METHODS AND COMPOSITIONS OF CARRIER SYSTEMS FOR THE PURPOSE OF INTRACELLULAR DRUG TARGETING

Abstract

The present invention relates to a carrier system, a carrier and a pharmaceutical composition comprising a pathogen entry protein or fragment thereof, which specifically binds to a molecule on the surface of a mammalian target cell of said pathogen and which is covalently linked to the surface of said carrier and at least one hydrophilic antipathogenic agent. It further relates to a method of manufacturing a carrier system and the carrier system or the pharmaceutical composition for the use as a medicament.

Claims

1. A carrier system, comprising (i) a carrier, (ii) a pathogen entry protein or fragment thereof, which specifically binds to a molecule on the surface of a mammalian target cell of said pathogen and which is covalently linked to the surface of said carrier, and (iii) at least one hydrophilic antipathogenic agent.

2. The carrier system according to claim 1, wherein said carrier is selected from the group consisting of nanoparticles, preferably matrices of solid-lipid nanoparticles (SLN); polymer particles, preferably nanocapsules; vesicles, preferably liposomes and other artifically-prepared spherical or non-spherical vesicles.

3. The carrier system according to claim 2, wherein the liposome is unilamellar or multilamellar and/or the overall charge of the liposome is positive, neutral or negative.

4. The carrier system according to claim 1, wherein the molecule on the surface of a mammalian target cell is a receptor protein, preferably a β.sub.1-integrin receptor.

5. The carrier system according to claim 1 wherein the pathogen entry protein is from a bacterium that sequesters in a non-phagocytic cell.

6. The carrier system according to claim 5, wherein the bacterium is a Gram-negative bacterium, preferably Chlamydia, Coxiella burnetii, Ehrlichia, Rickettsia, Legionella, Salmonella, Shigella, or Yersinia; or a Gram-positive bacterium, preferably Mycobacterium leprae, or Mycobacterium tuberculosis, more preferably Yersinia.

7. The carrier system according to claim 5, wherein the pathogen entry protein is selected from the group consisting of invasin, YadA, internalin and other inv-type and related adhesive bacterial outer membrane molecules.

8. The carrier system according to claim 1, wherein the covalent link between the carrier and the pathogen entry protein is direct or via a linker.

9. The carrier system according to claim 1, wherein the pathogen entry protein is linked via its C-terminus, its N-terminus or a side chain of an amino acid of said pathogen entry protein, preferably its N-terminus.

10. The carrier system according to claim 7, wherein the invasin has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or variants thereof with at least 70% amino acid sequence identity and which specifically bind to the extracellular domain of β.sub.1-integrin receptor.

11. The carrier system according to claim 1, wherein the fragment of the pathogen entry protein consists or essentially consists of the extracellular domain of pathogen entry protein.

12. The carrier system according to claim 11, wherein the hydrophilic antipathogenic agent is selected from the group consisting of small molecules; proteins; nucleic acids, preferably siRNA; nucleotides, preferably polynucleotides.

13. The carrier system according to claim 12, wherein the hydrophilic antipathogenic agent is an antibiotic or cytostatic.

14. The carrier according to claim 13, wherein (i) the antibiotic is selected from the group consisting of polypeptides, glycopeptides, aminoglycosides, lipopeptides, quinolones or β-lactam antibiotics and organic or anorganic salts thereof, (ii) the cytostatic is selected from the group consisting of alkylating substances, anti-metabolites, epothilones, nuclear receptor agonists and antagonists, anti-androgens, anti-estrogens, platinum compounds, hormones and antihormones, interferons and inhibitors of cell cycle-dependent protein kinases (CDKs), inhibitors of cyclooxygenases and/or lipoxygenases, biogenic fatty acids and fatty acid derivatives, including prostanoids and leukotrienes, inhibitors of protein kinases, inhibitors of protein phosphatases, inhibitors of lipid kinases, platinum coordination complexes, ethyleneamines, methylmelamines, trazines, vinca alkaloids, pyrimidine analogs, purine analogs, alkylsulfonates, folic acid analogs, anthracendiones, substituted urea, methylhydrazine derivatives, in particular acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, azathioprine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dapsone, daunorubicin, dibrompropamidine, diethylstilbestrol, docetaxel, doxorubicin, enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate, estrogen, ethinylestradiol, etoposide, flavopiridol, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fosfestrol, furazolidone, gemcitabine, gonadotropin releasing hormone analog, hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin, hydroxyprogesteronecaproate, hydroxyurea, idarubicin, idoxuridine, ifosfamide, interferon α, irinotecan, leuprolide, lomustine, lurtotecan, mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate, megastrol acetate, melphalan, mepacrine, mercaptopurine, methotrexate, metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone, mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine, nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards, oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine, phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin, procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib, rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustine, streptozocin, sulfacarb amide, sulfacetamide, sulfachlopyridazine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole, sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin, sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen, taxol, teniposide, tertiposide, testolactone, testosterone propionate, thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan, trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine, vinblastine, vinorelbine, and zorubicin and organic or anorganic salts thereof.

15. A carrier system according to claim 1, wherein the hydrophilic antipathogenic agent exhibits a variable release kinetic from the carrier system.

16. A pharmaceutical composition comprising a carrier system according to claim 1 and a pharmaceutical acceptable excipient.

17. A pharmaceutical composition according to claim 16, wherein the carrier system is released from the pharmaceutical composition with a variable release kinetic.

18. A pharmaceutical according to claim 17, wherein the release kinetic is selected from the group of rapid release kinetics, sustained release kinetics or delayed release kinetics.

19. A method of manufacturing a carrier system according to claim 1, comprising the step of covalently linking the pathogen entry protein or part thereof to the carrier either prior or after contacting the carrier with at least one hydrophilic antipathogenic agent.

20. The method of claim 19, wherein the pathogen entry protein and/or at least one constituent of the carrier comprises an activatable group prior to covalent linking.

21. The method of claim 20, wherein the activatable group is activated with an activating reagent selected from the group consisting of carbodiimides, preferably N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC), more preferably N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC); succinimidylesters, preferably sulfosuccinimide, N-hydroxybenzotriazole, more preferably N-hydroxysuccinimid (NHS); triazine-based coupling reagents, preferably 4-(4,6-Dimethoxy-1,3 ,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMNI); maleidesters; and glutaraldehyde.

