WO2023019389A1

WO2023019389A1 - Sealed bacterial ghosts, and compositions, methods and uses thereof

Info

Publication number: WO2023019389A1
Application number: PCT/CN2021/112727
Authority: WO
Inventors: Xianzhong Yu; Xiao-tian YANG; Xin ZHU; Bao ZHENG; Yanling Cheng; Wei Hua; Chuan Wang; Boqian SHI
Original assignee: Xianzhong Yu
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2023-02-23
Also published as: CN115666524A

Abstract

Provides are sealed bacterial ghosts, methods and compositions for producing sealed bacterial ghosts, and methods and compositions for using sealed bacterial ghosts for delivering drugs, treating or preventing diseases and other medical uses.

Description

SEALED BACTERIAL GHOSTS, AND COMPOSITIONS, METHODS AND USES THEREOF

Field of the Invention

The invention relates to sealed bacterial ghosts, methods and compositions for producing sealed bacterial ghosts, and methods and compositions for using sealed bacterial ghosts for delivering drugs, treating or preventing diseases and other medical uses.

Background of the Invention

Bacterial ghosts (BGs) are nonliving empty cell envelopes of bacteria generated by releasing the bacterial cytoplasm through channels in the cell envelope, which are generated via controlled production of the bacteriophage phi X174 lysis protein E in gram-negative bacteria ^1, 2, or by methods based on the critical concentrations of chemical compounds in gram-positive bacteria ^3, 4.

BGs still possess complete antigen structures on the bacterial cell surface, they can be used directly as vaccines ^5-7. BGs are also a good delivery platform for loading biomacromolecules such as antigens, drugs, and DNA ^8-11. More importantly, BGs contain well-known innate immune stimulating components, and thus have tremendous potential to act as adjuvants. Elicitation of immune response not only depends on the molecular properties of the antigen or on the immunogenic susceptibility of the host but also on the formulation of the antigen. Thus, most vaccine formulations contain immunomodulatory components, broadly termed as adjuvants, to augment the immune responses against the weakly immunogenic antigens. Adjuvants mostly potentiate the immunogenicity of vaccine antigens through the stimulation of innate immune receptors present on the cells of the host immune system ^12, 13. BGs preserve the antigenic nature of components such as lipopolysaccharide (LPS) , flagellin, peptidoglycan and many others found in the envelope of native bacteria, which are ligands for various pattern recognition receptors (PRRs) and are collectively known as pathogen-associated molecular patterns (PAMPs) ^14, 10, 11. These envelope structures are recognized and taken up by immune and non-immune cells ^15-17, which will mainly stimulate cells through TLR2 and TLR4 pathways ^18, 19, and the presence of multiple TLRs on a number of immune and non-immune cells forms the basis of their adjuvant activity. Thus, BGs are not only an excellent antigen delivery system but also an effective adjuvant for therapeutic or prophylactic vaccines. Furthermore, the particulate nature of BGs makes them ideal in vivo targeting vehicles for phagocytic antigen presenting cells such as dendritic cells and macrophages.

Summary of the Invention

The present disclosure provides sealed bacteria ghosts, as well as methods and compositions for producing sealed bacteria ghosts, and methods, compositions, and uses of sealed bacteria ghosts for delivering drugs, eliciting or augmenting immune responses, treating or preventing diseases etc. In particular, the present disclosure provides sealed bacterial ghosts of gram-positive bacteria. The present Inventor surprisingly found that bacterial ghosts of gram-positive bacteria can be effectively sealed by cationic liposomes. The molecules loaded into bacterial ghosts later sealed by cationic liposomes remain inside the bacterial ghosts even at 72 hours after the sealing. By contrast, most of the molecules loaded into bacterial ghosts that are not sealed after the loading leak out of the bacteria ghosts at 4 hours after loading. The present Inventor also found an effective and simple method for loading and/or sealing bacterial ghosts, by which at least 99%of bacteria used can be efficiently and effectively loaded and/or sealed. As shown by the present results, sealed bacterial ghosts can carry much more drugs or biomolecules than unsealed bacterial ghosts, and therefore have great advantages as a platform or vehicle for delivering drugs or biomolecules in vivo.

Brief Description of the Drawings

Figure 1: Schematic diagram of a non-thermal plasma jet discharge system.

