Patent Application
20220202996
One or more embodiments relate to a medical implant capable of inducing an anti-inflammatory response due to the surface-modification with a functional polypeptide. This application claims priority to Korea Patent Application No. 10-2019-0053898, filed on May 8, 2019, the disclosure of which is incorporated by reference herein in its entirety.
The most common local complication associated with implants made of silicon material is capsular contracture. Capsular contracture accounts for 10.6% of complications, which may occur especially in patients who have undergone breast augmentation. Capsular contracture may cause a fibrous foreign body reaction and pain, due to various factors that promote hardening and tightening of a film at the contact site between the tissue and the implant.
Although the pathogenesis of capsular contracture has not been fully identified, the cellular composition of the implant film, including macrophages, fibroblasts, and lymphocytes, appears to promote the progression of fibrous globular formation. Therefore, it is necessary to develop an implant that does not cause capsular contracture and reduces the possibility of inflammation by inducing anti-inflammatory responses.
One or more embodiments include a medical implant including: an implant base having a surface made of a silicon material; a linker having one end attached onto the surface of the implant base; and a cytokine bound to another end of the linker.
Other objectives and advantages of the present application will become more apparent from the following detailed description in conjunction with the appended claims and drawings. Content not described in this specification will be omitted because it can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present application.
Descriptions and embodiments provided in this application may also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it shall not be seen that the scope of the present application is limited by the detailed description described below.
One or more embodiments include a medical implant including: an implant base having a surface made of a silicon material; a linker having one end attached onto the surface of the implant base; and a functional polypeptide bound to another end of the linker.
The term “implant” used herein refers to all transplantable materials or implants that can be used for skin depressions or dents due to wrinkles, etc., and can be used to improve volume for cosmetic purposes. The term “medical implant” includes implants that restore human tissue when the human tissue is lost, and medical devices or instruments that are temporarily or permanently introduced into mammals to prevent or treat abnormal medical reactions. In addition, the implants may include any that is introduced subcutaneously, transdermally, or surgically and is left in organs such as arteries, the veins, ventricles, and the atria, or the tissues or lumens of organs.
The basic material of the implant may include one selected from ultra-high molecular weight polyethylene (UHMWPE), poly ether ether ketone (PEEK), polyurethane, silicon elastomer, bioabsorbable polymer, aluminum oxide, zirconium, physiologically active glass fiber, silicon nitrogen compounds, calcium phosphate, and carbon.
Silicone may be a polymer based on a bond between silicon and oxygen (—Si—O—Si—O—). A siloxane bond (—Si—O—Si—O—) is formed when methyl chloride (CH3Cl) is reacted with crystalline silicon to synthesize dimethyldichlorosilane and then hydrolyzed. According to this polymerization method, various kinds of polymers can be synthesized. A typical example is a silicone resin composed of linear polydimethylsiloxane and oligosiloxane molecules. Silicon is a colorless and odorless insulator that oxidizes slowly and is stable at high temperatures. Silicon may be used in lubricants, adhesives, gaskets, and molding artificial prostheses. The term “linker” used herein refers to a linkage that connects two different fusion partners (for example, biological polymers, etc.) using a covalent bond. The linker may be a peptide linker or a non-peptide linker, and in the case of a peptide linker, the linker may consist of one or more amino acids.
The term “functional polypeptide” used herein refers to a polypeptide having a biological function or activity that is identified through a definitive functional assay and is associated with a specific biological, morphological, or phenotypic change in a cell. The functional polypeptide may be derived from any species. The functional polypeptide may be either in the native or non-natural form thereof. A native functional polypeptide refers to a peptide that exists in nature. On the other hand, a non-natural functional polypeptide refers to a mutant polypeptide derived by introducing an appropriate mutation (addition, deletion, or substitution of amino acids) to an amino acid sequence as long as the unique function of the functional peptide is maintained based on the amino acid sequence of the native functional polypeptide. In an embodiment, the functional polypeptide may be one or more selected from proteins, cytokines and chemokines.