22. The method of claim 21, wherein the activating reagent is a mixture of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), preferably EDC in a concentration of 5-100 mM, preferably 48 mM and NHS in a molar concentration range of 1-50 mM, preferably 19 mM.

23. Carrier system according to claim 1 for use as medicament.

24. Carrier system according to claim 1 for the treatment or prophylaxis of infectious diseases, preferably systemic infection.

25. Carrier system according to claim 24, wherein the infectious disease is an infection with a bacterium that sequesters in a non-phagocytic cells, preferably a Gram-negative bacterium, more preferably Chlamydia, Coxiella burnetii, Ehrlichia, Rickettsia, Legionella, Salmonella, Shigella, or Yersinia; or Gram-positive bacterium, more preferably Mycobacterium leprae, or Mycobacterium tuberculosis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0069] The following figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0070] FIG. 1: Liposome preparation, characterization and protein covalent coupling andcell viability study

[0071] Monodisperse phospholipid fluorescent liposomes containing carboxylic groups were successfully prepared. Cholesterol in the bilayer was incorporated as a membrane stabilizer increasing the glass transition temperature. Liposomes were covalently coated with invasin or bovine serum albumin (BSA) without any observed aggregation. Size diameter of uncoated liposomes was around 142.5 nm, with a polydispersity index (PDI) of approx. 0.03. The zeta potential was in the range of −20 to −43mV. Increase in zeta potential was observed upon protein coating indicating higher stability due to steric hindrance by the protein corona. The protein coating efficiencies as judged by surface protein quantification using bicinchoninic acid (BCA) assay and western blot assay were comparable. Potential nanoparticle-dye interferences must be first considered to avoid false-positive and false-negative results. No interference was observed on measuring luminescence of ATP standards in presence of liposomes. FIG. 1 shows the viability of HEp-2 (a) and non-polarized (b) and polarized (c) Caco-2 cells after incubation with uncoated as well as invasin-coated liposomes. No significant difference in cell viability was observed for uncoated liposomes or liposomes coated with the bacterial surface protein, invasin compared to non-treated control cells.

[0072] FIG. 2.1: Invasin-coated liposomes promote tight adhesion to human epithelial cells I

[0073] Challenge experiments were designed to study the ability of invasin-coated (via physical adsorption (a) or covalent attachment (b)) liposomes to mediate adhesion to HEp-2 cells in presence of InvA-expressing Yersinia pseudotuberculosis acting onβ.sub.1-interin receptors. To do so, liposomes were added to the cells at 25° C., conditions where only cell adhesion but no bacterial invasion into host cells is observed. Two experimental sets were included in which the bacteria were added simultaneously or prior to the liposomes. This is in comparison to healthy state where cells were not exposed to bacteria. Control liposomes with adsorbed (c) or covalently-linked (d) BSA did not show significant adhesion to HEp-2 cells indicated via fluorescence imaging (FIG. 2-1-2.2).

[0074] FIG. 2.2: Invasin-coated liposomes promote tight adhesion to human epithelial cells II

[0075] Invasin coating resulted in a significant increase in cellular adhesion of about 2-5 (physically adsorbed InvA) and 32-38 fold (covalently-linked InvA) relative to control liposomes (FIG. 2-2). In presence of bacteria, a decrease of InvA-promoted adhesion of liposomes was observed. For instance, 2- and 6-fold decrease of cell-associated liposomes was detected when InvA-expressing bacteria were added simultaneously or prior to liposomes. This indicates that bacteria and liposomes use the same adhesion mechanism and compete for β.sub.1-interin receptors.

[0076] FIG. 3.1: Cell uptake kinetics and internalization mechanism into HEp-2 cells I

[0077] To analyze the capacity of InvA-coated liposomes to promote uptake into human cells, we investigated the number of intracellular uncoated (b, d) and invasin-coated (a, c) liposomes after 1 h (upper panel) and 4 h (lower panel) incubation of the liposomes with HEp-2 cells at 37° C. using confocal-multiphoton microscopy. Representative confocal images are shown in FIG. 3-1.

[0078] FIG. 3.2: Cell uptake kinetics and internalization mechanism into HEp-2 cells II

[0079] Results of image analysis of the sequestered z-stacks are demonstrated in FIG. 3-2. No significant cell uptake was observed for uncoated liposomes. Cell uptake was induced upon invasin coupling and the number of intracellular liposomes increased significantly over time. Notably, the uptake efficiency of uncoated liposomes and BSA-coated liposomes were similar and usually very low (FIG. 3-2). Therefore, only uncoated liposomes were used as control in all following uptake experiments.

[0080] FIG. 4: Uptake kinetics into HEp-2 cells

[0081] To determine uptake kinetics into HEp-2 cells, cell uptake into living cells was tracked over 4 h (FIG. 4i). The number of intracellular uncoated and InvA-coated liposomes increased over time. However, the overall number of internalized InvA-coated liposomes was significantly higher at each time point. Moreover, the average uptake rate was about 7-fold higher for invasin-coated (507 liposomes/h) relative to uncoated liposomes (70 liposomes/h) (FIG. 4ii). More time points in the first hour were not possible to realize in live cell imaging. This is to avoid continuous laser exposure of the treated cells on z-sectioning which may result in photobleaching and inaccuracy of the results. Therefore, to get more information on the first hour, 1 h fixed-cells experiments were performed similarly. Combining all time points, one could divide the uptake of invasin-coated liposomes into three phases: initial liposome uptake characterized by a fast exponential uptake rate leading to a plateau (saturation) which is typical for receptor-mediated uptake, followed by a process characterized by linear uptake rate.