The gas supply system 1 supplies gas to the gas adjusting system 2, which can adjust parameters like the gas flow rate, the ratio of different gases etc. as needed, and then introduce the gas into the jet forming device 4. 3 represents the direction of gas flow. The gas supply system 1 can supply different kinds of gases such as N ₂, He, H ₂, Ar, O ₂, air, CO ₂, and CO. Bacteria suspension 8 can be directly added to the liquid container 11, or it can be placed in another container (8a) and then introduced into the container 11. The treated bacteria suspension can be collected directly, or it can be directed to another container (8b) . The power supply system 9 applies a high voltage to both electrodes. The power adjusting system 10 adjusts the voltage, frequency, and current to optimal intervals in combination. The power supply system 9 also includes a feedback, display and recording systems for feeding back, displaying and recording changes in various parameters. The jet forming device 4 comprises a housing (or pipe) 6 with a nozzle at the distal end, a high-voltage electrode 5, and a grounded metal electrode 7. Gas passes through the cavity enclosed by the housing or pipe. The most distal end 4a of the jet forming device 4 can be at any position from 20 mm above the liquid surface of the bacteria suspension to 10 mm below the liquid surface.

Figure 2: Scanning electron microscope images of Lactococcus lactis with or without non-thermal plasma treatment.

A. Lactococcus lactis treated with non-thermal plasma. Some holes or channels on the surface of the bacteria are indicated by black arrows. B. Lactococcus lactis without non-thermal plasma treatment.

Figure 3: Viability test for Lactococcus lactis treated with non-thermal plasma.

Right plate: A suspension of Lactococcus lactis treated with non-thermal plasma was plated onto a MRS agar plate and cultured for 72 hours. Left plate: As a negative control, a suspension of growing Lactococcus lactis (log phase) was plated and incubated the same way as the bacteria treated with the non-thermal plasma.

Figure 4: Bacterial ghosts loaded with calcein and sealed by liposome.

A. Image of calcein filled and DOTAP-Chol liposome sealed bacterial ghosts of Lactococcus lactis observed by a fluorescent microscope. B. Analysis of calcein filled and DOTAP-Chol liposome sealed bacterial ghosts of Lactococcus lactis by a

easyCyte ^TM flow cytometer.

Figure 5: Calcein release from liposome sealed bacterial ghosts.

Cobalt ion (Co ²⁺) was added to calcein loaded bacterial ghosts sealed with DOTAP-Chol liposomes. The fluorescence intensity was measured in a Victor 1420 multilabel counter at different time points after cobalt addition.

Figure 6: Calcein release from unsealed bacterial ghosts.

At different time points, a portion of the calcein loaded bacterial ghost suspension was taken and washed three times with PBS to wash off any calcein outside bacterial ghosts. Washed bacterial ghosts were resuspended in 1 ml PBS, and fluorescence intensity was measured in a Victor 1420 multilabel counter.

Figure 7: Quenching of calcein by copper ion.

Copper ion (Cu ²⁺) was added into calcein filled and DOTAP-Chol liposome sealed bacterial ghost suspension at around 12 hour. Fluorescence intensity at different time points was measured in a Victor 1420 multilabel counter.

Detailed Description of the Invention

In one aspect, the present disclosure provides a method of producing sealed bacterial ghosts, comprising mixing bacterial ghosts with cationic liposomes. In some embodiments, the mixing may be carried out by adding cationic liposomes into bacterial ghosts suspension. In some embodiments, the mixing may further comprise adding CaCl ₂ solution into the suspension. In some embodiments, the mixing may further comprise incubating the suspension overnight at 37 ℃ with shaking.

In another aspect, the present disclosure provides a method of producing loaded and sealed bacterial ghosts, comprising mixing bacterial ghosts with a therapeutic, prophylactic, and/or diagnostic substance to obtain loaded bacterial ghosts, and mixing the loaded bacterial ghosts with cationic liposomes to obtain loaded and sealed bacterial ghosts. In some embodiments, the first mixing may be carried out by suspending freeze-dried bacterial ghosts in a buffer containing the therapeutic, prophylactic, and/or diagnostic substance. In some embodiments, the first mixing can further comprise incubating the suspension for about 2 hours at room temperature with shaking. In some embodiments, the second mixing may be carried out by adding cationic liposomes into the loaded bacterial ghosts suspension. In some embodiments, the second mixing may further comprise adding CaCl ₂ solution into the suspension. In some embodiments, the second mixing may further comprise incubating the suspension overnight at 37 ℃ with shaking.

