Patent Application
20240131222
This application claims the priorities of (1) filing with China National Intellectual Property Administration on Oct. 8, 2022, with application number 2022112245930, and (2) filing with China National Intellectual Property Administration on Apr. 3, 2023, with application number 2023103463184. The previous applications are incorporated herein by reference in their entireties.
The Sequence Listing associated with this application is provided in a Sequence Listing XML and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 81287-000005_SL.xml. The file is 3,538 bytes and was created on Nov. 17, 2023.
The present disclosure belongs to the technical field of bioengineering, in particular to a transdermal photocuring forming hydrogel with biological activity as well as a preparation method and an application thereof.
Maxillofacial soft tissue has a special anatomical physiological structure, which plays a crucial role in aspects of mastication, pronunciation, and aesthetics, etc., in human, and is very vulnerable to trauma, infection and congenital diseases, etc. at the same time. Abnormalities and deficiencies in the morphology and capacity of maxillofacial soft tissue, which are manifested as facial depression, asymmetry, and aging, etc., are very common in clinical practice. They not only cause functional problems for patients, but also seriously affect their appearance and mental health, bringing many troubles to their lives. The existing clinical repair methods mainly rely on autologous soft tissue transplantation, which can lead to secondary traumatization and is not conducive to shaping. In recent years, there is an increasing demand for aesthetic restoration. How to repair the appearance of damaged tissues as much as possible while restoring its normal function, reconstructing the function of damaged tissues, and achieving good aesthetic restoration results, has become the focus of maxillofacial soft tissue repair.
Soft tissue filling is a major non-surgical cosmetic treatment project, which involves implanting soft tissue filling materials into the body and occupying cavities and defect sites caused by tissue damage or lesions, replacing or enhancing their original functions, so as to achieve the purposes of tissue repair, deformity correction, and rejuvenation treatment. It meets the needs of patients for minimally invasive surgery and is also one of the current trends in the development of plastic surgery clinical technology. The application of injectable soft tissue filling materials for non-invasive facial filling has become the most popular means of operation. Injection filling technology is simple to operate, and results can be visible in just a few minutes of injection. The process is painless and convenient, without effects on the patients in aspects of daily life and work, etc, leaving no traces, and thus privacy of the customers is kept safe. However, the ideal injectable soft tissue filling materials must possess the characteristics like biocompatibility, safety, ease of operation, fixity, and durability, etc., at the same time, which has always been a highly challenging topic for scholars studying and developing plastic and cosmetic products.
The clinical repair effect of soft tissue defects is closely related to the sources and properties of materials. In order to achieve functional and aesthetic reconstructions of damaged tissues, it is not only required that injectable filling materials have good biocompatibility and immediate filling effect, but also that implanted materials have good tissue repair and regeneration functions, with a wide range of sources and ease of operation at the same time. Although injectable soft tissue filling agents such as hyaluronic acid and carboxymethyl cellulose, etc. are widely developed and applied, they lack of various biological signaling molecules required for cell adhesion, proliferation, and differentiation, and their ability to resist enzymolysis and free radical degradation is limited, making them difficult to regulate cell behaviors or promote tissue repair. For example, cross-linked hyaluronic acid and its derivatives as physical volume filling agents are not conducive to cell adhesion, with limited ability to induce tissue regeneration, and high metabolic rate, which result in limited effectiveness in maintaining the injection results. At the same time, the implantation of cross-linked hyaluronic acid products relies on the surgeon's shaping during the operation, which requires a high level of anatomical theoretical knowledge and surgical skills, meanwhile translation of the filling material may happen, which seriously affects the implantation outcome.
Therefore, the current injection materials have problems such as insufficient mechanical strength, short duration of injection effect maintenance, poor biocompatibility with human tissues, and unnatural effects after injection, etc., making it difficult to meet the needs of practical applications.
The object of the present invention is to provide a transdermal photocuring forming hydrogel with biological activity as well as a preparation method and a use thereof in view of the shortcomings of the present technology. The said hydrogel has good biocompatibility and can be formed in situ in a transdermal photo-crosslinking manner after injection, it can release recombinant collagen with biological activity while achieving the purpose of tissue filling, so as to promote the effect of tissue repair.
In order to achieve the above object, the present invention adopts the following technical solution:
Further, the N value is an integer ranging from 4 to 300.
Further, the N value is an integer ranging from 4 to 200. It includes but is not limited to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . . 200.
Further, the valuing of the N value can enable the molecular weight of recombinant collagen to reach from 1 kDa to 200 kDa, and further from 3 kDa to 150 kDa.
