Bacterial Membrane Vesicle-Based Chemotherapeutic Drug Delivery System

Abstract

The invention relates to a drug delivery system for use in healthcare, and particularly in pharmaceutical technology, aimed at enhancing therapeutic efficacy by ensuring the controlled delivery of chemotherapeutic drugs and their release at the desired rate and dose. Specifically, the invention relates to a drug delivery system consisting of at least one bacterial membrane vesicle (BMV) with a double-layered phospholipid membrane structure, obtained from Gram-negative or Gram-positive bacteria, containing at least one chemotherapeutic agent encapsulated within the BMV that has the ability to inhibit cellular proliferation or disrupt nucleic acid metabolism, and the method for preparing this drug delivery system.

Description

1 TARIFF Bacterial Membrane Vesicle-Based Chemotherapeutic Drug Delivery System Technical Area The invention is intended for use in chemotherapeutic applications in the health and especially pharmaceutical technology fields. the controlled, targeted delivery of drugs and the delivery of the drug at the desired speed and dose. 5 a drug delivery system aimed at increasing treatment effectiveness by enabling its release It is related. The invention specifically targets Gram-negative chemotherapeutic agents used in the treatment of various types of cancer. targeted via negative or Gram-positive bacterial membrane vesicles (BMV) a drug delivery system that enables the drug to be transported to the region and this drug delivery system 10 It relates to the method of preparation. State of the Art The use of chemotherapeutic agents in the treatment of various diseases, particularly cancers, Today, commonly used free drug formulations or drug delivery systems 15 This is accomplished through various means. Among these carrier systems are liposomal formulations. It holds an important place. One example used in clinical practice is Vyxoos (CPX- 351), the active substances cytarabine and daunorubicin in a specific molar ratio liposomal structure It is obtained by encapsulating it inside. However, current liposomal systems have several technical limitations. Liposomal 20 The limited physicochemical stability of drugs in formulations, in the circulatory system Their ability to undergo rapid clearance, and the variability in uptake efficiency by target cells. The fact that it is difficult to display and the production processes are high-cost is among the significant disadvantages. Furthermore, the exact interactions of liposomes with the immune system are not fully understood. Due to its unpredictability, it is difficult to direct patients to target tissues and therapeutically effective. 25 Varying results may occur. In recent years, BMWs have been used as alternative medicines due to their natural composition and biocompatibility properties. They are being investigated as transport systems. BMV-based systems are of biological origin. It is anticipated that this could provide an advantage in some applications. However, currently... Various technical problems persist in BMW-based applications. Especially in high-end 30s. Difficulties in achieving encapsulation efficiency, release of drugs loaded into the vesicle 2 inability to effectively control its kinetics, standardized and repeatable production the lack of methods and the controlled presence of multiple active substances in the same vesicle The inability to transport goods at appropriate rates is among these problems. Traditional experimental methods are used to optimize the drug release profile in BMVs. The approaches involve the simultaneous evaluation of numerous parameters. 5 This requires. This situation makes the processes time-consuming, increases costs, and results in... This leads to a decrease in the repeatability of the results obtained. Parameters Due to the complex and multifaceted relationships between them, the targeted emission rate, emissions Achieving the required quantity and therapeutic effect is often difficult. Therefore, in the current technique, chemotherapeutic agents can be delivered in BMV-based drug delivery systems in 10 Solutions for precise control of emission rate and amount released There is a need for multiple drug combinations to be effective against BMVs. the ability to load these drugs and their balanced and predictable release in accordance with clinical requirements. The ability to demonstrate these characteristics is also a problem that needs to be solved in the current technical field. It is among them. Current technologies adequately meet these requirements. 15 Since it cannot meet the demand, optimization of release behavior and therapeutic efficacy are necessary. Solutions are needed to increase [the number of successful participants]. In conclusion, due to the negative aspects described above and the current solutions, the subject matter... Due to its shortcomings, an improvement in the relevant technical field is necessary. 20 It has been made. Purpose of the Invention The present invention meets the aforementioned requirements while eliminating all disadvantages. 25 It relates to the drug delivery system for release. The primary aim of the invention is to enable the controlled, predictable and effective administration of chemotherapeutic agents. a BMV-based drug delivery system and related method that enables its transport to improve. 30 One aim of the invention is to identify the natural carriers of BMV of Gram-negative or Gram-positive bacterial origin. By taking advantage of their properties, chemotherapeutic agents can be encapsulated in vesicles with high efficiency. 