In some embodiments, in the methods described above, the bacterial ghosts are bacterial ghosts of gram-positive bacteria. In some embodiments, the bacterial ghosts are bacterial ghosts of lactic acid bacteria. In some embodiments, the lactic acid bacteria are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Leuconostoc, and Pediococcus. In some embodiments, the lactic acid bacteria are Lactobacillus or Lactococcus. In some embodiments, the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactococcus lactis. In some embodiments, the cationic liposomes comprise one or more cationic lipid selected from the group consisting of DOTAP, DC-Chol, DC-6-14, DOTMA, and DODAB. In some embodiments, the cationic liposomes comprise one or more neutral lipid selected from the group consisting of DOPE, DOPC, and cholesterol. In some embodiments, the cationic liposomes comprise DOTAP and cholesterol, or DC-Chol and DOPE. In some embodiments, the cationic liposomes are formed by about 1: 1 molar ratio of DOTAP and cholesterol. In some embodiments, the cationic liposomes are formed by about 30: 70 w/w ratio of DC-Chol and DOPE.

In another aspect, the present disclosure provides sealed bacterial ghosts, and loaded and sealed bacterial ghosts prepared by the above methods.

In another aspect, the present disclosure provides sealed bacterial ghosts, wherein the sealed bacterial ghosts comprise a synthetic cationic lipid, and bacterial ghosts of gram-positive bacteria. In some embodiments, the gram-positive bacteria are lactic acid bacteria. In some embodiments, the lactic acid bacteria are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Leuconostoc, and Pediococcus. In some embodiments, the lactic acid bacteria are Lactobacillus or Lactococcus. In some embodiments, the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactococcus lactis. In some embodiments, the synthetic cationic lipid is selected from the group consisting of DOTAP, DC-Chol, DC-6-14, DOTMA, and DODAB. In some embodiments, the sealed bacterial ghosts further comprise a neutral lipid. In some embodiments, the neutral lipid is selected from the group consisting of DOPE, DOPC, and cholesterol. In some embodiments, the sealed bacterial ghosts comprise DOTAP and cholesterol, or DC-Chol and DOPE. In some embodiments, the sealed bacterial ghosts can be used for delivering drugs; treating, preventing and/or diagnosing diseases; and/or acting as an adjuvant.

In another aspect, the present disclosure provides loaded and sealed bacterial ghosts, wherein the loaded and sealed bacterial ghosts comprise a synthetic cationic lipid, bacterial ghosts of gram-positive bacteria, and a therapeutic, prophylactic and/or diagnostic substance encapsulated by the bacterial ghosts. In some embodiments, the gram-positive bacteria are lactic acid bacteria. In some embodiments, the lactic acid bacteria are Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Leuconostoc, and Pediococcus. In some embodiments, the lactic acid bacteria are Lactobacillus or Lactococcus. In some embodiments, the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactococcus lactis. In some embodiments, the synthetic cationic lipid is selected from the group consisting of DOTAP, DC-Chol, DC-6-14, DOTMA, and DODAB. In some embodiments, the sealed bacterial ghosts further comprise a neutral lipid. In some embodiments, the neutral lipid is selected from the group consisting of DOPE, DOPC, and cholesterol. In some embodiments, the sealed bacterial ghosts comprise DOTAP and cholesterol, or DC-Chol and DOPE. In some embodiments, the loaded and sealed bacterial ghosts can be used for delivering drugs; treating, preventing and/or diagnosing diseases; and/or acting as an adjuvant. In some embodiments, the loaded and sealed bacterial ghosts can be used on human or animals.

In another aspect, the present disclosure provides a composition comprising the sealed bacterial ghosts, or the loaded and sealed bacterial ghosts for use in delivering drugs; treating, preventing and/or diagnosing diseases; and/or acting as an adjuvant.

In another aspect, the present disclosure provides a method of using the sealed bacterial ghosts, or the loaded and sealed bacterial ghosts for delivering drugs; or treating, preventing or diagnosing diseases, comprising administering to a subject a therapeutically, prophylactically or diagnostically effective amount of the sealed bacterial ghosts, or the loaded and sealed bacterial ghosts. In some embodiments, the subject can be human or animals. In another aspect, the present disclosure provides a method of using the sealed bacterial ghosts, or the loaded and sealed bacterial ghosts for acting as an adjuvant, comprising administering to a subject an effective amount of the sealed bacterial ghosts, or the loaded and sealed bacterial ghosts to enhance immune responses. In some embodiments, the subject can be human or animals.

In another aspect, the present disclosure provides uses of sealed bacterial ghosts, or loaded and sealed bacterial ghosts in preparing a medicament or composition for delivering drugs; treating, preventing and/or diagnosing diseases; and/or acting as an adjuvant.