[0082] FIG. 5: Further characterization of the uptake mechanism in HEp-2 cells

[0083] To further characterize the uptake mechanism, HEp-2 cells were incubated with uncoated or invasin-coated liposomes at 4° C. and 37° C. to determine relative liposomal uptake. At 4° C., energy-dependent uptake mechanisms (endocytosis) are greatly reduced. No significant difference in cell uptake of uncoated liposomes was observed at 37° C. when compared to 4° C. Reduction in temperature was however accompanied by significant decrease in cell uptake of invasin-coated liposomes (FIG. 5-1). Yet, still some invasin-coated liposomes were taken up at such low temperature, 4° C. Finally, to verify whether cell uptake mechanism of invasin-coated liposomes is a receptor-specific (62.sub.1-integrin) mechanism, cell uptake inhibition experiments were conducted. First, anti-integrin β.sub.1-antibody was added to HEp-2 cells before the addition of InvA-coated liposomes. As shown in FIG. 5-2, a significant reduction of liposome uptake was observed in the presence of the antibody, indicating that the InvA-triggered uptake of the liposome occurs via β-integrin receptors. Several inhibitors proven to reduce the InvA-triggered cell uptake of Yersinia pseudotuberculosis, Akt inhibitor VIII and NPC-15437, were examined. The serine threonine kinase Akt becomes activated in response to many β.sub.1-interin-initiated signaling processes. Activation of Akt is required for the invasin-mediated uptake of Y. pseudotuberculosis. Also protein kinase C (PKC) was shown to be implicated in the InvA-triggered uptake pathway. The selective PKC inhibitor NPC-15437, interacting at the regulatory domain of the enzyme, was effective in blocking the invasin-mediated bacterial invasion Inhibition experiments clearly indicate β.sub.1-interin receptor specific uptake whereas cell entry of invasin-coated liposomes was reduced to 22-29% compared to the untreated control (FIG. 5-II).

[0084] FIG. 6: Targeting of inflamed epithelium: Non-polarized versus polarized Caco-2 cells

[0085] InvA-mediated liposomes targeting to β.sub.1-integrin receptors could be exploited to develop drug delivery tools directed against an inflamed intestinal epithelium. Previous studies have demonstrated that β.sub.1-integrin receptors are not expressed on the apical side of enterocytes of the intestinal epithelial layer. Only the small number of M cells exposes this class of cell surface receptors on the apical side and are preferentially targeted by Yersinia pseudotuberculosis. However, under specific conditions, for instance during intestinal inflammation (e.g. inflammatory bowel disease), β.sub.1-integrins become more accessible on the apical side of the enterocytes. In order to mimic this situation liposome uptake into non-polarized and polarized Caco-2 cells was studied. Cells at 50% confluency express β.sub.1-integrins on their apical surface, whereas cells grown to over 90% confluency reduce the expression of the invasin receptor. To investigate whether invasin-coated liposomes are preferentially targeted to non-polarized 13.sub.1 integrin surface-exposing cells, InvA-coated and non-coated liposomes were used to challenge the non-polarized and polarized Caco-2 cells for 8 h. Only uptake of invasin-coated liposomes into sub-confluent Caco-2 cells was observed (FIG. 6a). Interestingly, some adhesion of InvA-coated liposomes on polarized cells was observed (FIG. 6c). However, this weak attachment was not sufficient to promote cell uptake. No uptake was observed for control uncoated liposomes into Caco-2 cells regardless of the confluency level (50% confluency FIG. 6b, 90% confluency FIG. 6d), demonstrating that the uptake process is specific for InvA.

[0086] FIG. 7: Size of gentamicin-containing liposomes

[0087] Liposomes containing gentamicin were firstly prepared by the lipid film hydration (LFH) method, by hydration of the lipid film with morpholine-4-ethanesulfonic acid hydrate (MES) buffer of pH 6 containing 10 mg/ml of gentamicin. Under these conditions, liposomes with a size of approximately 1000 nm were obtained. This size range does not fit with the desired size of ˜200 nm, as the cellular uptake is considered to be higher when particles are smaller. However, liposomes which were prepared in MES buffer without the addition of gentamicin showed an average size of ˜200 nm. Interestingly, when the amount of gentamicin added to the MES buffer was reduced, size of liposomes also decreased in a proportional manner with the amount of gentamicin. Therefore, pH of the hydration buffer was increased, to reduce the electrostatic interaction between liposomes and gentamicin, by instead using phosphate buffered saline (PBS) buffer of pH 7.4 which is close to the pKa of gentamicin (pKa=8.2). Using PBS buffer and 10 mg/ml of gentamicin, it was possible to obtain liposomes of 202.46+/−5.72 nm, an acceptable size.

[0088] FIG. 8: Colloidal characteristics of liposomes

[0089] In order to investigate the colloidal stability of liposome preparations, the size, PDI and zeta potential of liposomes prepared using three different methods were monitored over time. The size of liposomes prepared using the LFH method, the microencapsulation vesicle (MCV) method and the ammonium sulfate liposome (ASL) preparation method was found to be stable for a period of 45 days (FIG. 8a). For the PDI, all the three preparations showed an increase in PDI values, but never above a value of 0.2 (FIG. 8b). Concerning the zeta potential, the values of this parameter became more negative over the measured 45 day period (−20 mV to −35 mV) for LFH, MCV and ASL liposomes (FIG. 8c).

[0090] FIG. 9: Encapsulation and loading efficiency

[0091] The encapsulation efficiency is the amount of gentamicin which was encapsulated into liposomes versus the initial amount of gentamicin used for the preparation. LFH liposomes showed the highest encapsulation efficiency of 43.27%, whereas the encapsulation efficiency of MCV liposomes was 33.29%. 22.75% of the initially added gentamicin was found in ASL liposomes (FIG. 9b). Loading efficiency is also a parameter used to assess the efficiency of a drug loading into liposomes which depends on the actual (FIG. 9a) and the initial amount of gentamicin, phospholipids and cholesterol. In contrast to the encapsulation efficiency, the loading efficiency of ASL liposomes was found to be the highest among the three preparations 52.52%, while LFH liposomes showed 43.67% loading efficiency. 31.57% loading efficiency was measured in MCV liposomes (FIG. 9c).

[0092] FIG. 10: Chemical stability of liposomes

[0093] Stability was assessed in terms of size, PDI and zeta potential, as mentioned above, but also in terms of incorporated gentamicin. Encapsulation efficiency was evaluated (FIG. 10a) and the loading efficiency (FIG. 10b) of LFH, MCV and ASL liposomes on the day of preparation (day 0), as well as day 15 and day 21 after preparation. At day 0 the encapsulation efficiency ranged from 20 to 45% for the three preparations and the loading efficiency was found to be between 30 and 60%. After 15 days, both the encapsulation and loading efficiency of LFH liposomes had decreased to approximately 15%, and only 5% encapsulation efficiency and 2% loading efficiency were found for MCV liposomes. Surprisingly, the ASL preparation did not contain any gentamicin after 15 days. At day 21, only the LFH liposomes were seen to retain gentamicin (11% for both encapsulation and loading efficiencies). MCV and ASL liposomes did not contain any detectable gentamicin after 21 days.