3 to enable the loading of these agents and their interaction with the biological environment. Its purpose is to protect against enzymatic and chemical degradation. One of the aims of the invention is to increase the release rate of chemotherapeutic agents loaded into BMVs and By precisely controlling the amount of drug released, the desired therapeutic effect is achieved in the target tissue. The aim is to ensure that the correct concentration is achieved. This increases the effectiveness of the treatment and... The aim is to reduce the risk of systemic toxicity. Another objective of the invention is to address the low performance encountered in existing BMV-based drug delivery systems. encapsulation efficiency, uncontrolled release profile, lack of standardization in production processes 10 and technical problems such as the inability to transport multiple drug combinations in a balanced manner. It is the elimination of. Another aim of the invention is to clinically utilize multiple chemotherapeutic agents within the same BMV. encapsulation of the agents in appropriate molar ratios and controlled release of the agents in question. The goal is to increase the effectiveness of combination therapies by ensuring that the drugs are released in a controlled manner. Another aim of the invention is to regulate temperature, pH, membrane structure, vesicle size, and surface zeta. the effects of parameters such as potential and loading rate on drug release By evaluating the situation, it is possible to predict under what conditions and at what rate BMW will release the drug. The goal is to create a decision support mechanism that can do this. The invention also enables the transport of chemotherapeutic agents in BMVs with high stability, obtaining controlled and repeatable release profiles and optimizing the therapeutic window. by enabling higher local drug concentrations at lower doses 25 It aims to achieve this. To fulfill the purposes described above, the invention is used for the transportation of chemotherapeutic agents. and a drug delivery system for controlled release, for Gram-negative or Gram-positive bacteria. obtained from bacteria, having a double-layered phospholipid membrane structure, at least one bacterial 30 membrane vesicle (BMV), at least one of which is encapsulated within the internal volume of the BMV in question. It contains a chemotherapeutic agent. The structural and characteristic features and all the advantages of the invention are given in the figures below. Thanks to the detailed explanation written with references to the figures, it is clearer than ever. 35 4 This will be understood, and therefore the evaluation should also take these figures and detailed explanations into account. It must be done by taking it. Figures that will help understand the invention. Figure 1 shows a representative illustration of the drug delivery system, which is the subject of the invention. 5 Reference Numbers 1. Bacterial membrane vesicle (BMV) 2. Chemotherapeutic agent 3. Drug-loaded bacterial membrane vesicles 10 X: DNA, RNA, Enzyme, Toxin, Periplasmic protein, Peptidoglycan, cytoplasmic protein L: Lipopolysaccharide DMP: Outer membrane protein DM: Outer Membrane IM: Inner Membrane 15 PP: Periplasm PD: Peptidoglycan Q: Cytosol Detailed Description of the Invention 20 This detailed explanation of the invention is solely for the purpose of providing a better understanding of the subject. and is explained in a way that will not create any limiting effects. The invention aims to transport chemotherapeutic agents for use in cancer treatments and deliver them to the target within 25 seconds. It relates to a drug delivery system and its preparation method for controlled release. The subject of this invention is a drug delivery system derived from Gram-negative or Gram-positive bacteria. At least one BMV (1) with a double-layered phospholipid membrane structure, the inner part of the BMV (1) in question encapsulated in volume, inhibiting cellular proliferation or nucleic acid 30 It contains at least one chemotherapeutic agent (2) capable of disrupting its metabolism. The invention is drug-loaded bacterial membrane vesicles (3). The BMV (1) in question is preferably 90-120 nanometers in size. The chemotherapeutic agents in question (2) include, but are not limited to, cytarabine, daunorubicin, doxorubicin, cisplatin, paclitaxel, gemcitabine, dacarbazine, 5-fluorouracil, carboplatin, oxaliplatin, docetaxel, temozolomide, trastuzumab, or combinations thereof It has been selected. 5 In one application of the invention, BMV (1) contains more than one chemotherapeutic agent (2) together. It is encapsulated. BMV (1) Gram-negative or Gram-positive bacterial origin, double-layered phospholipid membrane 10 containing, released extracellularly, and preferably approximately 90-120 nanometers in size It is a vesicular structure. The BMV in question is embedded in or located on the surface of the membrane structure. Protein structures, along with their internal volume, allow for the encapsulation of various active substances. It acts as a natural carrier system providing this. The BMV shown in Figure 1 is a real Not on scale, but as a representation of structural elements and encapsulated therapeutic agents 15 It aims to demonstrate this. Chemotherapeutic agents used within the scope of the invention (2) inhibit cellular proliferation refers to pharmaceutical compounds that have the ability to induce or disrupt nucleic acid metabolism. These agents are encapsulated inside BMVs and present in biological fluids. It is protected against enzymatic and chemical degradation. This vesicle-based confinement... The mechanism increases the stability of the active ingredient in systemic circulation while delivering it to the target region. It minimizes the risk of toxicity until it reaches the target. The system described in this invention minimizes the risk of toxicity until these agents reach the target. by allowing the release rate to occur with programmable kinetics, therapeutic This ensures window optimization. In this way, high local 25 at low doses. By achieving the desired concentration, treatment effectiveness is maximized. Chemotherapeutic drug-loaded BMV (3), Gram-negative or Gram-negative included in the invention Naturally occurring, of positive bacterial origin, possessing a double-layered phospholipid membrane structure and internal volume. It is a vesicular carrier that allows the encapsulation of therapeutic agents within the same carrier system. 