Definitions:

“Bacterial Ghosts” or “BGs” as used herein refer to nonliving empty cell envelopes of bacteria generated by releasing the bacterial cytoplasm through a channel in the cell envelope. The cell envelope of gram-negative bacteria comprises the plasma membrane, the peptidoglycan cell wall, and the outer membrane. The cell envelope of gram-positive bacteria comprises the plasma membrane, and the peptidoglycan cell wall, which is much thicker than the cell wall of gram-negative bacteria. Methods for preparing BGs are well-known in the art. For example, BGs of gram-negative bacteria can be prepared through the controlled production of the bacteriophage phi X1 74 lysis protein E; BGs of gram-positive bacteria can be prepared through treating bacteria with minimum inhibition concentrations of certain chemicals (for example, see Wu, Xueyou et al. “Production of Bacterial Ghosts from Gram-Positive Pathogen Listeria monocytogenes. ” Foodborne pathogens and disease vol. 14, 1 (2017) : 1-7. doi: 10. 1089/fpd. 2016.2184) ; BGs of gram positive bacteria can also be prepared through treating bacteria with a non-thermal plasma as detailed in the Examples below.

“Sealed bacterial ghosts” as used herein refer to bacterial ghosts that are sealed by a synthetic lipid. Unlike bacterial ghosts, which have channels formed in the cell envelope leading to the cavity encapsulated by the cell envelope, sealed bacterial ghosts do not have such channels because they are closed by a synthetic lipid.

“Loaded bacterial ghosts” as used herein refer to bacterial ghosts loaded with a therapeutic, prophylactic and/or diagnostic molecule, wherein the molecule is inside the bacterial ghosts.

“Loaded and sealed bacterial ghosts” as used herein refer to bacterial ghosts loaded with a therapeutic, prophylactic and/or diagnostic molecule, and sealed by a synthetic lipid, wherein the molecule is inside the bacterial ghosts.

“Bacteria” as used herein encompass any gram-positive bacteria and gram-negative bacteria, and any naturally existing bacteria and genetically engineered bacteria, which have one or more genes added, deleted or altered as compared to naturally existing bacteria. For example, to reduce the amount of residual DNA of bacterial ghosts, a nuclease gene under the control of an inducible promoter can be genetically engineered into the bacterial cell, and the expression of the nuclease gene can be induced after the culturing of bacteria and before the process of generating bacterial ghosts (see, for example, Haidinger, W et al. “Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease. ” Applied and environmental microbiology vol. 69, 10 (2003) : 6106-13. doi: 10.1128/AEM. 69.10.6106-6113.2003) .

“Gram-positive bacteria” as used herein refer to bacteria that have a distinctive purple appearance when observed under a light microscope following Gram staining. This is due to retention of the purple crystal violet stain in the thick peptidoglycan layer of the cell wall. “Gram-negative bacteria” as used herein refer to bacteria that appear a pale reddish color when observed under a light microscope following Gram staining. This is because the structure of their cell wall is unable to retain the crystal violet stain so are colored only by the safranin counterstain.

“Lactic acid bacteria” or “Lactobacillales” as used herein refer to a group of gram-positive bacteria, non-respiring non-spore-forming, cocci or rods, which produce lactic acid as the major end product of the fermentation of carbohydrates. Lactic acid bacteria comprise the following genera: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Sporolactobacillus, Tetragenococcus, Vagococcus, and Weissella. Lactobacillus is a genus of gram-positive, aerotolerant anaerobes or microaerophilic, rod-shaped, non-spore-forming bacteria. The genus Lactobacillus comprises over 260 species, including Lactobacillus acidophilus, Lactobacillus delbrueckii (comprising subspecies (subsp. ) lactis, bulgaricus, delbrueckii, indicus etc. ) , Lactobacillus casei, Lactobacillus brevis, Lactobacillus plantarum etc. Lactococcus is a genus of gram-positive, catalase-negative, non-motile cocci bacteria that are found singly, in pairs, or in chains. The genus Lactococcus comprises at least 12 species currently recognized, including Lactococcus lactis (comprising subsp, lactis, cremoris, hordniae, and tructae) , Lactococcus garvieae, Lactococcus plantarum, Lactococcus raffinolactis etc.

“Liposome” as used herein refers to spherical or near-spherical vesicles made up of biodegradable natural or synthetic lipids. “Synthetic liposome” as used herein refers to liposomes that are partly or entirely made up of synthetic lipids.

