Method for analyzing and comparing biomacromolecule-based delivery carrier slow-release antibacterial drug and evaluating antibacterial effect on staphylococcus aureus by isothermal microcalorimetry

Abstract

The invention provides a method for analyzing and comparing biomacromolecule-based delivery carrier slow-release antibacterial drugs and evaluating the antibacterial effect on staphylococcus aureus based on an isothermal microcalorimetric method. The method comprises the following steps: (1) selecting an antibacterial drug sample; (2) carrying out test pretreatment; (3) carrying out an in-vitro drug release experiment; according to the invention, the in-vitro antibacterial activities of different biomacromolecule-based delivery carrier slow-release antibacterial drugs and antibiotic reference substances on staphylococcus aureus are compared, so that the related theoretical blank is filled, and the antibacterial activity of the biomacromolecule-based delivery carrier slow-release antibacterial drugs on pathogenic bacteria is rapidly evaluated; therefore, theoretical support is provided for screening and quality control processes of novel biological macromolecule-based delivery carrier medicine preparations.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis, specifically to an isothermal microcalorimetry method for analyzing and comparing sustained-release antibacterial drugs based on biomolecular delivery carriers and evaluating their antibacterial effects against Staphylococcus aureus. Background Technology

[0002] Roxithromycin (Rox) is a semi-synthetic 14-membered macrolide antibiotic, a derivative of erythromycin, with broad-spectrum antibacterial activity. It is characterized by good oral absorption, high blood concentrations, a long half-life, and stability against gastric acid, and has been widely used clinically. Biomacromolecules refer to biodegradable organic molecules such as proteins, peptides, and nucleic acids with large molecular weights. Biomacromolecule-based (drug) delivery carriers use biomacromolecules as a "coating" to encapsulate drugs and perform delivery. Protein-based drug delivery systems generally have retention effects and targeting properties, which can improve the effective payload pharmacokinetic characteristics and therapeutic index of drugs. There may be efficacy differences between free-state antibacterial drugs (such as roxithromycin) and drug delivery systems encapsulated with different biomacromolecule-based delivery carriers (such as zein and gliadin). Therefore, it is necessary to develop relevant analytical methods to evaluate the antibacterial effects of free-state antibacterial drugs and sustained-release antibacterial drugs with different biomacromolecule-based delivery carriers against Staphylococcus aureus.