According to an embodiment, the medical implant may suppress the inflammatory response that may occur in a subject upon contact with the implant because a functional polypeptide, such as IL-4, attached onto the surface of the implant induces the differentiation of anti-inflammatory cytokines. Therefore, the medical implant may replace the existing implant that may cause inflammation.
The term “cytokine” used herein is a small cell-signaling protein molecule secreted by a plurality of cells, and refers to a signaling molecule widely used for information exchange within a cell. Cytokine may include monokines, lymphokines, traditional polypeptide hormones, etc., and may include tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), transforming growth factor (TGF) (for example, TGF-α or TGF-β), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), Interleukin-1 (IL-1), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or IL-13. Cytokines may also include recombinant cell cultures and biologically active equivalents of a cytokine from natural sources or of native sequence cytokines.
The term “chemokine” used herein refers to a basic heparin-binding small-molecule protein that acts on leukocytic emigration and activation. There are four cysteine residues in the chemokine molecule, and the chemokine can be classified into four subfamilies: CXC(CXCL), CC(CCL), CX3C(CX3CL) and C(XCL) according to the type of existence of the first two cysteine residues in the molecule. Currently, more than 40 species have been identified.
In an embodiment, the surface of the implant base may include a shell made of a silicon material.
The term “shell” refers to an external part of an implant made to enable organ transplantation, and refers to a pouch in which a fluid material can be filled in the shell.
In an embodiment, the cytokine may include at least one selected from IL-4, IL-10, and IL-13.
IL-4 is a cytokine that induces differentiation of inactive helper T cells (Th0 cells) into Th2 cells. Th2 cells activated by IL-4 may produce additional IL-4. Th2 cells may secrete IL-4 to develop M2 macrophages. The “macrophages” used herein are cells that are distributed in all tissues in a living body and responsible for immunity, and refer to cells involved in the removal of invading pathogens, removal of virus-infected autologous cells and cancer cells, and induction of an inflammatory response. Macrophages can be categorized according to the process of development: tissue-resident macrophages differentiated from the yolk sac or fetal liver, and monocyte-derived macrophages differentiated from monocytes (differentiated from bone marrow cells) in the blood by inflammatory reactions or pathogen invasion. Tissue-resident macrophages and monocyte-derived macrophages may be differentiated, by cytokines, into M1 macrophages and M2 macrophages that act on different immune responses. M1 macrophages are induced by IFN-γ and TNF-α, which are cytokines of Th1 cells, and act on induction of Th1 response, induction of inflammatory response, inhibition of cancer growth, and the like. M2 macrophages are induced by IL-4, IL-10, etc., which are cytokines of Th2 cells, and may act in Th2 response induction, inflammatory response inhibition, damaged tissue repair, and the like. The increase in M2 macrophages is associated with the secretion of IL-10 and TGF-β, and as a result, pathological inflammation may be reduced.
IL-10 is a functional Th2 cell cytokine, which has the functions of replication of M1 macrophages, monocytes, and T-cell lymphocyte, and inhibition of secretion of inflammatory cytokines (IL-1, TNF-α, TGF-β, IL-6, IL-8, and IL-12). In an embodiment, the linker may be represented by Formula 1.
wherein, in Formula 1,
A is an implant with a silicon surface,
B is the end of the functional polypeptide, and
n is an integer from 5 to 15.
In an embodiment, the medical implant may induce the secretion of anti-inflammatory cytokines. Cytokines such as IL-4 or IL-10 may be attached onto the surface of the medical implant, which is associated with the activity of M2 macrophages as described above. When the M2 macrophages are increased, the secretion of anti-inflammatory cytokines such as IL-10 and TGF-β may be induced, so that the medical implant may suppress the inflammatory response.