[0094] FIG. 11: Stability of gentamicin-loaded, invasin-functionalized liposomes

[0095] For the invasin functionalization, the same conditions as used for preliminary BSA functionalization (two washing steps) were applied. The functionalization efficiency using LFH liposomes and invasin and measured via the BCA assay was approximately 60%, and as expected, the encapsulation efficiency was approximately 15%. These functionalized liposomes were subjected to a short-term stability study (designed to reflect the estimated duration of later cell experiments), where at day 0, day 2 and day 7 of storage the size, PDI, zeta potential, encapsulation efficiency and functionalization efficiency of liposomes was tested. After 2 and 7 days of storage, the size, PDI and zeta potential showed no appreciable change and stayed within the desired ranges (FIG. 11b, c, d). Whereas, the encapsulation efficiency at day 2 decreased to 7% and decreased even more to 5% after 7 days. The functionalization efficiency was also reduced after 2 days to 50%, and further to 40% after 7 days (FIG. 11a).

[0096] FIG. 12: Invasin functionalization efficiency measurement

[0097] The functionalization efficiency of invasin was measured by quantifying the amount of invasin in the liposomal preparation using the BCA assay. The results obtained with the BCA assay were then confirmed with SDS-PAGE using standard solutions of pure invasin and suspensions of invasin-functionalized gentamicin-containing liposomes (FIG. 12a). Results showed that the difference in measured functionalization efficiency between both methods was approximately 7%, which is to be expected given that the SDS method is more a qualitative technique than quantitative (FIG. 12b).

[0098] FIG. 13: Release test

[0099] Liposomes, invasin-functionalized or non-functionalized, were subjected to release testing to evaluate their ability to release the encapsulated gentamicin under a mechanical stress at 37° C. over a period of 3 h, using a dialysis membrane setup. A solution of gentamicin with same concentration to that contained within liposomes was used as a control. Cumulative percentages of gentamicin release values over 3 h, determined for three independent samples of each formulation, are shown in (FIG. 13). The gentamicin solution was detectable within the bulk release medium within 1 min, whereas gentamicin released from liposomes was not detectable until 3 min following initiation of the release test. After 1.5 h the gentamicin solution was shown to have completely permeated through the 10.000 MWCO cellulose membranes. In the case of liposomal gentamicin, complete release was achieved after 2.5 h, and both invasin-functionalized and non-functionalized liposomes showed similar kinetics of gentamicin release.

[0100] FIG. 14: Optimization of conditions for invasion assays

[0101] Epithelial cells of the HEp-2 cell line were infected with Salmonella enterica and Yersinia pseudotuberculosis using various different conditions, in order to determine the optimal parameters for testing the efficiency of invasin-functionalized liposomes loaded with gentamicin. The infection load of Salmonella enterica and Yersinia pseudotuberculosis, was assessed by using different multiplicities of infection (MOI) 1:10, 1:25, each with 1 h of infection time, and followed by either 1 h or 2 h of treatment with pure gentamicin in order to kill any remaining extracellular bacteria. Results showed that the 1:10 MOI and 1 h extracellular gentamicin treatment had the highest invasion rate for Salmonella enterica (FIG. 14a). For Yersinia pseudotuberculosis, the MOI of 1:25 and 1 h gentamicin treatment showed a similar invasion rate to that of the 1:10 MOI (FIG. 14b). Thus, for the conditions for liposomal invasion tests prior to the addition of liposomes, a 1:10 MOI and 1 h of pure gentamicin treatment was chosen for Salmonella enterica, and a 1:25 MOI with 1 h of pure gentamicin treatment was chosen for Yersinia pseudotuberculosis.

[0102] FIG. 15: Invasion assay—liposomal treatment of infected HEp-2 cells

[0103] As mentioned, gentamicin permeates very poorly through biological membranes due to its hydrophilicity. This limits its bactericidal action against intracellular bacteria. In this study, we tried to encapsulate gentamicin into liposomes and functionalize these particles with invasin to facilitate the interaction and the penetration of the liposomal gentamicin to infected HEp-2 cells. Salmonella enterica and Yersinia pseudotuberculosis were used to infect HEp-2 cells because they are intracellular bacteria. After infection with Salmonella enterica or Yersinia pseudotuberculosis, followed by incubation with pure gentamicin to kill extracellular bacteria, HEp-2 cells were treated with invasin-functionalized gentamicin-loaded liposomes (IGL) for 1 h. In Salmonella enterica-infected cells, we observed that treatment with IGL reduced significantly the intracellular bacterial load by 22%, when compared infected cells which were left untreated (blank −B) or which were treated with non-functionalized gentamicin-loaded liposomes (GL) (FIG. 15a). In the case of HEp-2 cells infected with Yersinia pseudotuberculosis, treatment with IGL also reduced the infection by 22% in comparison to control groups (FIG. 15c). Increasing the incubation time of IGL with infected cells from 1 h to 2 h resulted in a significant reduction of 30% in the infection load when compared to untreated cells as well as cells treated with empty liposomes (EL), invasin-functionalized empty liposomes (IEL) and GL. (FIG. 15b). The concentration of gentamicin used in the liposomal treatment was 50 mg/ml in 1.3 mM liposomes.

EXAMPLES

[0104] The following examples are for illustrative purposes only and do not limit the invention described above in any way.