Further, the characteristic amino acid sequences are arranged in the basic repetitive unit continuously or at intervals.
Further, the amino acid sequence of the recombinant collagen presents the following characteristics:
[-G-E-Xaa3-Yaa1-(G-Xaa1-Xaa2-G-E-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa3-(G-Xaa1-Xaa2-G-E-Xaa3)n3-Yaa4-(G-Xaa1-Xaa2-G-E-Xaa3)n4- . . . -Yaan-(G-Xaa1-Xaa2-G-E-Xaa3)n-Yaan+1-G-Xaa1-Xaa2-]N
Wherein, the Xaa1 is a non-polar hydrophobic amino acid; the Xaa2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); and the Xaa3 is an alkaline amino acid;
Preferably, the recombinant collagen sequence comprises no protein tag.
Preferably, the amino acid sequence of the recombinant collagen is SEQ ID NO. 1.
Preferably, the anhydride is one of cyclic unsaturated anhydride or carboxyl-containing unsaturated anhydride or 4-pentenoic anhydride, crotonic anhydride and methacrylic anhydride; further, the anhydride is one of maleic anhydride, citraconic anhydride, cis-aconitic anhydride, 4-pentenoic anhydride, crotonic anhydride and methacrylic anhydride; and still further, the anhydride is methacrylic anhydride.
Preferably, the natural polysaccharide is selected from the group consisting of hyaluronic acid, carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, alginic acid, glucan, agarose, heparin, chondroitin sulfate, ethylene glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan or chitosan quaternary ammonium salt; preferably, this natural polysaccharide is carboxymethylcellulose.
Preferably, the activating agent is NHS and EDC; Further, the amino vicinal-diol is 3-amino-1,2-propanediol. By introducing a side chain group containing vicinal-diol, aldehyde group can be generated via specific oxidation, effectively avoiding the destruction of the main chain structure of a natural polysaccharide.
The EDC in the present invention refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and NHS refers to N-hydroxysuccinimide.
Further, the oxidizing agent is selected from the group consisting of periodate and hypochlorite; preferably, the oxidizing agent is sodium periodate.
In a second aspect, the present invention provides a preparation method of a transdermal photocuring forming hydrogel with biological activity, and the hydrogel is obtained by a transdermal photocuring method;
Further, the specific steps are as follows:
The present invention regulates the composition and structure of the natural polysaccharide through appropriate chemical modification and reasonable formula. Upon chemical modification, a photoinitiating group is introduced into the natural polysaccharide, so that it can be subcutaneously injected to achieve the transdermal photocuring forming effect, and its mechanical and degradation properties are also significantly improved; such special structure can also support the survival and proliferation of cells, making the finally obtained tissue filling agent (hydrogel) with good thermal stability, mechanical strength, anti-enzymolysis properties and longer maintenance time in vivo.
In the field of soft tissue filling, carboxymethyl cellulose, like other polysaccharide macromolecule compounds, lacks cell adhesion sites, which makes it difficult for cells to adhere and spread in the hydrogel, leading to the difficulty for cells to migrate and grow in the hydrogel, and thus they can only proliferate and aggregate in situ. The present invention makes full use of the biological safety, high adhesive activity and biological effect of the recombinant collagen, which is conducive to the adhesion and growth of cells on the compound hydrogel. With the degradation of carboxymethyl cellulose, the recombinant collagen is continuously released, which regulates the biological behavior of cells and induces the expression of extracellular matrix, so as to achieve the repair effect of morphological maintenance in early stage and induction of soft tissue matrix regeneration in later stage.
The hydrogel of the present invention can be used to improve the contour defects of soft tissue, and is suitable for the filling of the dermis layer (such as the middle and deep layers) and the subcutaneous superficial to deep layers, for example restoration of skin subsidence areas such as wrinkles, scars, depressed defects or injuries, etc.
Preferably, the photo-crosslinking modification degree of the natural polysaccharide derivative in the step S1 is 30% to 60%; and the molar ratio is 1:(1-3):(2-5) (the natural polysaccharide derivative with photo-crosslinking properties:NHS:EDC);
Further, in the step S2, the molar ratio is 1:2:3 (the natural polysaccharide derivative with photo-crosslinking properties:NHS:EDC);
Preferably, in the step S3, the pH value of the solution for the activation reaction to which an amino vicinal-diol is added is 7 to 7.7, and the molar ratio is (1-10):1 (amino vicinal-diol:the natural polysaccharide derivative with photo-crosslinking properties); the dialysis time in the S3 and S4 is 20 hours to 72 hours, and the molecular weight retained by dialysis is 8000 kD to 14000 kDa.