“Cationic liposome” as used herein refers to liposomes that comprise positively charged lipids, or positively charged lipids and neutral lipids. Positively charged lipids that can be used to form cationic liposomes include 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) , 3β- [N- (N′, N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol) , O, O'-ditetradecanoyl-N- (a-trimethylammonioacetyl) diethanolamine chloride (DC-6-14) , 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) , and dimethyldioctadecylammonium bromide (DODAB) . Neutral lipids that can be used in combination with positively charged lipids to form cationic liposomes include dioleoyl phosphatidylethanolamine (DOPE) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , and cholesterol. Cationic liposomes can be prepared from any type of cationic lipids, any combination of any type of cationic lipids, and any combination of any type of cationic lipids and any type of neutral lipids at any possible molar ratios as desired and can be properly determined by those skilled in the art. For example, such combinations include but are not limited to: (1) DOTAP and DOPE; (2) DOTAP and DOPC; (3) DOTAP and Chol; (4) DOTAP, DOPE and Chol; (5) DOTAP, DOPC and Chol; (6) DOTAP, DOPC and DOPE; (7) DOTAP, DOPC, DOPE and Chol; (8) DC-Chol and DOPE; (9) DC-Chol and DOPC; (10) DC-Chol and Chol; (11) DC-Chol, DOPE and Chol; (12) DC-Chol, DOPC and Chol; (13) DC-Chol, DOPC and DOPE; (14) DC-Chol, DOPC, DOPE and Chol at any possible molar ratios as desired and can be properly determined by those skilled in the art.

“Therapeutic, prophylactic and/or diagnostic substance” as used herein refers to any molecule or mixture that has a therapeutic, prophylactic and/or diagnostic effect. Such molecules include but are not limited to any drug, small molecule (including lipids, fatty acids, glycolipids, sterols, monosaccharides, vitamins, hormones, neurotransmitters etc. ) , peptide, protein (including antibody, antibody-drug conjugate, enzyme etc. ) , DNA, RNA (including mRNA, rRNA, siRNA, miRNA, snoRNA etc. ) , polysaccharide, antigen (including peptide, protein, polysaccharide etc. ) etc. that have a therapeutic, prophylactic and/or diagnostic effect. Such mixture includes deactivated microorganisms (including bacteria and virus) that have a therapeutic, prophylactic and/or diagnostic effect.

The terms “a” , “an” , and “the” as used herein are intended to encompass both the singular and plural meaning of the following term, unless the context clearly suggests a singular meaning.

The term “and/or” as used herein are intended to include any and all possible combinations of one or more of the listed items.

The terms “comprises” and “comprising” as used herein are intended to indicate the presence of an element, component, feature, step etc., but not to exclude the presence of any other elements, components, features, steps etc. When the present disclosure describes that a product or system comprises certain components, or a method comprises certain steps, it should be understood that such descriptions also include a product or system consisting only of those components, or a method consisting only of those steps.

The term “about” as used herein intends to cover the range of ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%or ±0.5%of the number described.

Any numerical range described herein should be understood as encompassing the numbers or values of both endpoints, and any numbers or values in-between the endpoints.

Examples

Example 1: Preparation of bacterial ghosts.

Bacterial ghosts can be prepared by any methods known in the art, and a novel method utilizing non-thermal plasma as detailed below. Different preparation methods did not affect the results of loading and then sealing the bacterial ghosts with cationic liposomes as shown in the following examples.

(1) Preparation by a chemical method:

Bacterial ghosts were prepared from Lactobacillus acidophilus (DSM 20079, available as

4356 ^TM) , Lactobacillus delbrueckii (subsp. lactis) (DSM 20072, available as

12315 ^TM) , and Lactococcus lactis (subsp. lactis) (NCTC 6681, available as

19435 ^TM) according to the method disclosed in Wu, Xueyou et al. “Production of Bacterial Ghosts from Gram-Positive Pathogen Listeria monocytogenes. ” Foodborne pathogens and disease vol. 14, 1 (2017) : 1-7. doi: 10. 1089/fpd. 2016.2184. Briefly, standard experiments to determine the minimum inhibition concentrations for NaOH, SDS, CaCO ₃ and H ₂O ₂ were conducted, and the bacteria were first incubated with NaOH, SDS and CaCO ₃ at their minimum inhibition concentrations as determined, and then incubated with H ₂O ₂ at its minimum inhibition concentration as determined, and then incubated in 60%ethanol with gentle vertexing. Bacterial ghosts were harvested by centrifugation and washed thrice with 0.5%sterile saline. The final pellet was resuspended in sterilized distilled water. The bacterial ghost water suspension was kept at -80 ℃ for 4 hours, and then freeze-dried for 48 hours.