Example 1

Lipid Film Hydration Method

[0105] Liposomes were prepared by the lipid film hydration (LFH) technique as previously described by Bangham and his colleagues. In a round bottomed flask and in a molar ratio of (6:0.6:3), DPPC, DPPE and cholesterol respectively were dissolved in 5 ml of chloroform: methanol (2:1). 10 μg/ml of Rh-DPPE was added to color the liposomes for the imaging experiment. The flask was then connected to a rotary evaporator (Buchi Switzerland) equipped with a vacuum controller set at 200 mbar, and a heating bath set at 70° C. for 1 h. This led to the formation of a dry lipid film. The vacuum controller was then set at 40 mbar for another 30 min to remove any residual traces of the organic solvent. A 5 ml volume of gentamicin solution (10 mg/ml) in phosphate buffered saline (PBS pH=7.4) was added to the dry lipid film and rotation was recommended at 50° C. for 1 h, leading to the hydration of the lipid film and the formation of MLV. The resulting MLV were then extruded 10 times through 200 nm pore size polycarbonate membranes (Polycarbonate track-Etch Membrane, Sartorius Germany) at 70° C. The final dispersion of liposomes was diluted 1:10 and stored at 4° C. In detail, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Sigma-Aldrich, Steinheirn Germany) and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) (DPPE) (Avanti Polar Lipids, Inc., Alabaster, USA) in a molar ratio of 6:3:0.6 were dissolved in 5 ml chloroform/methanol mixture, 2:1. A 100 μl of 0.5 mg/ml chloroformic solution of the fluorescent dye 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamineB sulfonyl) (ammonium salt) (Rh-DPPE) (Avanti Polar Lipids, Inc., Alabaster, Ala.,USA) was added. The final lipid mixture (19.2 mM) was dried in a rotary evaporator (Büchi, Essen, Germany) at 70° C. 200 mbar and 145 rpm for 1 h to form a thin uniform lipid film. Complete evaporation of the remaining solvents was achieved by further heating at the same temperature under a pressure of 40 mbar with 145 rpm for further 30 min. The lipid film was then hydrated with 5 ml PBS buffer, pH 7.4 at a speed of 55 rpm for 1 h at 50° C. Unilamellar liposomes were prepared by extruding the resulting multilamellar vesicles through 200 nm polycarbonate membrane (AMD Manufacturing Inc., Ontario, Canada) at 60° C. under high pressure using nitrogen flow in a sealed stainless steel jacketed extruder (LiposoFast L-50, Avestin, Mannheim, Germany). Liposomal dispersions were diluted 1:10 with PBS and stored in the fridge.

Example 2

Microencapsulation Vesicle Method (MCV)

[0106] Liposomes containing gentamicin were prepared in two steps via this method; emulsification, and dispersion with mechanical agitation. The emulsification step was done by dissolving DPPC: DPPE: cholesterol a in molar ratio of 6:0.6:3 in 10 ml dichloromethane, then 5 ml of PBS containing 10 mg/ml gentamicin was added. The mixture was emulsified with a homogenizer (Polytron PT 2500 E, Germany) at 7000 rpm for 10 min resulting in the formation of water in oil emulsion (W/O). The first emulsion was then diluted 1:3 in PBS and mixed at 520 rpm and 30° C. to form water in oil in water emulsion (W/O/W). Stirring was continued until the organic solvent was completely evaporated (60 min). The liposomal dispersion was finally extruded through 200 nm pore size polycarbonate membranes to form liposomes of optimal size.

Example 3

Liposome Loading: Active Loading—ammonium sulfate liposomes (ASL)

[0107] In this method, gentamicin was introduced into liposomes by the use of a pH gradient as has been previously described for amphipathic drugs. Since the pKa of gentamicin is 8.2, gentamicin is uncharged when dissolved in basic solutions, allowing it to permeate through lipid membranes, such as those of liposomes. Once the gentamicin is inside the liposomes, it has to be transformed into a charged molecule which will not be able to leave the liposomes. Thus, liposomes are filled with an acidic solution to ensure the transformation of the uncharged gentamicin into a charged compound. Liposomes were prepared as described for LFH liposomes, but the hydration step was done with a 250 mM ammonium sulfate solution (pH 5.3) instead of PBS containing gentamicin. After liposome extrusion, the ammonium sulfate-containing liposomes were centrifuged at 13000 g for 45 min, and then the pelleted liposomes were re-suspended in carbonate buffer (pH 10.2) containing 10 mg/ml of gentamicin in the uncharged state, to facilitate its penetration into liposomes. The liposomes were incubated with gentamicin at 37° C. for 1 h with intermittent vortex mixing every 10 min.

Example 4

Functionalization of Liposomes

[0108] A covalent coupling of model or targeting protein to the surface of gentamicin-loaded liposomes was performed. A method has been developed that allows the crosslinking of the protein directly without incorporation of a crosslinking reagent in the final formulation. The employed crosslinking reagent EDC reacts with the surface-exposed carboxyl groups on liposomal DPPE, forming an unstable reactive O-acylsourea ester. NHS is then added in order to increase the stability and the coupling efficiency of EDC. This results in the formation of a semi-stable amine-reactive NHS ester; which can then react with the amine groups on the protein to be coupled resulting in the formation of a stable amide bond. After purification of liposome in order to remove the non-encapsulated gentamicin, gentamicin-loaded liposomes were functionalized using BSA as a model protein, or invasin. Briefly, 2 ml of the liposomal suspension was incubated with the crosslinking reagent solution of EDC and NHS in a molar ratio of (3:1) in an ice bath. For gentle mixing, the suspension was kept shaking (SM, Shaker Germany) for 3 h. Then liposomes were washed three times through Centrisart tubes to remove the excess of the crosslinking. Afterwards, 300 μl of BSA solution or invasin (1 mg/ml) was added to the liposomal suspension and the mixture was then kept in the ice bath overnight with gentle mixing. The degree of protein functionalization was then determined by BCA assay following liposomes purification.

Example 5

Liposome Purification

[0109] As a part of the functionalization procedure, and also before analysis of the different liposome content of both non-functionalized and functionalized liposomes, liposomal formulations were separated from any residual, non-incorporated components or other reagents which could affect the chemical characterization. In all cases, the separation process was carried out by centrifugal ultrafiltration using Centrisart tubes (Centrisart 1, Sartorius AG Germany) equipped with a 300 000 molecular weight cut off membrane (MWCO). Briefly, liposomal suspension was placed into a Centrisart tube followed by the filtration membrane, and then centrifuged at 3720 g and 4° C. for 30 min. The ultra-filtrate was then removed and the liposomes were re-suspended in fresh buffer. This procedure was repeated three times to ensure the complete removal of any residual non-liposomal material.