[0003] Currently, in vitro release simulation experiments are commonly used to predict the complex metabolic processes of drug delivery systems in the human body, which has significant reference value in drug formulation evaluation and quality control. HPLC exhibits advantages in detecting the release characteristics of drugs in drug delivery systems, offering high selectivity, sensitivity, and accuracy. Using HPLC to determine the cumulative release of drugs at specific time points allows for precise examination of the release properties of drug delivery systems at key time points (such as burst release and plateau phases). This enables comparative analysis of the release rates of free antimicrobial drugs and those of sustained-release drug delivery systems, further determining whether encapsulation of antimicrobial drugs with biomolecular-based delivery carriers facilitates sustained-release or burst release, achieving controlled-release effects. Furthermore, there are many methods for examining the impact of antimicrobial drugs on the metabolic activity of pathogens (Staphylococcus aureus) during growth and reproduction, such as the time-bactericidal curve method and the isothermal microcalorimetry method. The time-bactericidal curve method, as a common drug susceptibility testing method, can observe the metabolic activity of drugs after acting on pathogens within a required time range. However, it suffers from problems such as complex operation, susceptibility of results obtained by optical density measurements to the influence of sample color, solubility, and precipitation, and the long lead time required to obtain results. Isothermal microcalorimetry (IMC) is a technique for real-time monitoring of the thermal metabolic activity of microorganisms during their growth and reproduction. By comparing the growth and metabolic activity of pathogens, the antibacterial effects of different antimicrobial drugs can be reflected. The thermal power of the microorganism (…) P ) over time ( t The change curve of a microorganism can indicate its activity during growth and metabolism. The exponential growth phase of a microorganism satisfies this simple exponential model: P t = P 0exp( k t)——Equation (1), where, P 0 and P t These are the thermal power of the microorganism at the initial point t0 of exponential growth and at time t of growth, respectively. Equation (1) is converted to logarithmic form, i.e.: ln P t =ln P 0+ k t—Equation (2), the biologically significant growth rate constant can be directly obtained through linear regression. k The shape and trend of the heat power-time curve, along with thermodynamic information, can intuitively reflect the antibacterial activity of antimicrobial drugs. Specifically, the length of the stagnation period of microbial growth and the maximum heat power value ( ) are important indicators in the heat power-time curve. P max ) and maximum thermal power output time ( t maxThe relationship between these factors can, to some extent, reflect whether a biomolecular-based delivery carrier possesses the effect of sustained-release antibacterial drugs, which can be strongly verified through in vitro simulated release experiments. Numerous studies have reported the role of microcalorimetry in evaluating the effects of antibiotics on microbial growth and metabolism; however, the IMC method has not yet been used to evaluate the in vitro antibacterial efficacy of antibiotic drug delivery systems, especially sustained-release antibacterial drugs from biomolecular-based delivery carriers. The organic combination of HPLC and isothermal microcalorimetry, on the one hand, simulates the dynamic metabolic process of the drug system in vivo, and on the other hand, reflects the antibacterial activity of the drug delivery system against pathogens. This is of great significance for the screening of biomolecular-based delivery carriers and the quality control of drug formulations. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for evaluating the antibacterial activity of sustained-release antibacterial drugs based on biomolecular-based delivery carriers against Staphylococcus aureus, based on isothermal microcalorimetry, in order to fill the relevant theoretical gaps, achieve rapid evaluation of the antibacterial activity of sustained-release antibacterial drugs based on biomolecular-based delivery carriers against pathogens, and thus provide theoretical support for the screening and quality control of novel drug formulations based on biomolecular-based delivery carriers.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: An isothermal microcalorimetry method for evaluating the antibacterial effects of sustained-release antibacterial drugs delivered by biomolecule-based carriers against Staphylococcus aureus, comprising the following steps: (1) Selection of antimicrobial samples: including antimicrobial samples with biomolecular-based delivery carriers and antibiotic reference standards; (2) Pre-test treatment: including accurately weighing a certain amount of sample and control to prepare test solution, counting the colonies of overnight cultured bacterial culture, and taking overnight cultured bacterial culture to prepare test solution containing sample, control and no antibiotic respectively; (3) Conduct in vitro drug release experiments: At predetermined time points, the concentration of antibiotic drugs is detected by HPLC, and the drug accumulation and release curves of sustained-release antibacterial drugs and free antibiotics with different biomolecular base delivery carriers are obtained. (4) Conduct microcalorimetry experiments: compare the in vitro antibacterial activity of sustained-release antibacterial drugs and antibiotic reference standards against Staphylococcus aureus using different biomolecular delivery carriers.

[0006] The pre-test treatment includes the following steps: (2.1) Accurately weigh the antibacterial drug sample and antibiotic control sample based on the biomolecule delivery carrier, and dilute the sample and control with culture medium and anhydrous ethanol respectively to prepare the stock solution of the predetermined concentration; take a certain amount of the stock solution and add it to the culture medium, mix it thoroughly, and dilute it to prepare the sample and control test solution of the predetermined concentration. (2.2) Count the colonies of the overnight cultured microorganisms; (2.3) Dilute the overnight culture of bacteria with culture medium to obtain a bacterial suspension of a certain concentration, and then add it to the test solution containing antimicrobial drugs to prepare test solutions containing samples and control standards respectively. Combine the colony count results to calculate the concentration of microorganisms in the test solution at this time. (2.4) Dilute the overnight culture of bacteria with a culture medium to obtain a bacterial suspension of a certain concentration, and then add it to the culture medium to prepare a test solution containing bacteria without antibiotics.

