time, and coupling layer thickness, at least some collagen fragments achieve a detectable distribution below the stratum corneum. The goal of this stage is to complete the delivery of collagen precursors and maintain low viscosity, rather than prematurely forming a high-viscosity network.

[0025] Preferably, after component A has completed its primary delivery, component B is applied to the same area, and a second stage of penetration enhancement and local mass transfer is performed using low-frequency ultrasound (20-100 kHz), ultrasound (0.5-3 MHz), or a combination of both, to allow the aldehyde-modified polysaccharide to migrate into the skin tissue and fully contact the free amino groups of collagen. After the second stage, ultrasound is stopped, and the area is left to stand for 1-10 minutes to allow the amino-aldehyde dynamic covalent network to gradually form.

[0026] Preferably, after the second stage of ultrasound stops and the first network is formed or begins to form, blue light at 420-480 nm (preferably 440-470 nm) is used for irradiation to induce photochemical cross-linking of collagen mediated by riboflavin. If photoreactive precursors containing polymerizable unsaturated groups, such as methacryloyl hyaluronic acid and methacryloyl chondroitin sulfate, are present in the system, the free radical intermediates generated by riboflavin through type I reaction can initiate free radical polymerization of the above precursors, further forming additional covalent connections and constituting a second reinforcing network. Beneficial effects

[0027] (1) Separate the ultrasonic delivery of component A and component B from the cross-linking curing in time to avoid premature reaction of components A and B before delivery, and prevent the viscosity of the system from increasing and the diffusion of effective components from being restricted.

[0028] (2) Functional division of labor is achieved by the molecular weight difference of short-chain / long-chain collagen, while taking into account the transdermal delivery efficiency and the structural support capacity of the final network, thus solving the problems of insufficient network formation capacity of single low molecular weight components and excessive delivery resistance of single high molecular weight components.

[0029] (3) The cross-linked polysaccharide precursor is brought to the skin target area that is the same as or adjacent to the collagen precursor by the second stage of ultrasound, and then the amino-aldehyde dynamic first network is formed to improve the spatial matching, local positioning and initial structural stability of the two types of precursors.

[0030] (4) After stopping the ultrasound and forming the first network, a second enhancement network is constructed using riboflavin and 420-480 nm blue light to improve the modulus, swelling resistance, enzyme degradation resistance and structure retention of the composite network. Attached Figure Description

[0031] Figure 1 Schematic diagram of ultrasound-assisted permeation Figure 2 A schematic diagram illustrating the mechanism by which riboflavin-mediated blue light-induced photochemical cross-linking of collagen to form a second reinforcing network. Detailed Implementation

[0032] Example 1 Component A: Type I short-chain collagen (0.5-3 kDa) 1.2 g, Type I long-chain collagen (20-50 kDa) 1.2 g; long-chain collagen methacrylamide degree 22%, amino retention 72%; riboflavin-5-phosphate sodium, final concentration 0.05 wt%; pH 7.0 buffer to 100 mL. Short-chain:long-chain collagen mass ratio 50:50.

[0033] Component B: Aldehyaluronic acid (OHA) 1.2 g (aldehyde content 20%, Mw 12 kDa); Methacrylamide hyaluronic acid (HAMA) 0.8 g; pH 7.0 buffer to a final volume of 100 mL. Collagen:polysaccharide mass ratio = 1:0.83. Total solids content of the system is approximately 4.4% (w / v).

[0034] Operating steps: (1) Apply component A to the skin; first stage: 40 kHz pulsed ultrasound to promote penetration; wipe off excess component A from the skin surface; (2) Apply component B to the same area; second stage: use 1 MHz pulsed ultrasound to promote penetration and interface mixing; stop ultrasound; (3) Let stand for 3 min to form the first Schiff base network; irradiate with 455 nm blue light (12 J / cm²) to construct the second reinforcement network.