(2) Preparation by non-thermal plasma:

Bacterial ghosts were also prepared from lactic acid bacteria by treating the bacteria with non-thermal plasma, which, when generated by the right amount of energy, can puncture the cell envelope of gram-positive bacteria to release the internal components of the bacteria, while retaining the morphological, structural, and antigenic features of the cell envelope.

I. Material:

(i) A non-thermal plasma discharge system

(ii) Lactic acid bacteria strains: Lactobacillus acidophilus (DSM 20079, available as

4356 ^TM) , Lactobacillus delbrueckii (subsp. lactis) (DSM 20072, available as

12315 ^TM) , and Lactococcus lactis (subsp. lactis) (NCTC 6681, available as

19435 ^TM) .

(iii) MRS broth medium, which can be prepared by the following method:

10.0 g of peptone, 5.0 g of beef extract, 4.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 1.0 g of Tween-80, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, and 0.05 g of manganese sulfate were added to 1000 mL distilled water, and the pH was adjusted to 6.2; the mixture was stirred at room temperature for 15 min to completely dissolve the medium, and then sterilized by autoclaving at 115 ℃ for 30 min.

(iv) MRS agar plate, which can be prepared by the following method:

10.0 g of peptone, 5.0 g of beef extract, 4.0 g of yeast extract, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 1.0 g of Tween-80, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, and 15.0 g of agar were added to 1000 mL distilled water, and the pH was adjusted to 6.3; the mixture was stirred and heated to boiling to dissolve the medium completely, and then was sterilized by autoclaving at 121℃ for 15 minutes. The medium was then cooled to 45-50℃, well mixed, and poured into sterile Petri plates.

II. Procedure:

The lactic acid bacteria were cultured to the exponential growth phase in MRS broth medium according to standard methods known in the art, and then the bacteria were collected by centrifugation, washed with PBS buffer, and resuspended in sterilized distilled water to adjust the concentration of the bacteria to 1 × 10 ⁶-1 × 10 ⁷ CFU/ml.

The bacteria suspension obtained above was added to the liquid container of the non-thermal plasma discharge system (Figure 1) to be treated by non-thermal plasma. As a negative control, a portion of the bacteria suspension was set aside for later observation without any non-thermal plasma treatment. A non-thermal plasma was generated by using nitrogen as the working gas at a flow rate of 3 L/min, and applying to the gas an electric field provided by a power supply having a voltage of 8000-9500 volts, a frequency of 21000-23000 Hz and a power of 30-300 watts. The bacteria were treated by the non-thermal plasma for 20 seconds.

To prove that bacterial ghosts were formed, a scanning electron microscope was used to observe the treated bacteria (Figure 2A) and the non-treated bacteria (Figure 2B) . In addition, the DNA contents of the treated bacteria and the non-treated bacteria were respectively measured. Specifically, the bacteria were first subjected to enzymatic hydrolysis, and then a purification column (e.g., EZ-10 spin column) was used to absorb and elute off DNA, and the amount of eluted DNA was measured by a microplate reader.

Further, to verify that there were no live bacteria after the treatment, the treated bacteria were evenly spread onto a MRS agar plate (Figure 3, right plate) ; the plate was then sealed by parafilm, and placed in an incubator (37 ℃, 60%relative humidity) for 72 hours. As a negative control, a suspension of growing bacteria (log phase) was plated and incubated the same way (Figure 3, left plate) .

The rest of the treated bacteria (bacterial ghosts) were washed twice by sterilized distilled water and then resuspended in sterilized distilled water. The bacterial ghost water suspension was kept at -80℃ for 4 hours, and then freeze-dried for 48 hours.

III. Results:

Bacterial ghosts of lactic acid bacteria were formed after the non-thermal plasma treatment. It can be seen from Figure 2 that bacteria treated with the non-thermal plasma had a morphological structure similar to that of non-treated bacteria; however, the treated bacteria had holes or channels on their surface (Figure 2A) , which cannot be observed for non-treated bacteria (Figure 2B) . Moreover, the DNA content of the treated bacteria was less than 10%of that of the non-treated bacteria, indicating that the cytoplasmic contents had leaked out of the treated bacteria. Further, the treated bacteria did not contain any live bacteria (Figure 3, right plate) .

Example 2: Loading bacterial ghosts with small molecules.