Example 6

Liposome Characterization

[0110] Liposomes were prepared by different techniques resulting in liposomes of different physicochemical characteristics. These differences may have an impact on in vitro and in vivo behavior. Therefore, liposomal characterization for the purpose of conducting an evaluation of these different liposome preparation methods was carried out, and can be classified into three categories: physical, chemical and biological characterization. As part of physical characterization, the size distribution and also surface charge of liposomes were evaluated. Chemical characterization of the liposomes included evaluation of various liposomal constituents. The biological characterization focused on the impact of the liposomes in an in vitro cell model.

Example 7

Size and polydispersity index

[0111] The mean diameter and the polydispersity index (PDI) of liposomes were measured by the dynamic light scattering (DLS) technique using a Zetasizer (Nano ZS Malvern Instruments). This technique is based on the measurement of the intensity of light scattered by the molecules in the sample as a function of time. When light is scattered by a molecule or particle some of the incident light is scattered. If the molecule was stationary then the amount of light scattered would be a constant. Since all molecules in solution diffuse with Brownian motion in relation to the detector there will be interference (constructive or destructive) which causes a change in light intensity. By measuring the time scale of light intensity fluctuations, DLS can provide information regarding the average size, size distribution, and polydispersity of molecules and particles in solution. The zeta potential analysis is applied as a tool for the determining of particle surface charge in solution. This is an important parameter for understanding and predicting the long term stability of particle. Laser-doppler micro-electrophoresis was used to measure the zeta potential of liposomes using a Zetasizer (Nano ZS Malvern Instruments). This measurement is based on the application of an electric field to a solution of molecules or dispersion of particles, resulting in movement of the particles due to the interaction between their surface charge and the applied field. The direction and the velocity of particle motion is a function of particle charge, the suspending medium, and the electric field strength. Particle velocity is then measured using a laser interferometric technique called phase analysis light scattering (M3-PALS), as the particle velocity is proportional to the electrical potential of the particle at the shear plane—that is, the zeta potential. Thus, this optical measurement of the particle motion under an applied field can be used to the determine zeta potential.

Example 8

Liposome Imaging: Scanning Electron Microscopy

[0112] Scanning electron microscopy (SEM) is based on the use of a focused beam of high energy electrons in order to generate a variety of signals at the surface of solid specimens. The signals that derive from electron-sample interactions reveal information about the sample including external morphology, chemical composition, and crystalline structure of the materials that make up the sample. In order to characterize the surface morphology of gentamicin-loaded liposomes, SEM imaging was conducted using Zeiss EVO HD15 (Germany) SEM. Briefly, gentamicin-loaded liposomes were washed with water to remove any traces of buffer and then a dilution of 1:20 was carried out in order to avoid the formation of aggregates or any interactions between the particles. A volume of 10 μl was mounted on aluminum stubs, using double-sided adhesive carbon tape and silicon wafers in 5×5 mm chips (TED PELLA, Inc. Canada, USA). After drying, samples were sputter-coated with thick gold film using a Quorum Q150R ES (Gala Instrumente GmbH) sputter-coater, under argon atmosphere for secondary electron emissive SEM and then observed for morphology at an acceleration voltage of 5000 kV. Images were processed with SmartSEM® software.

Example 9

Fluorescence Microscopy

[0113] Gentamicin-loaded liposomes functionalized with invasin and containing rhodamine were produced. Rhodamine can emit fluorescence upon an excitation at 560 nm, giving the opportunity to visualize such liposomes using fluorescence microscopy. The preparation of samples for fluorescence imaging was done by linking liposomes to poly-L glutamic acid-coated glass. Briefly, glass bottom dish chambers (3.5×3.5 cm) were coated with 200 μl of 0.01% poly-L glutamic acid solution in distilled water for 5 min at room temperature. Chambers were washed with distilled water and incubated with 200 μl of 2 mM carbodiimide hydro-chloride (EDC) and 5 mM hydroxysuccinimide (NHS) in MES buffer (pH 6) for 15 min at room temperature to activate the carboxyl groups of poly-L glutamic acid. The non-bound crosslinking reagent EDC/NHS was removed and chambers were washed with MES buffer. Gentamicin-loaded liposomes functionalized with invasin, diluted 1:1, were then placed in the chambers and incubated for 2 h at room temperature in the dark. The crosslinking reaction was stopped using 50 mM TRIS-HCl buffer for 5 min, and then chambers were washed twice with MES buffer. Images were taken using Leica DMI6000B microscope, equipped with a metal halogenide lamp. The objective used was an oil immersion lens 63×, and images were processed using Leica Application Suite Advanced Fluorescence (LAS AF) software.