[0007] The in vitro drug release experiment includes the following steps: 2 mg / mL of a biomolecular-based delivery carrier antibacterial drug suspension (theoretical drug loading of 48.8 μg / mL) and 2 mL of free antibiotic solution are transferred into a dialysis bag (MWCO, 14 kDa). The dialysis bag is sealed and immersed in a release bottle containing 50 mL of PBS (pH=7.4) release buffer with 0.1% Tween 80. The bottle is then placed in a constant-temperature shaking incubator (37℃, 100 rpm) and continuously shaken. 1 mL of release medium is taken out at time points of 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h, respectively. Simultaneously, an equal volume of fresh release buffer is added at the same temperature. The test solution is then centrifuged (10000 rpm, 5 min) and analyzed by HPLC. The peak area is recorded, and the drug concentration is calculated based on the standard curve. The cumulative release amount of the drug (roxithromycin) is also calculated, and the corresponding drug release curve is plotted.

[0008] The isothermal microcalorimetry experiment includes the following steps: 1.875 mL of sterile LB medium is added to a sterile 4 mL ampoule, followed by 0.125 mL of antibacterial sample and antibiotic control at concentrations of 32, 16, 8, 4, and 2 μg / mL, respectively. A drug blank is used as the control group, resulting in final concentrations of the antibacterial test solution of 2, 1, 0.5, 0.25, 0.125, and 0 μg / mL. Then, the overnight bacterial culture is taken, and the bacterial suspension is adjusted to 0.5% MacFarland (concentration 1×10⁻⁶) using sterile LB medium. 8 (CFU / mL), then diluted tenfold to obtain a concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension: Add 100 μL of the diluted bacterial suspension to each ampoule containing the antibiotic test solution, to achieve a final bacterial concentration of approximately 5.0 × 10⁻⁶.5 CFU / mL. After sealing the ampoule tightly with a capping tool, place it in the equilibrated calorimeter chamber (37℃). Once the instrument baseline stabilizes, start recording the heat (Heat / J) change in the sample cell in real time. After the experiment, obtain a nonlinear curve of heat flow signal changing with time (i.e., thermal power-time curve). Based on the shape and trend of the curve and the thermodynamic information obtained by linear fitting of the exponential phase of the curve, compare the in vitro antibacterial effects of different biomolecular-based delivery carrier sustained-release antibacterial drugs and antibiotic reference standards against Staphylococcus aureus.

[0009] To further optimize the above-mentioned method for evaluating the antibacterial effects of sustained-release antibacterial drugs based on isothermal microcalorimetry as a means of analyzing and comparing their antibacterial efficacy against Staphylococcus aureus, the present invention adopts the following further technical measures: In some embodiments, in step (1), the biomacromolecule-based delivery carrier antibacterial drug sample refers to a nanodelivery system sample that uses naturally derived biomacromolecules as carrier units to encapsulate and deliver antibacterial active drugs, including but not limited to protein-based drug delivery systems (e.g., zein-roxithromycin nanoparticles, malt extract-roxithromycin nanoparticles).

[0010] In some embodiments, in step (2.1), the culture medium is 4.5~5 mL LB medium (pH=7.2~7.4), the concentration of the stock solution includes a standard concentration of 12.8 mg / mL and a sample concentration of 1% (w / v), and the concentration range of the sample and standard test solution is 0.125~2 μg / mL.

[0011] In some embodiments, in step (2.2), the bacterial culture is a Staphylococcus aureus culture, and the colony counting method is the method listed in General Chapter 1105 of the 2015 edition of the Chinese Pharmacopoeia.

[0012] In some embodiments, in step (2.3), the concentration of the bacterial suspension is 1.0 × 10⁻⁶. 7~8 The bacterial suspension volume is 10-300 μL, the final volume of the antibiotic-containing test solution is 2-3 mL, and the bacterial concentration in the test solution is 5 × 10⁻⁶ CFU / mL. 5 ~1.0×10 6 CFU / mL.

[0013] In some embodiments, in step (2.4), the concentration of the bacterial suspension is 1.0 × 10⁻⁶. 7~8The bacterial suspension volume is 10-300 μL, the final volume of the antibiotic-free test solution containing bacteria is 2-3 mL, and the bacterial concentration in the test solution is 5 × 10⁻⁶ CFU / mL. 5 ~1.0×10 6 CFU / mL.