Calcein is a highly negatively charged fluorescein derivative, with excitation/emission wavelengths of 494/517 nm at pH 8, and appears as yellow-orange solids that are soluble in DMSO, DMF or pH ＞ 6 water. Calcein is not membrane permeant, and is commonly used as an indicator of lipid vesicle leakage, and useful for the study of cell membrane integrity and fusion. The chemical structure of calcein is shown below:

Loading bacterial ghosts with calcein:

1 mg of freeze-dried bacterial ghost obtained in Example 1 was suspended in 900 mL of fusion buffer (100 mM NaCl, 10 mM sodium acetate, 10 mM HEPES, pH 5-7) containing calcein at a concentration of 0.5 mM. The bacterial ghost suspension was incubated with shaking at 150 rpm for 2 hours at room temperature.

Example 3: Preparing cationic liposome.

Several different cationic liposomes were prepared for bacterial ghost closure.

(1) DOTAP-Chol Liposomes:

1: 1 molar ratio of DOTAP (1, 2-dioleoyl-3-trimethylammonium-propane (methyl sulfate salt) ) (

Polar Lipids) and cholesterol in chloroform were mixed together in a glass vial. The vial was rotated and a dry nitrogen stream was used to evaporate the organic solvent to form a thin layer of lipids on the wall. ddH2O was added into the dried lipids at a concentration of 10 mg/ml (DOTAP) . The lipids were allowed to rehydrate overnight at 4 ℃. The rehydrated lipids were vortex mixed for 15 seconds on maximum speed several times. Several quick (10-15 seconds) bursts in a bath sonicator can be used if the lipids still remained attached to the side wall. The lipid suspension was vortexed again and heated at 65 ℃ in a water bath. The lipid suspension was then used to make liposomes in an Avanti Mini-Extruder with a 100 μm filter. The final concentration of the liposomes was 10 mg DOTAP + 5 mg Chol/mL.

(2) DC-Chol-DOPE Liposomes:

The DC-Chol-DOPE liposomes with a final concentration of 2 mM were prepared from DC-Chol/DOPE blend (30: 70, w/w) ) (

Polar Lipids) by the same procedure as above. DC-Chol/DOPE is a blend of a cationic lipid: 3β - [N- (N′, N′-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol) and a neutral lipid: dioleoyl phosphatidylethanolamine (DOPE) .

Example 4: Bacterial ghost and liposome fusion.

For fusion, 100 μl of the liposomes prepared in Example 3 was added into the calcein loaded bacterial ghost suspension (prepared from 1 mg of freeze-dried bacterial ghost) prepared above. CaCl ₂ solution at 1 M was added into the suspension to reach a final concentration of 25 mM. The suspension was incubated overnight at 37 ℃ with shaking at 250 rpm to induce fusion between the liposomes and the bacterial membrane around the channels.

Example 5: Detecting the packaging of calcein.

Bacterial ghosts loaded with calcein were examined under a fluorescence microscope, or analyzed by flow cytometry, after adding Co ²⁺ (50 mM) or Cu ²⁺ (1%w/v) . Cobalt ion (Co ²⁺) can efficiently quench the fluorescence of calcein but is not membrane permeant. Therefore, fluorescence persisting after addition of cobalt ion indicates successful sealing of bacterial ghosts. Copper ion (Cu ²⁺) can efficiently quench the fluorescence of calcein as well, but is membrane permeant. Therefore, fluorescence should be quenched after copper ion is added.

Calcein filled bacterial ghosts were sealed by DOTAP-Chol liposomes. After adding cobalt ion (Co ²⁺) to calcein loaded bacterial ghosts sealed with DOTAP-Chol liposomes as obtained in Example 4, bacterial ghosts were observed under a fluorescent microscope (Figure 4A) and analyzed using a

easyCyte ^TM flow cytometer (Figure 4B) . Calcein filled bacterial ghosts with DOTAP-Chol closure showed strong fluorescence, and nearly 100% (99.98%) of bacterial ghosts were filled with calcein.

Calcein filled bacterial ghosts were also sealed by DC-Chol-DOPE liposomes. After adding cobalt ion (Co ²⁺) to calcein loaded bacterial ghosts sealed with DC-Chol-DOPE liposomes as obtained in Example 4, bacterial ghosts were observed under a fluorescent microscope and analyzed using a

easyCyte ^TM flow cytometer. Calcein filled bacterial ghosts with DC-Chol-DOPE closure showed strong fluorescence, and at least 99%of bacterial ghosts were filled with calcein. Similar sealing results were obtained for all the lactic acid bacterium species used.