In an embodiment, the process of attaching the functional peptide onto the surface of the medical implant having a silicon material may be performed by the process illustrated in
In this regard, in the process of treating the oxygen plasma, the amount of energy and the treatment time may be appropriately changed as long as a sufficient amount of hydroxyl groups can be introduced onto the surface of the silicon material. For example, the amount of energy may be about 1000 W to about 50 W, about 900 W to about 150 W, about 800 W to about 250 W, about 700 W to about 350 W, or about 600 W to 450 W, and the like, and the treatment time may be from about 30 minutes to about 2 minutes, about 25 minutes to about 4 minutes, about 20 minutes to about 6 minutes, about 15 minutes to about 8 minutes, about 10 minutes to about 9 minutes, etc., but is not limited thereto. In addition, in the process of sequentially adding APTMS and Bis-dPEG®5-NHS ester to the surface of the silicon material, the reaction time and the concentration may also be suitably changed depending on the purpose and aspect of use.
In an embodiment, the medical implant may be a breast implant.
The ‘breast implant’ may be an implant that can be implanted into a patient in breast-related surgery and treatment. The inside of the shell of the breast implant may be filled with saline, hydrogel, or silicone gel as the filler. In addition, the breast implant may be a round-type implant and an anatomical-type implant depending on the shape thereof, and may be a smooth-type implant and a texture-type implant depending on the surface condition.
In an embodiment, the breast implant may inhibit the formation of breast capsular contracture. The term “capsular contracture” used herein refers to a condition in which the film around the transplanted implant becomes thick and hard due to excessive fibrosis, and is one of the side effects occurring during transplantation. Macrophages may be the main cause of this capsular contracture at the site of the implant. IL-4 or IL-10 attached onto the breast implant may induce the activation of M2 macrophages and the secretion of anti-inflammatory cytokines thereby. In this way, the breast implant may inhibit the formation of breast capsular contracture.
In an embodiment, the breast implant may have an Rq value of about 4 nm to about 10 nm. For example, the Rq value may be from about 4 nm to about 9 nm, about 4 nm to about 8 nm, about 4 nm to about 7 nm, about 4 nm to about 6 nm, about 4 nm to about 5 nm, about 5 nm to about 9 nm, about 5 nm to about 8 nm, about 5 nm to about 7 nm, about 5 nm to about 6 nm, about 6 nm to about 9 nm, about 6 nm to about 8 nm, or about 6 nm to about 7 nm. The Rq value refers to the mean square roughness value. After the surface of the implant is treated with oxygen plasma, 3-aminopropyltrimethoxysilane (APTMS) and cytokines are added and attached thereonto to give effective roughness to the surface of the breast implant. Due to this surface roughness, the movement of the implant after transplantation may be prevented through attachment to the breast tissue, and the formation of capsular contracture may be suppressed.
A medical implant according to an aspect can inhibit the formation of capsular contracture, which is one of the complications that may occur in patients undergoing breast implant transplantation, by inducing the differentiation of anti-inflammatory cytokines by a functional polypeptide, such as IL-4, attached onto the surface of the implant.
Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are for illustrative purposes of the present disclosure, and the scope of the present disclosure is not limited to these examples.
RAW 264.7 cells were purchased from the American Type Culture Collection (Rockville, Md., USA) and incubated in a medium-supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Gibco, Carlsbad, Calif.) under a humidified condition containing 5% CO2 at a temperature of 37° C.