The natural polysaccharide derivative of the present invention contains an aldehyde group, which can be combined with the recombinant collagen through the Schiff s reaction and gradually released in the hydrogel. Preferably, in the step S5, the mass-to-volume ratio of the natural polysaccharide derivative containing an aldehyde side chain is 1% to 5%; the mass-to-volume ratio of the recombinant collagen is 0.5% to 2%; and the mass-to-volume ratio of the photoinitiator is 0.01% to 0.05%.
In a third aspect, the present invention provides an application of the transdermal photocuring forming hydrogel with biological activity in soft tissue filling and repair.
The hydrogel of the present disclosure can also be used together with other functional materials as required, such as ingredients with antibacterial and bacteriostatic effects, anti-aging ingredients, anticoagulant ingredients, antioxidant ingredients, and growth factors, etc.
All features disclosed in this specification, or all methods or steps in the process disclosed herein can be combined in any way, except for mutually exclusive features and/or steps.
When the mass-to-volume ratio or other values or parameters are represented by a range, a preferred range, or a range limited by a series of upper- and lower-limit preferred values, it should be understood as specifically disclosing all ranges formed by any combination of any upper limits or preferred values of the range with any lower limits or preferred values of the range, regardless of whether the range is separately disclosed. For example, when the scope “1 to 5” is disclosed, the described scope should be interpreted as including ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, and “1 to 3 and 5”, etc. When a numerical range is described herein, it is intended to include its end value and all integers and fractions within that range, unless otherwise specified.
The present invention has the following beneficial effects:
The accompanying drawings illustrated here are used to provide a further understanding of the present invention, which form a part of the present invention. The illustrative embodiment and their descriptions of the present invention are used to explain the present invention, and do not constitute an improper limitation thereto. In the accompanying drawings:
In order to make the object, technical solution, and advantages of the present invention clearer and more obvious, the present invention will be further explained below in detail in conjunction with embodiment. It should be understood that the specific embodiment described here are only intended to explain the present invention and are not intended to limit that.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the technical field of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiment and are not intended to limit the present invention.
It should be clarified that the experimental methods used in the following embodiment are all conventional methods, unless otherwise specified. The materials and reagents, etc. used in the following embodiment are commercially available, unless otherwise specified.
The terms “comprising”, “including”, “having”, “containing” or any other variation thereof used herein are intended to cover non-exclusive inclusion. For example, compositions, steps, methods, or articles containing the listed elements need not be limited to those elements, but can include other elements that are not explicitly listed or inherent elements in such compositions, steps, methods, or articles.
(1) Preparation of Transdermal Photocuring Forming Soft Tissue Filling Matrix
Modification of carboxymethyl cellulose (CMC, 250 KD, DS=0.7) using methacrylic anhydride (MA): Weight 1 g of raw material CMC and dissolve in 100 mL of deionized water to a concentration of 1% (wt), the mixture is stirred for 6 hours with a mechanical stirrer and then placed in a refrigerator at 4° C. for 24 hours, then stir at room temperature for 2 hours to have well dissolved solution and the molecular chain is fully extended, which is conducive to the modification reaction. The dissolved 1% CMC solution is placed in a low-temperature water bath controlled by a circulating pump, and the reaction temperature is kept for 2 hours. At the beginning of the reaction, 2 mL of methacrylic anhydride is added dropwise to the CMC solution. During the reaction, the pH is adjusted with 5M NaOH to maintain the value between 8.0 and 8.5, after the pH of the reaction solution is stabilized, the solution is reacted overnight for 24 hours. Pour the reaction solution into 500 mL of ethanol to obtain the precipitate product, filter and remove some unreacted methacrylic anhydride and byproducts at the same time. Wash the product with ethanol for three times, then transfer into a dialysis bag of 8000 kDa to 14000 kDa and dialyzed with deionized water for 4 days, with the water changed three times a day. After the dialysis, proceed with freeze-dried to obtain the natural polysaccharide derivative CMC-MA with photo-crosslinking properties, store the product at 4° C.
(2) Preparation of Recombinant Collagen
1. Preparation of Recombinant Collagen by Escherichia coli. Fermentation
Gene recombination and transcription: Collagen DNA fragments are subjected to codon optimization and splice recombination using a PCR method. The expression vector is constructed using pET-32a and then transferred to the Escherichia coli. expression strain BL21. After cultivation and screening, Escherichia coli. genetic engineering strains with high protein expression are obtained.