[0014] In some embodiments, in step (3), the dialysis bag is cut to an appropriate length (approximately 10 cm), soaked in deionized water and boiled for 10 min, then soaked in deionized water for 30 min, tested for leakage by adding water, and then soaked in release buffer (PBS) for later use; the HPLC analysis uses a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), a mobile phase of acetonitrile-phosphate buffer (pH 3.0, 60:40 v / v), a flow rate of 1.0 mL / min, an injection volume of 10 μL, a detection wavelength of 210 nm, and a column temperature of 25 °C; the standard curve is prepared by preparing a roxithromycin standard solution (0.1~50 μg / mL), injecting and analyzing the sample, and plotting a peak area-concentration standard curve (R²). 2 ≥0.999); the drug concentration is obtained by injecting and analyzing antibacterial drug samples and antibiotic reference standards under the same chromatographic conditions, recording the peak area, and calculating the drug concentration based on the standard curve; the drug release curve is plotted with time as the abscissa and cumulative release percentage as the ordinate.

[0015] In some implementations, in step (4), the exponential phase of the linear fitting curve is achieved by utilizing the fact that the natural logarithm is the inverse function of the exponential function (lne). x =x), to increase thermal power P Over time t Convert the change curve to logarithmic form ln P t The growth rate constant with biological significance was obtained by using a linear regression model based on the natural logarithm of the curve representing the change over time t. k The thermodynamic information includes the maximum heat flow output ( P max ), maximum heat output time ( t max ), total heat at time T ( Q T ), growth rate constant ( k ), generation time ( G ), inhibition rate ( I ) and half-inhibitory concentration ( Ic 50The parameters can be obtained through thermal power-time curves, linear fitting, and related calculation formulas, and are used as evaluation parameters for antibacterial effects, thereby comparing the antibacterial activity of sustained-release antibacterial drug samples and antibiotic reference standards based on biomolecular delivery carriers.

[0016] In some embodiments, the bacterial strain is Staphylococcus aureus ATCC6538. A single clone culture is picked and placed into 10 mL of fresh LB medium and cultured at 37℃±0.5℃ for 18 h before use.

[0017] In some embodiments, the culture medium is Luria-Bertani medium with pH 7.2-7.4. The liquid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride. A solid culture medium is prepared by adding 1.5% agar to the liquid culture medium.

[0018] In some implementations, the instruments used for microcalorimetry testing are sterilized at 121°C for 15 minutes.

[0019] The beneficial effects of this invention are: Based on isothermal microcalorimetry, this invention provides a rapid and intuitive comparison and evaluation of the inhibitory effects of different biomolecular-based delivery carriers on the metabolic activity of sustained-release antibacterial drugs during the growth and reproduction of Staphylococcus aureus. In practical production applications, evaluating sustained-release antibacterial drugs with good metabolic release processes (controlled release) and superior antibacterial activity using biomolecular-based delivery carriers can play an important role in the screening and quality control of novel drug formulations (materials). Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall process flow of the present invention.

[0021] Figure 2a ~c is the heat power-time curve of the sustained-release antibacterial drug of the biomolecule-based delivery carrier acting on the growth and metabolism of Staphylococcus aureus in Example 1 of the present invention.

[0022] Figure 3 This is a heat power-time curve of the sustained-release antibacterial drug by the biomolecule-based delivery carrier acting on the growth and metabolism of Staphylococcus aureus in Example 2 of the present invention.

[0023] Figure 4 This is an electron micrograph of the cell morphology of Staphylococcus aureus after treatment with a sustained-release antibacterial drug using a biomolecular-based delivery carrier in Example 3 of the present invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be described in detail, clearly, and completely below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Furthermore, based on the embodiments of the present invention, any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to all other embodiments obtained without creative effort should be included within the protection scope of the present invention.