Example 10

Phospholipid, Cholesterol and Gentamicin Quantification

[0114] The Stewart assay, a simple and sensitive colorimetric method for the quantitative determination of phospholipids in liposomes was utilized in this study. This method is based on the ability of phospholipids to form a complex with ammonium ferrothiocyanate. Ferrothiocyanate reagent was prepared by dissolving 27.03 g of ferric 3-chloride-hexahydrate (FeCl.sub.3.6H.sub.2O) and 30.4 g of ammonium thiocyanate (NH4SCN) in 1 1 of distilled water. A lipid stock solution was prepared by dissolving 10 mg of DPPC in 100 ml chloroform (0.1 mg/ml). Duplicate volumes of this solution between 0.1 and 1 ml were then added to the volume of chloroform required to make the final volume to 2 ml. A 2 ml volume of the ammonium ferrothiocyanate solution was then added to each, in order to create a range of standard solutions in duplicate. Tubes of standard solutions were then vigorously vortexed for 20 sec and centrifuged for 10 min at 130 g (Rotina Centrifuge 420). A standard curve was constructed by measuring the optical density of the lower layer consisting of phospholipids and chloroform at 485 nm using a spectrophotometer (Lambda 35 UV/VIS Spectrophotometer, Perkin Elmer). The same procedure was used to determine the amount of phospholipids in liposomes by mixing 0.1 ml of liposomes with 1.9 ml of chloroform and 2 ml of the ferrothiocyanate reagent. The obtained absorbance was applied in the calibration equation to calculate the phospholipids concentration in liposomes. High performance liquid chromatography (HPLC) method for cholesterol quantification was used, with some modifications. Briefly, the Dionex HPLC system was used (Thermo Scientific, Germany) composed of a P680 pump, an Elite degassing System, an Asta-medica AG 80 column oven and a UV detector. The analytical column used was a LiChrospher® 100, RP-18 (5 μm), 125×4 column (Merck KGaA, Darmstadt, Germany). The oven temperature was set at 30° C. A mobile phase of acetonitrile: methanol (70:30 v/v) with a flow rate of 2 ml/min was used, with an analysis time of 15 min and an injection volume of 100 μl. All samples were analyzed in triplicate. Cholesterol was detected at a wavelength of 210 nm. Identification of the cholesterol peak in HPLC chromatograms was done by comparison of the retention times of the sample peak with those of the standards. Quantification of cholesterol in liposomes was done by comparison of sample peak area under the curve (AUC) with AUC values of standards. The standard curve was constructed using 7 standard concentrations, prepared using a stock solution of 200 μg/ml of cholesterol in 50:50 vol/vol of acetonitrile: methanol/ethylacetate (1:1), which was diluted in order to produce concentrations varying from 0 to 200 μg/ml cholesterol. For liposomes, a 400 μl volume of liposome formulation was mixed with 1 ml of 50:50 vol/vol acetonitrile: methanol/ethylacetate (1:1). A fluorometric procedure was used for gentamicin quantification. This method is based on the reaction of primary amine groups of gentamicin with the utilized reagent, O-phthaldialdehyde (OPA). Under basic pH conditions, this reaction produces a fluorescence which has a linear relationship with the gentamicin concentration, and which can be read directly on a simple fluorimeter (Tecan, Infinite M200, Germany) at an excitation wavelength of 344 nm and an emission wavelength of 450 nm. The preparation of the OPA reagent was performed by dissolving 0.2 g of OPA in 1 ml methanol and then adding 19 ml of boric acid (0.4 M, pH 10.4). The mixture was then stirred and 0.4 ml of 2-mercaptoethanol (14.3 M) was added. The pH was then re-adjusted to 10.4 using potassium hydroxide. Both boric acid and 2-mercaptoethanol were used in order to achieve high reaction efficiency and to stabilize the fluorescent product. Standards were prepared using 1 ml of gentamicin solution ranging in concentration from 0 to 30 μg/ml in PBS (pH 7.4). 0.6 ml of methanol was then mixed with each standard followed by the addition of 0.9 ml of the reagent solution (0.1 ml OPA reagent and 0.8 ml methanol). Quantification of the gentamicin in liposomes first required an extraction of the lipids due to their interference with this method. The extraction was done by adding 250 μl of dichloromethane to 200 μl of washed liposomal dispersion, followed by 500 μl of methanol. The mixture was then vigorously mixed until a clear solution was obtained. Afterwards 250 μl of NaOH solution (0.2 M) followed by 250 μl of dichloromethane were added to the mixture and mixed again. The resulting biphasic system was then centrifuged at 3720 g for 5 minutes, and 480 μl of the remaining upper layer was used for gentamicin quantification. This extracted 480 μl was made to a volume of 1 ml by adding PBS; 0.6 ml of methanol was then added, followed by 0.9 ml of the reagent solution (0.1 ml OPA reagent and 0.8 ml methanol). Standards and samples were then incubated for 10 min in dark, following which the fluorescence was measured in a plate reader with an excitation of 344 nm and emission of 450 nm. The amount of gentamicin entrapped within liposomes was then calculated by comparing the measured fluorescence of samples to that of standard solutions. The entrapped amount of gentamicin was then expressed as an Encapsulation Efficiency, in which the amount of entrapped gentamicin is given as a percentage of the initial amount of gentamicin added during liposome preparation (Equation 1). Using the measured amounts of gentamicin and the measured amounts of lipid components (actual loading) as well as the initial amounts of gentamicin and lipid components (initial loading), the Loading Efficiency of liposomes was also calculated (Equation 2):

[00001]$.Math.\mathrm{Equation}.Math..Math.1..Math..Math.\mathrm{Encapsulation}.Math..Math.\mathrm{efficiency}$$\mathrm{Encapsulation}.Math..Math.\mathrm{Efficiency}.Math..Math.\%=\frac{\mathrm{Actual}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\mathrm{Gentamicin}}{\mathrm{Initial}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\mathrm{Gentamicin}}100.$$.Math.\mathrm{Equation}.Math..Math.2..Math..Math.\mathrm{Loading}.Math..Math.\mathrm{efficiency}$$\mathrm{Loading}.Math..Math.\mathrm{Efficiency}.Math..Math.\%=\frac{\mathrm{Actual}.Math..Math.{\mathrm{Loading}}^{*}}{\mathrm{Initial}.Math..Math.{\mathrm{Loading}}^{**}}100.$${\hspace{0.17em}}^{*}.Math.\mathrm{Actual}.Math..Math.\mathrm{Loading}=\frac{\mathrm{Actual}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\mathrm{Gentamicin}.Math..Math.\left(\mu .Math..Math.\mathrm{mol}.Math.\text{/}.Math.100.Math..Math.\mu .Math..Math.l\right)}{\mathrm{Actual}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\left(\mathrm{Phosph}+\mathrm{Chol}\right).Math..Math.\left(\mu .Math..Math.\mathrm{mol}.Math.\text{/}.Math.100.Math..Math.\mu .Math..Math.l\right)}$${\hspace{0.17em}}^{**}.Math.\mathrm{Initial}.Math..Math.\mathrm{Loading}=\frac{\mathrm{Initial}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\mathrm{Gentamicin}.Math..Math.\left(\mu .Math..Math.\mathrm{mol}.Math.\text{/}.Math.100.Math..Math.\mu .Math..Math.l\right)}{\mathrm{Initial}.Math..Math.\mathrm{amount}.Math..Math.\mathrm{of}.Math..Math.\left(\mathrm{Phosph}+\mathrm{Chol}\right).Math..Math.\left(\mu .Math..Math.\mathrm{mol}.Math.\text{/}.Math.100.Math..Math.\mu .Math..Math.l\right)}$