After 24 hours, RAW 264.7 cells were dissociated with cold PBS and homogenized on ice using cell lysis buffer (Cell Signaling Technology, Danvers, Mass., USA). After heating the sample at a temperature of 95° C. for 5 minutes and cooling briefly on ice, 30 μg of protein was loaded onto a 10% SDS-PAGE polyacrylamide gel. After gel electrophoresis, the gel was transferred to a nitrocellulose membrane (GE Healthcare, Piscataway, N.J., USA). The membrane was blocked with 5% BSA in PBS for 2 hours at room temperature, and primary antibodies against iNOS (Abcam, Cambridge, UK), Arg-I (Santa Cruz, Calif., USA) and control GAPDH were incubated overnight at a temperature of 4° C. After washing 4 times with PBS-T (pH 7.4), the cell membrane was diluted 1:2,000 with HRP-conjugated an anti-mouse or anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) and stored at room temperature for 2 hours. Next, the membrane was washed 4 times with PBS-T. A Western blot detection kit (EZ-Western Lumi pico, Dogen, Korea) was used for protein detection. Finally, protein was quantified in the blot, and analysis for densitometry of the blot was performed in Image J (Image J, National Institutes of Health, USA). Relative quantitation was calculated after being converted to GAPDH levels. The above analysis method was repeated twice.
Cells were washed 3 times with PBS (pH 7.4) for 5 min each. Then, the slides were treated with a blocking solution (0.2% Triton X-100, 1% BSA in PBS) for 1 hour to block non-specific antigen bindings. The slides were then incubated overnight with diluted primary antibody. The next day, after washing 3 times with PBS, the plate was incubated at room temperature for 1 hour with a secondary antibody diluted 1:2000. Then, the slides were thoroughly washed with PBS and then staining was performed thereon using DAPI (DAPI, VECTASHIELD, Vector Laboratories, USA) to stain cell nuclei. Images were then taken using a z-stack with a confocal microscope.
RNA of RAW 264.7 cells was extracted according to the instructions of the RNA extraction kit (easy-BLUE RNA extraction kit, iNtRON Biotechnology, Gyeonggi-do, Korea). RNA was quantified with a spectrometer (Nanodrop 1000, Wilmington, Del.). From 2 μg of RNA, 20 μl of cDNA was synthesized using reverse transcriptase (AccuPower® RT PreMix, Bioneeer Corporation, Daejeon, Korea) according to the manufacturer's instructions. The reaction was performed using an ABI 7500 Real-Time PCR System (Applied Biosystems). The expression level of the gene was normalized using GAPDH mRNA. Expression levels presented were the mean values of each sample. In the case of RT-PCR, the annealing temperature for IL-6 and GAPDH was 62° C. The resultant product was electrophoresed on a 2% agarose gel and stained with ethidium bromide.
Each data was presented as mean±standard error (SEM). One-way ANOVA was used for multi-group comparisons after Tukey's test. Power analysis was applied to determine the difference between the control group and the treatment group. P<0.05 was considered as being significant.
This embodiment was performed to manufacture a medical implant according to an aspect.
As shown in
After coating APTMS, bis-dPEG®5 NHS ester was added to modify the surface. As shown in
In this example, the contact angle was measured to measure the degree of modification of the silicon surface prepared by the method of Example 1. Specifically, as the contact angle, a water contact angle (WCA) was measured using a program (First Ten Angstroms FTA 1000 C Class) in which Sessile drop technology was combined with drop shape analysis software. To measure static advancing contact angles, 2.0 μL of water droplet was added to the droplet every 2 seconds to grow the droplet, and then the droplet was added and within 5 seconds, images thereof were captured. This procedure was repeated 20 times. For the concrete reliability of WCA, the contact angle of a non-ideal surface such as APTMS SAM was calculated using the tangent-leaning method. The WCA of each of: a sample including Si/O2 plasma/APTMS/bis-dPEG®5 NHS ester/IL-4, which was a surface-modified silicon prepared by the method of Preparation Example 1; a control sample including Si, Si/O2 plasma, Si/02 plasma/APTMS, Si/O2 plasma/APTMS/bis-dPEG®5 NHS ester, or Si/02 plasma/APTMS/bis-dPEG®5 NHS ester/(IL-4 or IL-13), was measured. The WCA value is the average of at least three measurements. The measurement results are shown in Table 1.