[0035] Example 2 Component A: Short-chain collagen (0.3-1.5 kDa) accounts for 70% of the total collagen mass, and long-chain collagen (20-50 kDa) accounts for 30%; the degree of methacrylation of long-chain collagen is 12%, and the amino retention is 81%; the final concentration of riboflavin-5-phosphate sodium is 0.03 wt%; 2% glycerol is added for wetting; the total solid content of the system is 2.2 wt%.

[0036] Component B: The ratio of OHA (aldehyde degree 12%, Mw 8 kDa) and HAMA is the same as in Example 1; Collagen: Polysaccharide = 1:0.4.

[0037] Operation: The two-stage ultrasound time is shortened by 50%, and the rest is the same as in Example 1.

[0038] Example 3

[0039] Component A: short chain 0.5-5 kDa, long chain 20-80 kDa; short chain: long chain = 45:55; long chain functionalization 30%, amino retention 63%; riboflavin final concentration 0.06 wt%.

[0040] Component B: OHA aldehyde degree 32%, combined with HAMA in a ratio of 4:1; collagen:polysaccharide = 1:1.2.

[0041] Procedure: The first stage is 25 kHz low-frequency ultrasound to open the stratum corneum, and the second stage is 2 MHz high-frequency ultrasound for mass transfer. The rest is the same as in Example 1.

[0042] Example 4

[0043] Component A: Type I short-chain collagen (0.5-3 kDa) 1.2 g, Type I long-chain collagen (20-50 kDa) 1.0 g, long-chain collagen was not functionalized; riboflavin final concentration 0.05 wt%; Component B: OHA (Mw 25 kDa) 1.0 g, HAMA 0.8 g; Collagen: Polysaccharide = 1:0.82.

[0044] Operation: First stage: 30-60 kHz low-frequency ultrasound for stratum corneum modulation; second stage: 1.5 MHz high-frequency ultrasound for mass transfer; the rest is the same as in Example 1.

[0045] Example 5

[0046] Component A: Short chains account for 80% of the total collagen mass, and long chains account for 20%; long chain functionalization is 18%, and amino groups are retained at 75%; riboflavin final concentration is 0.05 wt%.

[0047] Component B: The ratio of OHA to HAMA is 4:1; Collagen: Polysaccharide = 1:0.75.

[0048] Operation: Same as the standard two-stage ultrasound procedure in Example 1.

[0049] Example 6

[0050] Component A: 0.8 g short-chain collagen, 2.0 g long-chain collagen; long chain accounts for 71%; long-chain collagen methacrylamide degree 33%, amino retention 58%; riboflavin final concentration 0.05 wt%.

[0051] Component B: OHA 1.5 g, HAMA 1.0 g; Collagen: Polysaccharide = 1:0.89.

[0052] Procedure: Standard two-stage ultrasound; due to the high viscosity of A, the ultrasound time for the first stage was extended by 30% compared to Example 1.

[0053] Example 7 Component A: 1.0 g short chain, 1.3 g long chain; type I: type III collagen ratio of 7:3 within the long chain; functionalization of the long chain 25%, amino retention 68%; final concentration of riboflavin-5-phosphate sodium 0.06 wt%.

[0054] Component B: OHA 0.8 g, aldehyde-modified chondroitin sulfate (OCS) 0.5 g, HAMA 0.8 g; polysaccharide complex.

[0055] Procedure: Standard two-stage ultrasound, same as in Example 1.

[0056] Example 8

[0057] Component A: Type I short-chain collagen (0.5-3 kDa) 1.0 g, Type I long-chain collagen (20-50 kDa) 1.0 g, long-chain collagen was not functionalized; riboflavin final concentration 0.05 wt%.

[0058] Component B: 1.2 g of low molecular weight OHA (Mw 7 kDa), 0.5 g of HAMA.

[0059] Procedure: The first stage A ultrasound remains unchanged; the second stage ultrasound time is shortened to promote the penetration of low molecular weight polysaccharides, and the rest is the same as in Example 1.