Example 6: Detecting calcein release from liposome sealed bacterial ghosts, in comparison with unsealed bacterial ghosts.

Cobalt ion (Co ²⁺) was added to calcein loaded bacterial ghosts sealed with DOTAP-Chol liposomes as obtained in Example 4. The fluorescence intensity was measured in a Victor 1420 multilabel counter at different time points after cobalt addition (Figure 5) . The results show that there was no significant changes of fluorescence intensity even at 72 hours after cobalt ion was added. Similar results were also obtained for calcein loaded bacterial ghosts sealed with DC-Chol-DOPE. Similar results were obtained for all the lactic acid bacterium species used.

For comparison, the loaded bacterial ghost suspension as obtained in Example 2 was used. At 0, 1, 2, 3 and 4 hour, a portion of the suspension was taken and washed three times with PBS to wash off any calcein outside bacterial ghosts. Washed bacterial ghosts were resuspended in 1 ml PBS, and then the fluorescence intensity was measured (Figure 6) .

Example 7: Quenching of calcein by copper ion.

Copper ion (Cu ²⁺) was added into calcein filled and DOTAP-Chol liposome sealed bacterial ghost suspension as obtained in Example 4 at around 12 hour; the fluorescence intensity was measured in a Victor 1420 multilabel counter at different time points (Figure 7) . The results show that the fluorescence of calcein was quenched after Cu ²⁺ was added, and bacterial ghosts only exhibited week fluorescence. Similar results were also obtained for calcein loaded bacterial ghosts sealed with DC-Chol-DOPE. Similar results were obtained for all the lactic acid bacterium species used.

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Claims

A method of producing sealed bacterial ghosts, comprising mixing bacterial ghosts with cationic liposomes.
A method of producing loaded and sealed bacterial ghosts, comprising mixing bacterial ghosts with a therapeutic, prophylactic, and/or diagnostic substance to obtain loaded bacterial ghosts, and mixing the loaded bacterial ghosts with cationic liposomes to obtain loaded and sealed bacterial ghosts.
The method of claim 1 or 2, wherein the bacterial ghosts are bacterial ghosts of gram-positive bacteria.
The method of any one of claims 1-3, wherein the bacterial ghosts are bacterial ghosts of lactic acid bacteria.
The method of any one of claims 1-4, wherein the cationic liposomes comprise one or more cationic lipid selected from the group consisting of DOTAP, DC-Chol, DC-6-14, DOTMA, and DODAB.
The method of claim 5, wherein the cationic liposomes comprise one or more neutral lipid selected from the group consisting of DOPE, DOPC, and cholesterol.
The method of any one of claims 1-6, wherein the lactic acid bacteria are Lactobacillus or Lactococcus.
The method of claim 7, wherein the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus delbrueckii, or Lactococcus lactis.
The method of any one of claims 1-8, wherein the cationic liposomes comprise DOTAP and cholesterol, or DC-Chol and DOPE.
Sealed bacterial ghosts prepared by the method of any one of claims 1 and 3-9.
Loaded and sealed bacterial ghosts prepared by the method of any one of claims 2-9.
Sealed bacterial ghosts, comprising a synthetic cationic lipid, and bacterial ghosts of gram-positive bacteria.
The sealed bacterial ghosts of claim 12, wherein the synthetic cationic lipid is selected from the group consisting of DOTAP, DC-Chol, DC-6-14, DOTMA, and DODAB.
The sealed bacterial ghosts of claim 12 or 13, wherein the sealed bacterial ghosts further comprise a neutral lipid.
The sealed bacterial ghosts of claim 14, wherein the neutral lipid is selected from the group consisting of DOPE, DOPC, and cholesterol.
The sealed bacterial ghosts of any one of claims 12-15, wherein the gram-positive bacteria are lactic acid bacteria.
The sealed bacterial ghosts of claim 16, wherein the lactic acid bacteria are Lactobacillus or Lactococcus.
The sealed bacterial ghosts of claim 17, wherein the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus delbrueckii, or Lactococcus lactis.
The sealed bacterial ghosts of any one of claims 10-18, wherein the sealed bacterial ghosts comprise DOTAP and cholesterol, or DC-Chol and DOPE.
Loaded and sealed bacterial ghosts, comprising the sealed bacterial ghosts of any one of claims 12-19, and a therapeutic, prophylactic, and/or diagnostic substance, wherein the therapeutic, prophylactic, and/or diagnostic substance is encapsulated by the sealed bacterial ghosts.