As shown in Table 1, the untreated silicon surface had strong hydrophobicity, and thus, a large WCA value was measured therefor. When the silicon surface was formed with oxygen plasma, due to the enhanced hydrophilicity caused by the presence of OH— functional groups, the WCA value was decreased rapidly to 0.16°. In addition, as APTMS was introduced to the surface, the WCA value was 97.80°, that is, the hydrophobicity became stronger. After introduction of bis-diPEG®5 NHS ester, the WCA value was 100.60°, that is, the hydrophobicity was further enhanced. Additionally, the WCA values corresponding to the case in which functional polypeptides, IL-4 or IL-13 were introduced, were 78.1° C. and 76.6° C., respectively. That is, hydrophobicity was slightly attenuated, which is considered to be due to the hydrophilicity of cytokines. These WCA values and the changes thereof indicate that the silicon surface was modified step by step.
In this example, atomic force microscopy (AFM) analysis was performed in order to obtain an image of the surface layer of each sample used in Example 2. XE-100 AFM (Park Systems) was used for biofilm imaging, the resonant frequency was 200 kHz to 400 kHz, and the nominal force constant was set to be 42 N/m. Surface imaging was obtained in non-contact mode by using a silicone tip of a 125 μm-long nitride lever coated cantilever (PPP-NCHR 10M; Park Systems). The scan frequency was typically 1 Hz per line. Roughness was calculated with 3 μm×3 μm images. The results are shown in
Regarding the results of
This experimental example was performed to measure the cytotoxicity of a medical implant according to an aspect. In order to measure cell viability as an indicator of cytotoxicity, RAW 264.7 cells were prepared by the method described in Reference Example 1. Cells were divided into two groups, which were then brought into contact with a smooth silicon surface which was not unmodified (smooth), or a silicon surface which was modified with IL-4 prepared according to Example 1 (smooth+IL-4). After detaching cells from each group at time points of 24, 48, and 72 hours, the cells were washed once with PBS. In DMEM medium, 0.5 mg/mL of MTT was added to each well, then the cells were incubated at a temperature of 37° C. for 4 hours, and then the MTT solution was removed. Finally, formazan crystals were dissolved in DMSO and the absorbance thereof at 560 nm was read in a microplate reader (EPOCH2, BioTek). The results are shown in
According to the results shown in
This experimental example was performed to identify whether IL-4 introduced to the silicon surface affects the healing of M2 or M1 wounds and tissue recovery of macrophages and affects the inflammatory immune response. After preparing RAW 264.7 cells by the method described in Reference Example 1, the cells were divided into two groups, which were respectively brought into contact with a non-modified smooth silicon surface, or a silicon surface modified with IL-4 prepared according to Example 1 (smooth+IL-4). Then, the immunofluorescence staining method of Reference Example 3 and Western blotting of Reference Example 2 were performed to confirm the expression levels of genes and proteins. The results are shown in
As shown in
In this experimental example, the production of proinflammatory cytokines IL-6 and TNF-α was measured. Enzyme-linked immunosorbent assay (ELISA) was performed. Captured antibodies were diluted with PBS and coated on a 96-well plate at room temperature for 24 hours. Then, the plate was washed twice with PBS, and blocked with PBS with 10% FBS for 2 hours. After adding the sample extracted from the cell culture supernatant of each of the smooth group and the smooth+IL-4 group thereto, the reaction was performed at room temperature for 2 hours. After treatment with secondary antibodies, substrate reagents were reacted and reading was carried out at a 405 nm wavelength in an ELISA reader (EPOCH2, BioTek). The results are shown in
As shown in
This experimental example was performed to measure the production of IL-4 and IL-10, which are anti-inflammatory cytokines. The measurement method was performed by the methods of RT-PCR and qRT-PCR described in Reference Example 4. The results are shown in the graph of
As shown in
Activation of the STAT6 pathway is an important factor in differentiating macrophages to the M2 type. Accordingly, in this Experimental Example, Western blotting according to Reference Example 2. was performed on STAT6 and pSTAT6 to determine whether IL-4-modified silicon alone could activate the STAT6 pathway. The results are shown in
As shown in
In this experimental example, an animal experiment was performed to measure the in vivo effect of the medical implant. For animal experiments, 10 Sprague-Dawley mice weighing 250 g to 300 g at 9 weeks of age were used. Five animals in each group were randomly distributed into each of two groups. Animals were exposed in a 12/12 h light/dark cycle in specific-pathogen-free (SPF) conditions with free access to food and water. Approval for this protocol was approved by the Bundang Seoul National University Hospital Animal Experiment and Use Committee (approval number: BA1801-240/011-01), and all procedures were in accordance with the guidelines of the NIH. There were an animal group in which an intact silicon was inserted as an implant and an animal group in which silicon modified with IL-4 was inserted as an implant. The former group was used as a control group.