[0060] Example 9

[0061] The formulation was exactly the same as in Example 1; only the final concentration of riboflavin-5-phosphate sodium was reduced to 0.02 wt%.

[0062] Operation: The two-stage ultrasound remained unchanged; the blue light irradiation time was extended, and the light dose was increased to 18 J / cm² to compensate for the decrease in photosensitizer.

[0063] Example 10

[0064] formula: Component A: Type I short-chain collagen (0.5-3 kDa) 2.4 g, without long-chain collagen or methacrylylation modification; riboflavin-5-phosphate sodium, final concentration 0.05 wt%; pH 7.0 buffer to 100 mL.

[0065] Component B: Aldehyaluronic acid (OHA) 2.0 g (aldehyde degree 20%, Mw 12 kDa); HAMA-free or other modified polysaccharides containing polymerizable unsaturated groups; pH 7.0 buffer to 100 mL. Collagen:polysaccharide mass ratio = 1:0.83. Total solids content of the system is approximately 4.4% (w / v).

[0066] Operation: Same as the standard two-stage ultrasound procedure in Example 1.

[0067] Example 11

[0068] formula: Component A: Same as in Example 1.

[0069] Component B: Aldehyaluronic acid (OHA) 1.2 g (aldehyde content 20%, Mw 12 kDa); chondroitin sulfate (CSMA) 0.8 g (functionality 25%); pH 7.0 buffer to a final volume of 100 mL. Collagen:polysaccharide mass ratio = 1:0.83.

[0070] Operation: Same as the standard two-stage ultrasound procedure in Example 1.

[0071] Comparative Example 1 The total amount of the formulation materials is exactly the same as in Example 1; components A and B are thoroughly mixed before application to the skin; ultrasound with total energy equivalent to that in Example 1 is administered, and subsequent static placement plus blue light remains unchanged.

[0072] Comparative Example 2 The formulation is the same as in Example 1. Components A and B are not ultrasonicated; they are simply coated and left to stand.

[0073] Comparative Example 3 The formulation is the same as in Example 1, except that component A is sonicated in the first stage, while component B is not sonicated.

[0074] Comparative Example 4: The formulation was the same as in Example 1, except that component B was sonicated in the second stage, while component A was not sonicated.

[0075] Comparative Example 5 Component A is the same as in Example 1; Component B removes aldehyde-modified hyaluronic acid (OHA) and retains only HAMA and buffer; the two-stage ultrasound and blue light process remains unchanged.

[0076] Comparative Example 6 It contains riboflavin, is not exposed to blue light, and is otherwise the same as in Example 1.

[0077] Comparative Example 7 Component A, without the addition of riboflavin, was subjected to the same blue light irradiation as in Example 1, and otherwise remained the same.

[0078] Comparative Example 8 The total collagen mass remained the same as in Example 1; Component A consisted entirely of 0.3-5 kDa short-chain collagen, without the addition of 10-100 kDa long-chain collagen; Component B, the two-stage ultrasound, and the blue light process remained unchanged.

[0079] In the above embodiments, percutaneous delivery performance was determined using a Franz diffusion cell combined with an in vitro porcine skin model, with cumulative permeation and intradermal retention over 24 hours as indicators. Intradermal colocalization effect was observed using FITC-labeled collagen and Rhodamine B-labeled polysaccharide combined with laser confocal scanning microscopy. Gel mechanical properties were measured using a rheometer to determine the first network storage modulus G'1 after standing and the second network storage modulus G'2 after blue light irradiation. Enzyme degradation resistance was expressed as the mass retention rate after degradation by type I collagenase (1 U / mL) at 37°C for 24 hours. Intradermal retention was measured in μg / g (wet weight per gram of skin tissue). Maximum distribution depth was determined using FITC-labeled collagen combined with laser confocal scanning microscopy, expressed as the maximum skin depth detectable by fluorescence signal. All tests were performed with three parallel samples, and data are expressed as mean ± standard deviation. It should be noted that the gel mechanical and enzyme degradation resistance data in the current embodiments are mainly based on in vitro equivalent gel model measurements, and the in situ intradermal performance can be further verified through histological staining and intradermal microrheology.

