CN116328047B

CN116328047B - Precise medical catheter

Info

Publication number: CN116328047B
Application number: CN202211659796.2A
Authority: CN
Inventors: 赵俊春; 曹媚; 张慧敏; 王甲松; 黄健洪
Original assignee: Shanghai Qizhi Medical Technology Co ltd
Current assignee: Shanghai Qizhi Medical Technology Co ltd
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-10-10
Anticipated expiration: 2042-12-22
Also published as: CN116328047A

Abstract

The invention provides a precise medical catheter, which is prepared through the following steps: 1) Preparing a coating mixture: adding carboxylated multiwall carbon nanotubes, zinc oxide and a cross-linking agent into a mixture of silicon rubber and a vulcanizing agent, and uniformly stirring; 2) Coating the coating mixture on the inner surface and/or the outer surface of a common medical catheter, and curing at normal temperature to obtain a coated medical catheter; and 3) modifying the coated medical catheter with a short peptide, wherein the short peptide has an amino acid number between 6 and 20 and contains 2-3 acidic amino acids. The precise medical catheter provided by the invention has the advantages of protein adsorption resistance, antibacterial effect and good biocompatibility.

Description

Precise medical catheter

Technical Field

This disclosure relates to medical catheters, and more particularly to precision medical catheters that prevent bacterial adsorption and protein adsorption.

Background

In clinic, silicone tubing is often used in human body cavity surgery, but the silicone tubing can contact human tissue or body fluid when it is inserted into the human body, resulting in adsorption of proteins or other substances. In addition, bacteria are easy to adsorb and grow on the surface of the silica gel tube material, so that infection can be caused, and the tube diameter is seriously reduced or even blocked (such as a catheter or a nasal intestinal tube which resides in a human body for a long time).

In chinese patent publication CN108463489a, a silica gel tube containing Carbon Nanotubes (CNT) and zinc oxide (ZnO) with bacteriostatic ability is described, and when we studied this silica gel tube, it was found that bacteriostatic ability was significantly affected when it was contacted with a protein-containing solution, and biocompatibility was still to be improved.

Disclosure of Invention

Provided herein is a precision medical catheter prepared by the steps of:

1) Preparing a coating mixture: adding carboxylated multiwall carbon nanotubes, zinc oxide and a cross-linking agent into a mixture of silicon rubber and a vulcanizing agent, and uniformly stirring;

2) Coating the coating mixture on the inner surface and/or the outer surface of a common silica gel catheter, and curing at normal temperature to obtain a coated medical catheter; and

3) The coated medical catheter is modified with a short peptide, wherein the number of amino acids of the short peptide is between 6 and 20 and contains 2-3 acidic amino acids.

In some embodiments, the carboxylated multi-walled carbon nanotubes and zinc oxide in step 1) are both 2% by weight of the mixture of silicone rubber and vulcanizing agent.

In some embodiments, step 1) comprises: the Dow Corning C6-150 silicone rubber and benzoyl peroxide as vulcanizing agent are mixed in a weight ratio of 100 to 1.5, and 4% of multi-wall carbon nano tube and zinc oxide mixed in a weight ratio of 1 to 1 and 2.5% of methyl trimethoxysilane as cross-linking agent are added into the mixture.

In some embodiments, the acidic amino acid is glutamic acid.

In some embodiments, the short peptide does not contain a basic amino acid.

In some embodiments, the short peptide contains only glycine and glutamic acid.

In some embodiments, the short peptide has a sequence selected from the group consisting of SEQ ID NOs: 18. 19 and 22.

The term "conventional silicone tubing" as used herein is intended to distinguish it from the precision medical tubing of the present invention by having no or less antibacterial and/or protein-adsorbing capacity than the precision medical tubing of the present invention, and includes various commercially available medical silicone tubing, or even non-medical silicone tubing.

The precise medical catheter provided by the invention has the effects of resisting protein adsorption and inhibiting bacteria, and can be used as a medical catheter in clinic, such as a catheter, a nose intestinal canal, a hemodialysis catheter (short-term or long-term), a sheath tube for intravascular interventional treatment and the like.

Drawings

FIG. 1 is a graph of cell proliferation plotted against time of incubation and absorbance values reflecting the biocompatibility of various silica gel tubes with Jurkat cells.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The present invention generally provides a precision medical catheter having anti-protein adsorption and antibacterial properties (e.g., inhibiting the growth of E.coli and P.aeruginosa on its surface to form a biofilm) that is not amenable to occlusion during prolonged residence in the body. The precise medical catheter has good biocompatibility and can be applied to various clinical aspects.