[0025] Example 1 Using free roxithromycin, zein-roxithromycin nanoparticles, and wheat zein-roxithromycin nanoparticles as research objects, isothermal microcalorimetry was applied to study the differences in antibacterial activity of sustained-release antibacterial drugs with different biomolecular-based delivery carriers in inhibiting the growth and metabolism of Staphylococcus aureus under concentration gradient conditions.

[0026] Weigh 0.128 g of roxithromycin and dissolve it completely in 5 mL of anhydrous ethanol. Then add 5 mL of sterile water and vortex for 30 s to obtain a homogeneous 12.8 mg / mL stock solution of the reference standard. Take 50 μL of this stock solution and add it to 4.95 mL of LB medium. Mix well to obtain a 128 μg / mL reference standard solution. Then, continue to use LB medium to prepare reference standard stock solutions of 32, 16, 8, 4, and 2 μg / mL using the two-fold dilution method, respectively, for later use. Separately, weigh 0.05 g of zein-roxithromycin nanoparticles and wheat zein-roxithromycin nanoparticles and dissolve them in 5 mL of LB medium. Vortex for 30 s to obtain a homogeneous 1% (w / v) sample solution. Calculate the encapsulation efficiency of the zein nanoparticles for further dilution. Take 2.623 mL of the 1% (w / v) sample solution and add it to 2.377 mL of LB medium. In LB medium, shake for 30 seconds to obtain a homogeneous sample solution of 128 μg / mL. Then, continue to use LB medium to prepare sample stock solutions of 32, 16, 8, 4, and 2 μg / mL by a two-fold dilution method for later use.

[0027] The Staphylococcus aureus culture medium was removed from the -80℃ freezer and revived by streaking on LB agar plates, followed by two subcultures for activation. Then, the overnight cultured Staphylococcus aureus colonies were counted according to the method listed in General Chapter 1105 of the 2015 edition of the Chinese Pharmacopoeia.

[0028] A certain volume of overnight Staphylococcus aureus culture was diluted using LB medium to adjust the bacterial suspension to 0.5 MacFarland (concentration 1×10⁻⁶). 8 (CFU / mL), then diluted tenfold to obtain a concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension, for later use.

[0029] Add 1.875 mL of sterile LB medium to a sterile 4 mL glass ampoule, then add 0.125 mL of control standard and antimicrobial stock solution at concentrations of 32, 16, 8, 4, and 2 μg / mL, respectively, to achieve final antimicrobial test solution concentrations of 2, 1, 0.5, 0.25, and 0.125 μg / mL. Then, add 100 μL of the diluted bacterial suspension to each ampoule containing the antimicrobial test solution. Calculate the microbial concentration in the test solution based on the colony count results. In this example, the Staphylococcus aureus concentration in the test solution is 5.0 × 10⁻⁶. 5 CFU / mL.

[0030] Take 100 μL of the diluted bacterial suspension and place it in a sterile ampoule containing 2 mL of LB medium to prepare a test solution containing bacteria without antibiotics (blank control).

[0031] The experiment was divided into three treatment groups: Group 1: free roxithromycin; Group 2: zein-roxithromycin nanoparticles; Group 3: zein-roxithromycin nanoparticles. Each group had an antibacterial drug concentration gradient of 0 (Control), 0.125, 0.25, 0.5, 1, and 2 μg / mL.

[0032] After sealing the ampoule tightly with a capping tool, place it in the equilibrated calorimeter chamber (37℃). Once the instrument baseline stabilizes, start recording the heat (Heat / J) change in the sample cell in real time. The thermal power-time curve is shown in Figure 2(ac). The results show that regardless of whether the antibacterial drug roxithromycin is encapsulated by a biomolecular carrier, its antibacterial effect is concentration-dependent, that is, the higher the concentration (0~0.5 μg / mL), the more obvious the antibacterial activity. Moreover, compared with free roxithromycin, roxithromycin nanoparticles encapsulated with gliadin showed better antibacterial effect.