[0080] Table 1. Experimental results of a photoresponsive collagen-polysaccharide transdermal in-situ crosslinking material. Example 1 42 18.6 142 260 320 980 71 Example 2 25 20.2 132 240 285 760 64 Example 3 38 21.5 164 300 345 980 72 Example 4 40 23.2 176 330 365 1090 77 Example 5 30 24.8 156 320 290 790 64 Example 6 66 13.8 164 215 480 1360 85 Example 7 45 20.1 162 285 410 1160 80 Example 8 39 20.0 178 305 455 1210 82 Example 9 42 18.4 148 268 338 810 66 Example 10 28 23.5 128 310 210 620 52 Example 11 42 18.4 140 258 318 975 70 Comparative Example 1 410 6.1 70 100 470 920 68 Comparative Example 2 42 10.2 85 135 205 720 56 Comparative Example 3 42 18.1 118 245 245 830 61 Comparative Example 4 42 12.0 121 165 305 900 66 Comparative Example 5 40 18.2 115 255 85 560 46 Comparative Example 6 42 18.5 145 260 320 338 50 Comparative Example 7 42 18.4 143 260 318 420 52 Comparative Example 8 24 26.5 128 350 200 530 42 Example 1 employs a process flow of "first-stage ultrasonic permeation of component A and second-stage ultrasonic permeation of component B, followed by stopping ultrasonication and allowing the mixture to stand to form a Schiff base first network, and then inducing a second enhanced network through blue light". This process can achieve a comprehensive balance among multiple properties such as transdermal delivery, intradermal retention, gel mechanics, and resistance to enzyme degradation.

[0081] As can be seen from Examples 2-11, the viscosity of component A, the ratio of long-chain to short-chain collagen, the molecular weight of polysaccharides, and the concentration of riboflavin significantly regulate the overall performance of the system. Low viscosity and a high proportion of short-chain collagen are beneficial for transdermal penetration. In Example 5, the proportion of short-chain collagen was increased to 80%, and the cumulative permeation amount after 24 h reached 24.8 μg / cm², but the network mechanical strength and resistance to enzyme degradation decreased. Increasing the proportion of long-chain collagen (Example 6) increases the viscosity of component A and reduces the transdermal permeation amount to some extent, but it can significantly improve the strength of the gel skeleton. After blue light, G'2 can reach 1360 Pa, and the mass retention rate after enzyme degradation reaches 85%. Optimizing the ultrasound mode and polysaccharide molecular weight can further enhance the intradermal co-localization effect. Example 4 used dual-frequency sequential ultrasound, and Example 8 used low molecular weight aldehyde-modified polysaccharide combined with a second-stage ultrasound. The intradermal retention reached 176 μg / g and 178 μg / g, respectively, and the first network G'1 reached 365 Pa and 455 Pa, respectively. However, the total action time of the dual-frequency mode was correspondingly prolonged, and the entanglement ability of the low molecular weight polysaccharide chain was weaker. Riboflavin concentration mainly regulates the enhancement effect of the second network. In Example 9, after reducing riboflavin to 0.02 wt%, the transdermal delivery index was basically the same as that of Example 1, but after blue light, G'2 decreased to 810 Pa and the 24-hour enzymatic degradation mass retention rate decreased to 66%. In the simplest formula containing only short-chain collagen, riboflavin and aldehyde-modified polysaccharide (Example 10), a dual network can still be formed in the skin and the transdermal delivery efficiency is better (24-hour cumulative permeation 23.5 μg / cm²), but the first network G'1 is only 210 Pa and G'2 is only 620 Pa after blue light. Pa, mechanical strength and resistance to enzyme degradation decreased significantly; after replacing HAMA with CSMA (Example 11), the properties were close to those of Example 1, indicating that the modified polysaccharide containing polymerizable unsaturated groups is replaceable.