The present invention will be described in detail with reference to the following examples.

EXAMPLE 1 preparation of silica gel tube containing carbon nanotube-ZnO

A silica gel tube sample was prepared with reference to the method described in chinese patent publication CN108463489 a. The preparation method is briefly described as follows: the daokanning 184 is mixed with: curing agent 10:1, adding 4% by weight of multiwall carbon nanotube (MWCNT, CNT104 of Beijing Kodak island gold technology Co., ltd.) and zinc oxide (ZnO) mixed in a weight ratio of 1:1, uniformly stirring, and curing and molding at 75 ℃ for 2 hours in a mold to prepare a sample of MWCNT-ZnO silica gel tube (inner diameter 4mm, tube wall thickness 0.6 mm). Meanwhile, a silica gel tube sample containing no MWCNT and ZnO was prepared as a control.

Example 2 Effect of proteins on bacteriostasis of nanotube-ZnO containing silica gel tube samples

When we studied the bacteriostatic action of the MWCNT-ZnO silicone tube prepared in example 1, it was found that it had a inhibitory action on bacterial adsorption, but at the same time, it was found that the bacteriostatic effect of the MWCNT-ZnO silicone tube exhibited a significant difference when the experimental bacteria used were cultured with LB medium and with M9 basal medium, respectively. Considering that one of the main differences in the components of these two media is that the LB medium contains peptone component and the M9 basal medium does not contain this component, we therefore suspected that the protein component in the medium introduced on the MWCNT-ZnO silicone tube may affect its bacteriostatic effect when determining the bacteriostatic effect, and subsequently confirmed the idea by the following experiment.

The experimental groupings and the bacteria used (E.coli (E. Coli) or Pseudomonas aeruginosa (P. Aeromonas)) and the culture medium types are shown in Table 1 below. When Albumin (ALB) was added, its concentration in M9 medium was 30mg/L.

TABLE 1

Group of	Type of silicone tube	Bacterial species	Type of culture medium
				E1#	MWCNT-ZnO silica gel tube	E.coli	LB
E2#	MWCNT-ZnO silica gel tube	E.coli	M9
				E3#	MWCNT-ZnO silica gel tube	E.coli	M9+ALB
E4#	Control silica gel tube	E.coli	LB
				P1#	MWCNT-ZnO silica gel tube	P.aeruginosa	LB
P2#	MWCNT-ZnO silica gel tube	P.aeruginosa	M9
				P3#	MWCNT-ZnO silica gel tube	P.aeruginosa	M9+ALB
P4#	Control silica gel tube	P.aeruginosa	LB

The test method comprises the following steps: the experimental silicone tube and culture medium were steam sterilized or bag filter sterilized prior to the experiment. To different wells of a 24-well flat bottom cell culture plate, 2mL of different kinds of media was added, and 1 silica gel tube (sheared to a length of 10 mm) prepared in example 1 was placed into each well. 10. Mu.L of bacterial suspension was added to each well to give a final concentration of 3.5X10 of bacteria in the well ⁵ cfu/mL, cultured at 37 ℃. Half of the medium was replaced every 24 hours (1 mL of culture was aspirated and 1mL of fresh corresponding medium was added). After 5 days of incubation, the silicone tubes were removed, each tube was rinsed 4 times with 2mL of 0.9% physiological saline, the non-adherent bacteria and other impurities were washed off, and placed in a tube containing 1mL of M9 medium, and sonicated (120W, 15 minutes) to release the bacteria adhering to the inner and outer surfaces of the tube from the tube into the M9 medium. 0.2mL of the fungus-containing M9 culture medium is taken to be subjected to 10-time gradient dilution by PBS buffer solution, 0.1mL of the dilution solution is taken to be uniformly mixed with LB agar culture medium cooled to about 40 ℃, the mixture is placed at 37 ℃ for culture for 24 hours, the growth condition of colonies is observed by naked eyes, and the number of the colonies is counted.

Experimental results: the amount of bacteria adsorbed on the surface of the silicone tube (average value of 3 wells) is shown in Table 2.