Fermentation culture: the optimized single colony of Escherichia coli. genetic engineering strains is selected from LB plate and placed in a 100 mL Erlenmeyer flask containing 10 mL of LB medium, cultured at 37° C. and 220 rpm for 12 hours to 16 hours, the bacterial solution is inoculated into a fermentation tank containing LB medium at a volume ratio of 1:100 for scale-up cultivation, and cultured at 37° C. and 220 rpm for 3 hours until the OD600 is about 0.6, then add 0.5 mM of IPTG, and culture at 16° C. for 20 hours, centrifuge to collect the thalli.
Protein separation and purification: suspend the thalli with tris buffer, stir at high speed to completely dissolve the thalli, then centrifuge, the supernatant is collected and cooled to 4° C. and then filtered with 1 μm, 0.45 μm and 0.22 μm of membrane sequentially, and further purified by affinity chromatography to obtain the recombinant collagen.
The characteristic of the amino acid sequence of recombinant collagen: a repetitive unit with a combination of multiple characteristic amino acid sequences, i.e. GER GAP GFR GPA GPN GIP GEK GPA GER GAP (SEQ ID NO: 2), which is connected 16 times to obtain the recombinant collagen of the present invention, and the amino acid sequence is shown in SEQ ID NO. 1:
2. Cell Culture Method
Prepare recombinant collagen solution having a certain concentration (such as 0.5 mg/mL) using PBS, and animal-derived collagen solution is to the same concentration (such as 0.5 mg/mL) using PBS, while PBS is used as the blank;
(3) Preparation of Recombinant Collagen Activated Transdermal Photocuring Forming Soft Tissue Filling Agent
Cytotoxicity Assay of CMC-MA
According to the provisions on cytotoxicity in GB/T 16886.5-2017, Biological Evaluation of Medical Devices—Part 5: in vitro Cytotoxicity Assay, the safety of CMC-MA compound materials is acceptable.
Cytotoxicity Assay of CMC-MA-AP-CHO-rhColIII Hydrogel
CMC-MA-AP-CHO-rhCol III hydrogel is tested for toxicity according to the method in CMC-MA cytotoxicity Assay. According to the provisions on cytotoxicity in GB/T 16886.5-2017, Biological Evaluation of Medical Devices—Part 5: in vitro Cytotoxicity Assay, the safety of hydrogel compound materials is acceptable (
It can be seen from
The results are shown in
The injectability of injectable materials is related to the convenience for surgeon's use. And the presence of obstacles such as blockages and discontinuities, etc. in the extrusion process or not, is an important physicochemical indicator. To evaluate the injectable performance, the loading force of compound materials in the syringe is measured with a universal material testing machine (AGS-X, SHIMADZU, Japan), and the specific method is as follows:
As shown in
In summary, the natural polysaccharide derivatives prepared in the present invention exhibit significant gelation in vitro and in vivo upon transdermal injection and light illumination for 1 minute, having quick gelation time and no damage to skin tissues. The natural polysaccharide derivative with photo-crosslinking properties CMC-MA reacts with 3-amino-1,2-propanediol and is oxidized to obtain a natural polysaccharide derivative CMC-MA-AP-CHO, which reduces the damage to the main chain of the sugar ring during the oxidation process, and can reduce the decline of mechanical strength to a certain extent. At the same time, the test results also show that the hydrogel of such modified natural polysaccharide derivative shows good safety and also has good injectability and operability. With the degradation of carboxymethyl cellulose, the recombinant collagen activated CMC-MA-AP-CHO-rhCol III hydrogel can continuously release rhCol III, and induce the biological behavior of extracellular matrix, thus achieve the repair effect of morphological maintenance in early stage and induction of soft tissue matrix regeneration in later stage, which has a more long-lasting action time.
The aforementioned examples are only illustrative and are used to explain some features of the method described in the present invention. The attached claims are intended to claim the widest possible ranges that is conceivable, and the embodiment presented herein are only an explanation of the chosen embodiments based on all possible combinations of the embodiment. Therefore, the applicant's intention is that the attached claims are not limited by the selection of examples which illustrate the features of the present invention. Some numerical ranges used in the claims also include subranges within them, and changes within these ranges should be interpreted as being covered by the attached claims where possible.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022112245930 | Oct 2022 | CN | national |
| 2023103463184 | Apr 2023 | CN | national |