[0082] Combined with a comparative proportion, a univariate control analysis was conducted: (1) If components A and B are premixed before application, the viscosity of the system increases from 42 mPa·s to 410 mPa·s; even with the same total ultrasound energy, the cumulative penetration and intradermal retention in 24 h are significantly reduced. Components A and B must be delivered separately at low viscosity and cross-linking occurs only after ultrasound is stopped; pre-gelling before delivery will severely hinder the migration of components to the deeper layers of the skin.

[0083] (2) When only component A is sonicated (Comparative Example 3), collagen delivery is improved, but a large amount of polysaccharide remains on the skin surface and the first network modulus is insufficient; when only component B is sonicated (Comparative Example 4), polysaccharide can penetrate into the skin, but the intradermal collagen supply is insufficient; when components A and B are sonicated separately in two stages (Example 1), collagen and aldehyde polysaccharide can be delivered to the same skin target area at the same time, achieving intradermal co-localization, and the intradermal retention and first network modulus are optimal.

[0084] (3) Removing aldehyde-modified polysaccharide OHA almost does not affect the transdermal penetration of collagen, but the lack of amino-aldehyde dynamic covalent interaction exacerbates the outward loss of intradermal components, significantly reduces the first network G'1, and weakens the subsequent enhancement effect of the second blue light network. The first network can both anchor and retain collagen fragments that have entered the skin and provide a cross-linking reaction substrate for photochemical reactions.

[0085] (4) The gel modulus is limited by relying solely on the Schiff base first network; riboflavin photosensitizer and 420-480 nm blue light must be present simultaneously to achieve a significant increase in modulus. Riboflavin without blue light (Comparative Example 6) and blue light without riboflavin (Comparative Example 7) cannot achieve effective network enhancement, and the resistance to enzyme degradation is simultaneously low.

[0086] (5) When all short-chain collagen is used, the transdermal permeability is optimal, but the lack of chain entanglement and bridging backbone brought by long-chain collagen significantly reduces the mechanical properties and resistance to enzyme degradation after gelation. This confirms that the design of using a combination of short-chain and long-chain collagen in a mass ratio of (20:80)-(80:20) is the key to balancing transdermal delivery and network stability.

[0087] In summary, the various technical features of the complete process of this invention form a synergistic effect: the sequential separation of components A and B combined with two-stage ultrasound ensures low-viscosity delivery and intradermal co-localization; the first anchoring network is constructed based on the aldehyde-aminoSchiff base reaction; the second covalently enhanced network is induced by riboflavin-blue light; and the short-chain-long-chain bimodal collagen complex balances transdermal delivery and scaffold support. Omitting any of the above key technical features will result in significant deterioration of one or more of the following: transdermal performance, intradermal retention, gel mechanics, or resistance to enzyme degradation.

Claims

1. A photoresponsive transdermal in-situ crosslinking material, characterized in that, It includes a storage-stage separated component A and a component B; component A contains short-chain collagen fragments with a molecular weight of 0.3-10 kDa, long-chain collagen fragments with a molecular weight of 10-100 kDa, and riboflavin or riboflavin derivatives; component B contains aldehyde-modified polysaccharides. Components A and B do not form a three-dimensional cross-linked network during the storage stage.

2. The photoresponsive transdermal in-situ crosslinking material according to claim 1, characterized in that, The mass ratio of short-chain collagen fragments to long-chain collagen fragments in component A is (20:80)-(80:20) based on the total dry weight of collagen fragments.