TABLE 2

As can be seen from the results in Table 2, E4 group had approximately 8 times the number of E1 group as compared to E1 group (1.55x10 ⁷ /1.87x10 ⁶ ) The method comprises the steps of carrying out a first treatment on the surface of the The number of P4 colonies was about 4 times that of P1 colonies compared with P1 colonies (9.36x10 ⁶ /2.37x10 ⁶ ) The MWCNT-ZnO silicone tube has obvious antibacterial or bacteria adsorption preventing effect compared with a control silicone tube. However, comparison of the results of E1 and E2 groups and P1 and P2 groups showed that E.coli and P.aeruginosa could still adsorb on the surface of the silica gel tube in large amounts when grown in LB medium, and the number of colonies produced was much more than that of M9 medium, 13 times that of the latter (1.87x10 ⁶ /1.43x10 ⁵ ) And 67 times (2.37x10) ⁶ /3.53x10 ⁴ ). We found in additional experiments that E.coli and P.aeruginosa were grown at different rates in LB and M9 media, and were inoculated in equal amounts (final concentration 3.5X10) ⁵ cfu/mL), the amount of E.coli in LB medium was about 1.5 times the amount in M9 medium, at 24 hours, when cultured at 37℃for 12 hours and detected by OD600The number of the culture medium is about 1.7 times of that in the M9 culture medium at all times; when cultured at 37℃for 12 hours and detected by OD600, the Pseudomonas aeruginosa was found to be about 7 times the amount of M9 medium in LB medium, and about 5 times the amount of M9 medium in 24 hours. Obviously, the great difference of the amount of bacteria adsorbed by the silica gel tube in different culture mediums cannot be completely attributed to the difference of the amount of bacteria in the culture mediums, wherein the silica gel tube has stronger antibacterial effect in the M9 culture medium, which indicates that the bacteria cultured in the M9 culture medium are not easy to be adsorbed on the silica gel tube. However, comparing the E2 group with the E3 group and the P2 group with the P3 group, it was found that the number of bacteria adsorbed by the silica gel tube was significantly increased by about 4 times the number of M9 medium (5.56x10 ⁵ /1.43x10 ⁵ ) And about 17 times (6.08 x10 ⁵ /3.53x10 ⁴ ) This suggests that the presence of albumin in the medium does promote the adsorption of bacteria by the silicone tube or inhibits the antibacterial effect that the silicone tube would otherwise have.

We analyzed that the carbon nanotubes themselves had an adsorption effect on proteins, and when the silicone tube was in contact with proteins present in the medium, the proteins were rapidly adsorbed to the surface thereof to constitute a thin protein layer, which inhibited the antibacterial effect of the silicone tube itself.

EXAMPLE 3 bacteriostatic action of short peptide modified MWCNT-ZnO Silicone tube

When used in a patient, the catheter is in contact with blood, interstitial fluid, urine or the like of the patient, wherein the protein present therein affects the bacteriostatic effect of the MWCNT-ZnO silica gel catheter after being adsorbed thereto, and therefore, it is necessary to inhibit the adsorption of the protein by the MWCNT silica gel catheter. The MWCNT-ZnO silica gel tube prepared in example 1 was surface-modified with various polymer materials, such as polyethylene glycol, chitosan, polydopamine, polyacrylate, etc., in hope of inhibiting protein adsorption, thereby eliminating the influence of protein on bacteriostasis. As a result, it was found that the bacteriostatic ability of the surface-modified MWCNT-ZnO silicone tube was almost lost (compared with the control silicone tube without carbon nanotube-ZnO). However, in subsequent experiments, we have unexpectedly found that the effect of albumin on the bacteriostatic action of MWCNT-ZnO silica gel tubes can be reduced when the MWCNT-ZnO silica gel tubes are modified with short peptides. Subsequently, we designed a number of different short peptides in order to be able to screen for short peptides that fully retain the bacteriostatic ability of MWCNT-ZnO silicone tubes.

MWCNT-ZnO silica gel tube sample preparation

The MWCNT-ZnO silica gel tube sample preparation was performed essentially as in example 1, except that carboxylated multiwall carbon nanotubes (CNT 304, of the gold technology ltd of beijing) were selected for the convenience of short peptide coupling.

Preparation of short peptides

A variety of short peptides were prepared by artificial synthesis, and the amino acid sequences thereof are shown in Table 3 below.

TABLE 3 Table 3

Preparation of MWCNT-ZnO silica gel tube sample modified by short peptide

The MWCNT carboxyl on the surface of the MWCNT-ZnO silica gel tube is activated by EDC-NHS, then excessive short peptide is added, stirring reaction is carried out for 24 hours, and then unreacted polypeptide is washed off.