30 This provides an opportunity. The natural membrane structure of BMV allows for the biological penetration of chemotherapeutic agents. It contributes to the preservation, transport, and delivery of the substance to target cells in the environment. The invention concerns the delivery and targeted controlled release of chemotherapeutic agents for pharmaceuticals. The method for preparing the support system includes the following steps: 35 6 a. minimum inhibitory concentration (MIC) of bacteria against chemotherapeutic agents (2) determining their values, b. bacteria, with chemotherapeutic agents (2) in accordance with the determined MIC values incubation, c. Removal of bacterial cells from the medium after incubation, 5 d. Filtering and purifying the supernatant after removing the cells, e. BMVs in the purified supernatant (1) by ultracentrifugation method precipitation, f. Washing and suspension of the precipitated drug-loaded BMVs (3) In step a of the method that is the subject of the invention, the bacteria are at minimum resistance to chemotherapeutic agents (2). Inhibitory concentration (MIC) values ​​are preferably obtained using the liquid microdilution method. The MIC value, OMV efficiency (particles / mL or mg / L), and drug concentrations are determined. incubation time, centrifugation and ultracentrifugation conditions (g force, time, temperature) examination of parameters 15 Minimum inhibitory concentration (MIC) of bacteria against chemotherapeutic agents (2) Liquid values ​​recommended by the Clinical and Laboratory Standards Institute (CLSI) It is determined by the microdilution method, and the experimental method is for sampling purposes. This is explained below. 20  Liquid recommended by the Clinical and Laboratory Standards Institute (CLSI) using the microdilution method,  Preparation of drug stock solutions  The prepared stock solutions are added to 96-well microplates and Mueller–Hinton liquid solutions are added. It will be tested with a two-fold (1 / 2) serial dilution in the culture medium. 25  Bacteria should be added to each well to achieve a final bacterial density of 5 × 10⁵ CFU / mL. The suspension will be added. Then the microplates will be incubated at 37°C for 16–20 hours. will be done. Measurement conditions: - Culture medium: Mueller–Hinton liquid agar. 30 - Microplate: 96-well microplate. - Dilution method: Two-fold (1 / 2) series dilution. - Bacterial inoculum: 5 × 10⁵ CFU / mL. - Incubation conditions: 16–20 hours at 37°C. 7 Evaluation criteria MIC definition and evaluation: The lowest drug concentration in which there is no visible bacterial growth. Concentration will be evaluated as the MIC value. In the method that is the subject of the invention; Chemotherapeutic agents (2) bacterial system and BMV (OMV) 5 the transfer / packaging into it, the addition of drugs to bacterial culture medium, and the bacteria Passive loading / endogenous packing occurring during natural OMV biogenesis It occurs through this mechanism. The filter type used in the filtration process in the method described in the invention is cellulose acetate. The pore diameters are 10 their thicknesses are 0.22 µm and 0.45 µm. In the method described in the invention, BMVs (OMVs) undergo filtering processes after It is preferably precipitated by ultracentrifugation at 150,000 × g, 4 °C, for 3 hours. In the application of the method described in the invention, BMVs obtained after ultracentrifugation (OMV) pellets of HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid) and DTT It is washed with a buffer containing (dithiothreitol) and suspended in a volume of 200 µL. Finally... To remove pollutants, the suspension was heated at 16,000 × g, 4 °C, 30 centrifuged for 80 minutes, then the supernatant is collected and BMV (OMV) stocks are reduced to 80 20 It is stored at °C. The invention method also covers the production and release conditions, loading efficiency, and vesicle of BMVs (1). Evaluation based on size and surface zeta potential parameters, and this Optimizing BMV production and emission conditions in line with the parameters 25 It includes. Chemotherapeutic agents (2) are contained within the drug-loaded BMV system (3), inside the BMV's internal volume. It is transported in an encapsulated form. The natural double-layered phospholipid membrane structure of BMV allows this. 30 Protection of active ingredients against enzymatic and chemical degradation in a biological environment While providing this, it also increases stability in systemic circulation. Thanks to this structure, Sudden, high peak concentrations are frequently observed in the administration of free drugs. Instead of uncontrolled emission profiles, a more balanced, controlled, and time-spread emission profile. is obtained. BMVs have naturally occurring membrane proteins and lipid components on their surfaces. 35 It can interact with mammalian cells and be internalized by them. 8 This cellular uptake mechanism allows for the processing of encapsulated chemotherapeutic agents. This enables more efficient transport to target cells and affects different cancer cells. It contributes to increasing therapeutic efficacy in these types. The BMV-based drug delivery system offers 5% more advantages compared to existing liposomal drug delivery systems. It exhibits high biological stability, a characteristic that can be achieved with lower doses and less frequently. It allows for effective treatment to be provided with controlled release intervals. When its behavior and the mechanism of effective interaction with target cells are evaluated together, to reduce systemic side effects, especially in resistant or recurrent cancer cases The aim is to increase therapeutic efficacy. 10 Within the framework of this working principle, the drug delivery capacity, release kinetics, and Cellular interaction behaviors are being experimentally analyzed; the resulting multidimensional findings... The data is processed using machine learning algorithms. This AI-powered approach. Thanks to this, the relationships between drug delivery efficiency and cellular responses are modeled, and Dosage strategies are determined with optimum release profiles specific to the target cell. Thus, 15 The BMV-based system enables the development of more effective cancer treatment protocols. It is positioned as an innovative pharmaceutical delivery platform. 25