The process of inserting the implant is specifically as follows. The subject mice were anesthetized by inhalation of isoflurane (Hana Pharm, Korea), the hair on the back was shaved, and the surgical site was disinfected with 70% alcohol and betadine. Then, a 2-3 cm incision was made in the back with a #15 scalpel blade, and the implant was inserted into the cortical pouch. The incision site was closed with surgical sutures (Nylon 4/0, Ethicon, USA). The surgical site was disinfected again with 70% alcohol and betadine and a light dressing was applied thereon.
Animals were monitored for 12 weeks after transplantation, confirming the development of cascade inflammation. Therefore, at predetermined time points of 1, 2, 4, 8 and 12 weeks, all animals in each group were tissue biopsied. For biopsies, selected animals were euthanized with carbon dioxide, and tissues and implants in the dorsal region with epidermis, dermis, posterior and anterior capsules were removed.
6.1. Evaluation of Capsular Thickness and Collagen Density In Vivo
The thickness of the capsule tends to be increased over time due to the accumulation of collagen. Accordingly, the in vivo capsular thickness and collagen density were investigated. Capsular thickness was determined by analyzing tissue slides which were H&E-stained using a microscope (LSM 700, Carl Zeiss, Oberkochen, Germany) at 40× magnification. The capsular range was defined from the top of the silicone insertion area to the bottom of the dorsal subcutaneous muscle. To evaluate the overall capsular thickness from the tissue slides, three different parts of the capsule were randomly photographed, and the capsular thickness was measured with ZEN software. The results thereof are shown in
Collagen density was analyzed using image analysis of 5 randomly selected regions on slides stained with MT staining at 40× magnification. Collagen bundles were stained as being blue to analyze the density of collagen over the entire microscopic area with Image J software. The results thereof are shown in
Regarding the results of
The present experimental example was based on the IL-10 surface-modified silicon implant prepared in the same manner as in Example 1, and the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-13 produced in RAW 264.7 cells and the levels of IL-10 and IL-4 were measured. In addition, cytotoxicity evaluation with respect to IL-10 introduced into the silicon surface was performed, and the differentiation level of M2 or M1 macrophages was evaluated, and the effect on wound healing and tissue recovery of macrophages was confirmed. This experiment was performed in the same manner as in Experimental Examples 1 to 3, and the control group was a group (smooth) in which the subject was in contact with a smooth silicon surface, which was not modified.
As a result, as shown in
In the present experimental example, regarding the IL-13 surface-modified silicon implant prepared in the same manner as in Example 1, the level of proinflammatory cytokines TNF-α, and IL-1β produced in RAW 264.7 cells and the level of IL-10 were measured, and the level of Arg-1, a marker of M2 macrophages, was measured. In addition, cytotoxicity evaluation with respect to IL-13 introduced to the silicon surface was performed. This experiment was performed in the same manner as in Experimental Examples 1 to 3, and the control group was a group (smooth) in which the subject was in contact with a smooth silicon surface, which was not modified.
As a result, as shown in
The description of the present disclosure described above is for illustration, and those of ordinary skill in the art to which the present disclosure pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present disclosure. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0053898 | May 2019 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/017471 | 12/11/2019 | WO | 00 |