[0033] Based on a simple exponential model that microorganisms satisfy during the exponential growth phase ( P t = P 0 exp( k t), select the curve for this period and perform logarithmic transformation (ln P t =ln P 0+ k t), and obtain the biologically meaningful growth rate constant through linear regression. k Calculate the growth rate constant of the bacterial test solution without antibiotics in the three test groups. k Take the average value, and denote it as k 0, the growth rate constant of the bacterial test solution containing antibiotics in each group of tests is denoted as .k c .

[0034] Then, through G =ln2 / k Calculating the generation time of microorganisms ( G );pass I =( k 0- k c ) / k 0 Calculate the inhibition rate ( I The half-inhibition concentration was calculated using linear regression analysis (logarithmic function to base 10). I c 50 In addition, other thermodynamic information, such as the maximum thermal power value, was recorded directly from the thermal power-time curves of each group of test samples. P max ), Maximum thermal power output time ( t max ), total heat at time T ( Q T The thermodynamic information for each sample group is shown in Table 1.

[0035] Table 1. Differences in thermodynamic parameters of roxithromycin antibiotics with and without encapsulation during the antibacterial process.

[0036] The results above show that as the concentration of antibacterial drugs increases (0.125~0.5 μg / mL), the growth rate constant of Staphylococcus aureus (…) decreases. k ), Maximum heat output ( P max ) and total calories in 24 hours ( Q 24h All showed a decreasing trend, with the peak time ( t max ) and generational time ( G The duration of inhibition was prolonged, indicating that the growth of Staphylococcus aureus was inhibited. When the concentration of the antimicrobial agent was greater than 1 μg / mL, the growth and metabolism of Staphylococcus aureus were completely inhibited within 24 hours. The results showed that compared with the control and the zein-based antimicrobial agent, the zein-based antimicrobial agent had a more significant antibacterial effect, with an inhibition rate (…). I The highest half-inhibitory concentration (WIC) was achieved. Ic 50 Only 0.129 μg / mL is needed.

[0037] Implementation 2 The same technical solution as in Example 1 was adopted, except that only the antibacterial drug samples of 1 μg / mL and 2 μg / mL and the control were compared in step (4) with microcalorimetry monitoring for more than 80 hours to detect their antibacterial activity against Staphylococcus aureus. Figure 3 The results showed that both the 1 μg / mL zein-roxithromycin nanoparticle antibacterial agent and the control could inhibit the growth of Staphylococcus aureus approximately 40 h before the test, while the zein-roxithromycin nanoparticles could inhibit the growth of Staphylococcus aureus approximately 55 h before the test. Furthermore, the maximum heat flux output of the zein-based antibacterial agent was less than 25 μW (23.80 μW), followed by the zein-based antibacterial agent (31.66 μW). The thermokinetic information is shown in Table 2.

[0038] Table 2. Differences in thermodynamic parameters of roxithromycin antibiotics with and without encapsulation during the antibacterial process.

[0039] The results showed that sustained-release antibacterial drugs delivered via biomolecular-based delivery carriers can effectively enhance antibacterial activity. This also demonstrates that isothermal microcalorimetry can accurately and effectively analyze and compare the antibacterial effects of sustained-release antibacterial drugs delivered via biomolecular-based delivery carriers against Gram-positive bacteria.

[0040] Example 3 Using the same technical approach as in Example 1, Staphylococcus aureus was cultured and sample stock solutions were prepared. The difference was that Staphylococcus aureus cultured overnight was treated with a culture medium containing 1 μg / mL of the drug sample and a control (referring to their final concentration in LB medium). After incubation at 37°C with shaking for 12 hours, centrifugation, washing, cell fixation, ethanol gradient dehydration, and critical point carbon dioxide drying were performed. Finally, cell morphology was observed using a SU8100 scanning electron microscope (Hitachi, Japan). The results showed that untreated Staphylococcus aureus cells exhibited a regular, plump, spherical morphology and a complete, smooth cell surface. Figure 4 A); After treatment with 1 μg / mL free roxithromycin, some bacterial cells deformed, and localized cell disintegration occurred within cell clusters. Figure 4 B); In strains treated with 1 μg / mL zeaxanthin-roxithromycin nanoparticles, most cells deformed and their contents leaked out of the cell surface, and multiple instances of cell lysis and cytoplasmic content aggregation were observed in cell clusters. Figure 4 C); After treatment with 1 μg / mL gliadin-roxithromycin nanoparticles, most bacterial cells shrank, resulting in large-scale cell disintegration within cell clusters, and significant aggregation of cell contents. Figure 4D). The results further demonstrate the broad market application potential of using isothermal microcalorimetry to analyze and compare the sustained-release antibacterial drugs delivered by biomolecular-based delivery carriers and to evaluate their antibacterial efficacy against Gram-positive bacteria.