Antibacterial effect of MWCNT-ZnO silica gel tube sample modified by short peptide

The amount of bacteria adsorbed on the surface of the silica gel tube sample (in cfu/mL of the bacteria-containing M9 medium obtained by ultrasonic cleaning) was measured as described in example 2, and the fold difference in the amount of bacteria adsorbed on the surface of the silica gel tube in the M9 medium and the M9 medium supplemented with albumin (30 mg/L) was calculated, and the results are shown in Table 3. When designing a short peptide sequence, the influence of acidic amino acid (glutamic acid), basic amino acid (lysine) and chain length on bacteriostasis is focused on. As can be seen from the results in Table 3, when a basic amino acid is contained in the peptide chain, the adsorption of Escherichia coli can be generally inhibited, but the adsorption of P.aeromonas is not substantially affected or tends to be promoted. When acidic amino acids are contained, it is generally advantageous to inhibit adsorption of both bacteria, but as the chain length increases (especially over 25 amino acids), the inhibition ability decreases. Advantageous for bacteriostasis are short peptides with chain lengths between 5 and 20 amino acids, in particular between 6 and 14 amino acids, and containing 2 to 3 acidic amino acids. The most preferred sequences are: GGGGGGGGGEGEGE (SEQ ID NO: 19), GGGEGEGE (SEQ ID NO: 18), and GEGEGEGEGEGEGE (SEQ ID NO: 22).

We then determined the amount of protein adsorbed on the surface of the silica gel tube by the biquinolinecarboxylic acid method (BCA). The silica gel tube was incubated with M9 medium added with albumin (30 mg/L) at 37℃for 3 days, the silica gel tube was taken out, washed 3 times with PBS buffer, the washed silica gel tube was placed in a test tube containing 1mL of PBS, and subjected to ultrasonic vibration (120W, 15 minutes), the silica gel tube was taken out, a solution of biquinolinecarboxylic acid and copper sulfate was added to the test tube, the incubation was performed at 37℃for 30 minutes, the absorbance value was measured at 560nm, and the protein adsorption amount was converted by a standard curve. The amount of albumin adsorbed by the MWCNT-ZnO silica gel tube without the short peptide modification was calculated as 100%, and the amount of albumin adsorbed by the MWCNT-ZnO silica gel tube with the short peptide modification is shown in Table 3.

The results in table 3 show that the silica gel tube samples can substantially inhibit the adsorption of albumin on the surface of the silica gel tube samples after being modified by short peptides with proper lengths, and the adsorption capacity of the albumin is enhanced along with the increase of the chain length of the short peptides. However, as shown in the results of Table 3, the bacteriostatic ability is not directly related to the adsorption amount of albumin, and when the peptide chain is too long, for example, more than 20 amino acids, the adsorption amount of albumin can be inhibited, but the adsorbed bacterial amount is not reduced, which means that the bacteriostatic ability of the MWCNT-ZnO silica gel tube is inhibited when the chain length is increased; too short a peptide chain, e.g., less than 5 amino acids, often makes it difficult to inhibit adsorption of albumin on the surface of a silica gel tube. The effect of short peptides containing basic amino acids on e.coli and p.aerobosa adsorption is different and may be related to the difference in surface substances of the bacteria themselves.

Example 4 Silicone tube with coating

In view of the high price of carbon nanotubes themselves, the cost of the final product is too high when used for manufacturing medical catheters, and we have attempted to reduce the cost by adding a thin coating on the surface of a conventional silicone tube.

The preparation process of the silica gel tube sample with the coating is as follows:

1) Silica gel tube without MWCNT-ZnO was prepared as described in example 1; alternatively, silicone catheters (Weili medical, 16 FR) purchased in the market are used, all sheared to a length of 1 mm;

2) Preparing a coating mixture: mixing silicone rubber (dakaning, C6-150) with vulcanizing agent (benzoyl peroxide) at a weight ratio of 100:1.5, adding 4% by weight of multiwall carbon nanotubes (MWCNT, CNT104 of Beijing Kodado island gold technology Co., ltd.) and zinc oxide (ZnO) mixed at a weight ratio of 1:1, and 2.5% by weight of crosslinking agent (methyltrimethoxysilane (MTMS)) to the mixture, and stirring the mixture uniformly;

3) Uniformly coating the prepared coating mixture on the inner surface and the outer surface of the silica gel tube prepared in 1), standing for more than 2 hours at room temperature, and finally forming a coating with the thickness of about 0.1 mm;

4) The short peptide modification was performed as described in example 3 using a short peptide of GGGGGGGGGEGEGE (SEQ ID NO: 19).