3. The photoresponsive transdermal in-situ crosslinking material according to claim 2, characterized in that, The collagen fragments are selected from type I collagen, type III collagen, or a combination of both; at least a portion of the long-chain collagen fragments are attached with methacryloyl and / or acryloyl groups, with a functionalization degree of 5%-40%, and the remaining free amino groups after functionalization account for 30%-90% of the initial free amino groups.

4. The photoresponsive transdermal in-situ crosslinking material according to claim 1, characterized in that, The aldehyde-modified polysaccharide is selected from one or more of aldehyde-modified hyaluronic acid, aldehyde-modified chondroitin sulfate, aldehyde-modified dextran, and aldehyde-modified pullulan, with a degree of aldehyde modification of 5%-45% and a molecular weight of 1-80 kDa.

5. The photoresponsive transdermal in-situ crosslinking material according to claim 1, characterized in that, Component B further comprises a modified polysaccharide capable of polymerizing unsaturated groups, wherein the modified polysaccharide is selected from one or more of methacrylamide hyaluronic acid, methacrylamide chondroitin sulfate, acrylamide hyaluronic acid, and acrylamide chondroitin sulfate, and has a functionalization degree of 5%-50%.

6. The photoresponsive transdermal in-situ crosslinking material according to claim 1, characterized in that, The total solid content of the system after mixing components A and B is 0.5%-15% (w / w), the mass ratio of collagen fragments to total polysaccharides is 1:(0.1-2.0), the pH value of the system is 6.0-7.8, and the final concentration of riboflavin or its derivatives is 0.005%-0.20% (w / w).

7. The photoresponsive transdermal in-situ crosslinking material according to claim 1, characterized in that, The components A and B are packaged independently in a double-chamber container, double bottle, double-cylinder injector, double bag, or multi-chamber packaging with a release film; the material also contains one or more of the following additives: glycerol, propylene glycol, trehalose, inorganic salts, buffer components, amino acids, and preservatives.

8. A method of using the photoresponsive transdermal in-situ crosslinking material according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Apply component A to the target area of ​​the skin and perform the first stage of ultrasound-assisted penetration to allow collagen fragments to migrate to the target area of ​​the skin tissue; (2) Component B is applied to the same area and a second-stage ultrasonic permeation is performed to allow the aldehyde-modified polysaccharide to migrate to the target area and come into contact with the collagen fragments. (3) Stop the ultrasound and let it stand for 1 min-10 min to allow the free amino groups of collagen to react with the polysaccharide aldehyde groups to form a dynamic covalent first network; (4) After the first network is formed or begins to form, irradiate with 420-480 nm blue light to form a second reinforcing network of collagen photochemical crosslinking mediated by riboflavin or its derivatives.

9. The method of using the photoresponsive transdermal in-situ crosslinking material according to claim 8, characterized in that, The first stage of ultrasound uses low-frequency ultrasound of 20-100 kHz, and the second stage of ultrasound uses ultrasound of 0.5-3 MHz. Both stages use continuous or pulsed operating modes. The blue light irradiation wavelength is 420-480 nm and is implemented after the second stage of ultrasound stops.

10. A method for preparing a photoresponsive transdermal in-situ crosslinked material according to any one of claims 1-7, characterized in that, It includes the following steps: S1. Restricted hydrolysis of type I and / or type III collagen, combined with ultrafiltration, dialysis or membrane separation fractionation, to obtain short-chain collagen fragments with a molecular weight of 0.3-10 kDa and long-chain collagen fragments with a molecular weight of 10-100 kDa. S2. Controllable functionalization of some long-chain collagen fragments while retaining free amino groups, combined with short-chain collagen fragments, long-chain collagen fragments and riboflavin or its derivatives, to prepare component A. The preparation process is carried out in the dark. S3. Aldehyde-modified polysaccharides are prepared by controlled oxidation of periodate, and optionally modified polysaccharides containing polymerizable unsaturated groups are prepared by unsaturated functionalizing reagents, and component B is obtained by formulation. S4. Dispose of components A and B separately by sterilization, light protection, and isolation.

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