Claims

9 REQUESTS 1. A drug delivery system for the transport and controlled release of chemotherapeutic agents. feature;  Double-layered phospholipid 5 obtained from Gram-negative or Gram-positive bacteria. at least one bacterial membrane vesicle (BMV) with membrane structure (1),  at least one chemotherapeutic agent encapsulated in the internal volume of the BMV in question (1) (2) It includes.

2. It is a drug delivery system that conforms to claim 1 and is characterized by its ability to deliver drugs to the bacterial membrane vesicle (1) Its size is between 90-120 nanometers.

3. A drug delivery system that conforms to Claim 1 and is characterized by the fact that the chemotherapeutic agent (2) cytarabine, daunorubicin, doxorubicin, cisplatin, paclitaxel, gemcitabine, dacarbazine, 5-fluorouracil, 15 carboplatin, oxaliplatin, docetaxel, temozolomide, trastuzumab or any of these It is selected from combinations.

4. It is a drug delivery system that complies with Claim 1 and has the characteristic of having more than one BMV (1) inside. The chemotherapeutic agent (2) is encapsulated together. 20 5. Drug carriers for the delivery and targeted controlled release of chemotherapeutic agents. It is a method for preparing the system, and its characteristic is; a. minimum inhibitory concentration (MIC) of bacteria against chemotherapeutic agents (2) Determining their values, 25 b. Chemotherapeutic agents (2) in accordance with the determined MIC values ​​of bacteria by incubation, the chemotherapeutic agent is transferred to OMVs during OMV biogenesis. ensuring the loading c. Removal of bacterial cells from the medium after incubation, d. 0.45 μm and then 0.22 μm membrane of cell-free supernatant 30 purification by removing bacteria through filters, e. purification of BMVs in the supernatant by (1) ultracentrifugation method () precipitation, f. Suspension of precipitated drug-loaded BMVs (3) in HEPES buffer containing DTT and centrifugation of the final pollutants to obtain the final product 35 It includes the steps.

6. A method that complies with Claim 5, characterized by the fact that in step a, bacteria are subjected to chemotherapeutic treatment. Minimum inhibitory concentration (MIC) against agents (2) by liquid microdilution method It is the determination of. 5 7. A method that complies with claim 5, characterized by the fact that the supernatant is purified in step e. BMVs (1) inside were ultracentrifuged at 150,000 × g, 4 °C for 3 hours It is the precipitation.

8. A method compliant with Claim 5, characterized by the fact that drug-loaded BMVs are precipitated in step f. (3) Suspension in 50 mM HEPES buffer containing 0.5 mM DTT at pH 6.8 and final The pollutants are centrifuged at 16,000 × g for 30 minutes to obtain the final product.

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