And simultaneously preparing a coated silicone tube which is not modified in the step 4) as a control.

The antibacterial ability of the prepared coated MWCNT-ZnO silicone tube was tested using the method described in example 3, and the results are shown in tables 4 and 5.

TABLE 4 Table 4

TABLE 5 amount of bacteria adsorbed on the surface of silica gel tube (average value of 3 wells) expressed as cfu/mL in the M9 medium containing bacteria obtained by ultrasonic cleaning

The short peptide modified silica gel tube produced about 1.6 times (E.coli) and about 2.1 times (P.aeroginosa) the number of colonies in the M9 medium containing albumin compared to the M9 medium without the short peptide modified silica gel tube, and similar to the results of example 3, the prepared coated silica gel tube still had strong antibacterial ability and was able to resist the influence of albumin in the environment (Table 4).

From the results shown in Table 5, the coated silicone tube prepared had extremely remarkable antibacterial ability as compared with the conventional silicone tube, and the adsorbed Escherichia coli and Pseudomonas aeruginosa were about 1/20 of those of the commercially available silicone tube (3.03x10, respectively ⁵ /6.16x10 ⁶ ) And about 1/28 (8.77 x 10) ⁴ /2.47x10 ⁶ )。

Example 5 biocompatibility testing

The MWCNT-ZnO silicone tube prepared in example 1 and the silicone tube without MWCNT-ZnO, the short peptide-modified MWCNT-ZnO silicone tube prepared in example 3 and the commercially available silicone tube modified with short peptide and having a coating prepared in example 4 were evaluated for biocompatibility by MTT assay method.

The experimental process comprises the following steps: into the wells of a 24-well flat bottom cell culture plate, a silicone tube (10 mm) was placed, 1mL of cell culture medium (RPMI 1640 containing 10% fetal bovine serum) was added, and 8X10 was inoculated into each well ⁴ Jurkat cells (100. Mu.L) at 37℃and 5% CO ₂ Culturing under the condition. Each group was provided with 5 duplicate wells and the experiment was repeated 3 times. Absorbance values (570 nm) of each well were measured by MTT method at 0, 12, 24, 36 and 48 hours of culture, respectively, to reflect the proliferation of cells. Wells without silicone tubes were used as controls. Cell proliferation curves were plotted with incubation time and absorbance values, and the results are shown in fig. 1.

As can be seen from FIG. 1, the MWCNT-ZnO silica gel tube significantly affected Jurkat cell proliferation, and the MWCNT-ZnO silica gel tube and coated silica gel tube modified with the short peptide substantially restored cell proliferation capacity, which was similar to that of the control group, relative to the silica gel tube without MWCNT-ZnO.

The experimental proteins in the above examples were all albumin, and those skilled in the art will appreciate that similar results can be obtained when experiments were performed with other proteins.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; those of ordinary skill in the art will appreciate that: the technical schemes described in the foregoing embodiments may be modified, or some or all of the technical features may be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the invention, and they should be included in the scope of the present disclosure.

Claims

1. The precise medical catheter is characterized by being prepared by the following steps:

2) Coating the coating mixture on the inner surface and/or the outer surface of a common medical catheter, and curing at normal temperature to obtain a coated medical catheter; and

2. The precision medical catheter according to claim 1, wherein the carboxylated multi-walled carbon nanotubes and zinc oxide in step 1) are each 2% by weight of the mixture of silicone rubber and vulcanizing agent.

3. The precision medical catheter according to claim 1, wherein step 1) comprises: the Dow Corning C6-150 silicone rubber was mixed with the vulcanizing agent benzoyl peroxide in a weight ratio of 100 to 1.5, to which 4% by weight of the silicone rubber in a weight ratio of 1:1 weight percent of mixed multi-wall carbon nano tube and zinc oxide and 2.5 weight percent of cross-linking agent methyltrimethoxysilane.

4. The precision medical catheter of claim 1, wherein the acidic amino acid is glutamic acid.

5. The precision medical catheter according to claim 1, wherein the short peptide is free of basic amino acids.

6. The precision medical catheter according to claim 1, wherein the short peptide contains only glycine and glutamic acid.

7. The precision medical catheter of claim 1, wherein the short peptide has a sequence selected from the group consisting of SEQ ID NOs: 18. 19 and 22.