[0041] This invention utilizes isothermal microcalorimetry to analyze and compare the sustained-release antibacterial effects of biomolecular-based delivery carriers and to evaluate the antibacterial efficacy against Staphylococcus aureus, achieving real-time rapid measurement and qualitative and quantitative evaluation. This technology features fully automated dynamic recording during monitoring, a precise and stable temperature control system, and extremely high sensitivity (down to the nanowatt level) of the probe calorimeter. It can be used for real-time, continuous, and dynamic analysis and comparison of the in vitro antibacterial effects of sustained-release antibacterial drugs based on biomolecular-based delivery carriers, which is of great significance for the screening of novel pharmaceutical materials and the development and quality control of formulations.

[0042] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An isothermal microcalorimetry method for analyzing and comparing the sustained-release antibacterial effects of biomolecular-based delivery carriers against Staphylococcus aureus, comprising the following steps: (1) Select antibacterial drug samples and antibiotic reference standards based on biomolecular delivery carriers; (2) Perform pre-test treatment; (2.1) Accurately weigh the antibacterial drug sample and antibiotic control sample based on the biomolecule delivery carrier, and dilute the sample and control with culture medium and anhydrous ethanol respectively to prepare stock solutions of predetermined concentrations; take a certain amount of stock solution and add it to the culture medium, mix thoroughly, and dilute to prepare stock solutions of sample and control at predetermined concentrations. (2.2) Count the colonies of the overnight cultured microorganisms; (2.3) Dilute the overnight culture of bacteria with culture medium to obtain a bacterial suspension of a certain concentration, and then add it to the test solution containing antimicrobial drugs to prepare test solutions containing samples and control standards respectively. Combine the colony count results to calculate the concentration of microorganisms in the test solution at this time. (2.4) Dilute the overnight culture of bacteria with culture medium to obtain a bacterial suspension of a certain concentration, and then add it to the culture medium to prepare a test solution containing bacteria without antibiotics; (3) Conduct in vitro drug release experiments: At predetermined time points, the concentration of antibiotic drugs is detected by HPLC, and the drug accumulation release curves of sustained-release antibacterial drugs and free antibiotics with different biomolecular-based delivery carriers are obtained; The steps of the in vitro drug release experiment are as follows: the suspension of antibacterial drugs with biomolecular-based delivery carriers and the solution of free antibiotics are transferred into a dialysis bag, the dialysis bag is sealed and immersed in a release bottle of release buffer, and then placed in a constant temperature shaking shaker for continuous shaking. The release medium is taken out at multiple different time points, and fresh release buffer of equal temperature and volume is immediately added. Then the test solution is centrifuged and analyzed by HPLC, the peak area is recorded, the drug concentration is calculated according to the standard curve, the cumulative drug release is calculated and the corresponding drug release curve is plotted. (4) Microcalorimetry experiment: Compare the in vitro antibacterial activity of sustained-release antibacterial drugs and antibiotic reference standards on Staphylococcus aureus using different biomolecular-based delivery carriers; The steps of the microcalorimetry experiment are as follows: Add sterile LB medium to sterilized glass ampoules, and then add different concentrations of antibacterial drug samples and antibiotic reference standards respectively, with a drug blank as the control group. Then take the bacterial culture that has been cultured overnight, adjust the bacterial suspension with sterile LB medium, and then dilute the bacterial suspension by a tenfold dilution method. Take the diluted bacterial suspension and add it to each ampoule containing the antibacterial drug test solution; after sealing the cap tightly with a capping device, place the ampoule in the balanced calorimeter chamber, and start recording the heat change in the sample cell in real time after the instrument baseline stabilizes. After the experiment, obtain the nonlinear curve of the heat flow signal changing with time. Based on the shape trend of the curve and the thermodynamic information obtained by linear fitting of the exponential stage of the curve, compare the in vitro antibacterial effects of sustained-release antibacterial drugs and antibiotic reference standards on Staphylococcus aureus using different biomolecular-based delivery carriers.

2. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that, In step (1), the antibacterial drug sample based on the biomolecular delivery carrier is zein-roxithromycin nanoparticle or zein-roxithromycin nanoparticle; the antibiotic reference standard is roxithromycin.

3. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... In step (2.1), the culture medium is 4.5~5 mL LB medium, the concentration of the stock solution includes a standard concentration of 12.8 mg / mL and a sample concentration of 1% (w / v), and the concentration range of the sample and standard test solution is 0.125~2 μg / mL.

4. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... In step (2.3), the concentration of the bacterial suspension is 1×10⁻⁶. 7~8 The bacterial suspension volume is 10-300 μL, the final volume of the antibiotic-containing test solution is 2-3 mL, and the bacterial concentration in the test solution is 5.0 × 10⁻⁶ CFU / mL. 5 ~1.0×10 6 CFU / mL.

5. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... In step (2.4), the concentration of the bacterial suspension is 1×10⁻⁶. 7~8 The bacterial suspension volume is 10-300 μL, the final volume of the antibiotic-free test solution containing bacteria is 2-3 mL, and the bacterial concentration in the test solution is 5.0 × 10⁻⁶ CFU / mL. 5 ~1.0×10 6 CFU / mL.

6. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... In step (3), the dialysis bag is cut to an appropriate length, soaked in deionized water and boiled for 10 minutes, then soaked in deionized water for 30 minutes, and a water test is performed to check for leakage. It is then soaked in release buffer PBS for later use. The HPLC analysis uses a C18 reversed-phase column, with acetonitrile-phosphate buffer as the mobile phase, a flow rate of 1.0 mL / min, an injection volume of 10 μL, a detection wavelength of 210 nm, and a column temperature of 25℃. The standard curve is prepared by injecting a roxithromycin standard solution, analyzing the sample, and plotting a peak area-concentration standard curve. 2 ≥0.999; The drug concentration is obtained by injecting and analyzing antibacterial drug samples and antibiotic reference standards under the same chromatographic conditions, recording the peak area, and calculating the drug concentration based on the standard curve; The drug release curve is plotted with time as the abscissa and cumulative release percentage as the ordinate.

7. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... In step (4), the exponential phase of the linear fitting curve is achieved by utilizing the fact that the natural logarithm is the inverse function of the exponential function (lne). x =x), to increase thermal power P Over time t Convert the change curve to logarithmic form ln P t Over time t The growth rate constant with biological significance was obtained by using a linear regression natural logarithm linear model to analyze the change curve. k The thermodynamic information includes the maximum heat flow output. P max Maximum heat output time t max Total heat at time T Q T Growth rate constant k Generations G Inhibition rate I and half-inhibitory concentration Ic 50 The parameters can be obtained through thermal power-time curves, linear fitting, and related calculation formulas, and are used as evaluation parameters for antibacterial effects, thereby comparing the antibacterial activity of sustained-release antibacterial drug samples and antibiotic reference standards based on biomolecular delivery carriers.

8. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... The strain was Staphylococcus aureus ATCC6538. A single clone of the strain was picked and cultured in 10 mL of fresh LB medium and incubated at 37℃±0.5℃ for 18 h before use.

9. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that... The culture medium is Luria-Bertani medium with pH 7.2-7.

4. The liquid culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L sodium chloride. A solid culture medium is prepared by adding 1.5% agar to the liquid culture medium.

10. The method for evaluating the antibacterial effect of sustained-release antibacterial drugs based on isothermal microcalorimetry as described in claim 1, characterized in that, All instruments used in the microcalorimetry test were sterilized at 121°C for 15 minutes.

Images