Example 11

Protein Quantification—BCA Assay

[0115] The amount of protein attached to the liposomes was quantified using the bicinchoninic acid protein assay (BCA). The BCA assay combines a protein-induced biuret reaction with the highly sensitive and selective colorimetric detection of the resulting cuprous cation (Cu.sup.1+) by bicinchoninic acid. A Cu.sup.2+protein complex is formed under alkaline conditions, followed by reduction of the Cu.sup.2+to Cu.sup.1+. A purple-colored reaction product is formed by chelation of two molecules of bicinchoninic acid with one cuprous ion. The bicinchoninic acid-copper complex is water soluble and exhibits a linear absorbance at 562 nm over a board range of protein concentrations. This absorbance is proportional to the protein concentration. Standard curves were prepared in accordance with the utilized BCA assay kit (Quantipro BCA Assay Kit, Sigma-Aldrich). Standards were made using different concentrations from stock solutions of either invasin or BSA (50 μg/ml). The Quantipro Working Reagent was prepared by mixing 25 parts of Reagent QA (Solution of sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.2 NaOH, pH 11.25) with 25 parts of Reagent QB (Solution of bicinchoninic acid 4% w/v, pH 8.5). After Reagents QA and QB were combined, 1 part of Reagent QC (4% w/v cupric sulfate and pentahydrate solution) was added and mixed until a homogenous green color was obtained. In glass tubes, 1 ml of the standards was mixed with 1 ml of the Quantipro Working Reagent. Mixtures were incubated at 60° C. for 1 hour. The UV absorbance was recorded in 96-well plates in a plate reader at 562 nm. As in the case of standards, 1 ml of liposome samples in glass tubes was combined with 1 ml of Quantipro working Reagent, and the UV absorbance measured at 562 nm. The concentration of liposome-bound protein was then calculated in reference to the created standard curve. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in order to confirm the results of the protein quantification by BCA assay. After loading protein-functionalized liposome samples, protein standards and a protein ladder (Thermo Scientific™ Spectra™ Multicolor Broad Range Protein Ladder), electrophoresis was carried out in electrode running buffer at 30 mA constant voltage for 45 min. The gel was washed and stained with Page Blue Protein Staining Solution (Fermentas, Lithuania). Images from the gel were taken by Gel Doc™ EZ Imager (Bio-Rad, Germany) and processed with Image Lab Software (Bio-Rad, Germany).

Example 12

Stability and Release Studies

[0116] Liposomes were stored at 4° C. for a period of 45 days, and at set time intervals samples were taken and analyzed in terms of size, PDI, zeta potential, as well as gentamicin and invasin content. In vitro release of gentamicin from the liposomes was investigated over a period of 3 h. Dialysis cellulose-ester membranes of 11.5 mm diameter and 10.000 MWCO (Biotech, USA) were soaked for 1 h before use in distilled water at room temperature to remove the preservative, followed by rinsing thoroughly in distilled water. Dialysis membranes containing 5 ml of gentamicin-loaded liposomes, invasin-functionalized, gentamicin-loaded liposomes or gentamicin solution were kept stirring at 200 rpm in separate beakers containing 60 ml of PBS (pH 7.4) and incubated at 37° C. (Binder Incubator, Germany) for 3 h. At predetermined time intervals, 1 ml aliquots of PBS solution were removed and substituted with an equal volume of fresh PBS. The amount of gentamicin in removed PBS aliquots was then quantified.

Example 13

Biological Characterization

[0117] Human Larynx Carcinoma cell line (HEp-2) cells were cultured in a 75 cm.sup.2 flask using Roswell Park Memorial Institute (RPMI 1640) medium, supplemented with 7.5% newborn calf serum (NCS). Cells were incubated in a humidified incubator (Heraeus CO2 Thermo Scientific Incubator) at 37° C. and 5% CO.sub.2. Medium was changed every two days and cells were split when confluency was reached. For cellular invasion experiments, cells grown in 75 cm.sup.2 flasks were washed with PBS (PBS Dulbecco, Biochrom Germany) and incubated with 3 ml of trypsin 0.5 g/l for 10 min to detach the cells. Afterwards, 7 ml RPMI medium supplemented with 7.5% NCS was added to the flask to inhibit the trypsin activity. Cells were then plated in 24 well plates at a density of 1×10.sup.5 cells per well and incubated in a humidified incubator at 37° C. and 5% CO2 for 18 h to allow cells to adhere to the plate. HEp-2 cells infected with the pathogens Salmonella enterica serovar Typhimurium SL1344 and Yersinia pseudotuberculosis YPIII were used as an in vitro model to test the invasive ability and resulting efficacy of gentamicin-loaded liposomes. Bacteria were cultured 24 h prior to experiments in overnight tubes containing 5 ml of Lennox broth (LB) medium (Carl Roth GmbH, Germany). Tubes were kept overnight in a shaking incubator (Infors HT, Multitron) at 37° C. in the case of Salmonella enterica, and 25° C. for Yersinia pseudotuberculosis. Prior to invasion experiments, Salmonella enterica was freshly diluted 1:100 with LB medium and incubated at 37° C. for a further 3 h growing to late exponential phase in order to induce expression of pathogenicity island I (SPI1) proteins important for cell invasion. Afterwards, both bacteria were washed once and suspended in PBS (PBS tablets. Medicago, Sweden). The culture medium of HEp-2 cells (seeded one day before in a 24 well-plate) was then exchanged with binding buffer (RPMI 1640 medium with 20 mM Hydroxyethyl-piperazineethane-sulfonic acid buffer (HEPES) and 0.4% BSA) containing Salmonella enterica or Yersinia pseudotuberculosis at ratios of 1/10 and 1/25 Multiplicity of Infection (MOI); which is the ratio of infection targets to infectious agents (cell/bacteria). 24 well plates were then centrifuged at 1000 rpm for 5 min (Eppendorf 5810 R Centrifuge) to sediment the bacteria onto the cells. Cells and bacteria were then incubated for 1 h in a humidified incubator at 37° C. and 5% CO2 atmosphere to allow binding and penetration of the bacteria into the cells. Cells were then washed twice with PBS and incubated for 1 h or 2 h with binding buffer containing 50 μg/ml of gentamicin (Sigma-Aldrich, Germany) to kill any extracellular located bacteria. The infected cells were then washed twice with PBS to eliminate the extracellular gentamicin and killed extracellular bacteria, leaving HEp-2 cells containing either intracellular Salmonella or intracellular Yersinia. Following the invasion protocol as above, infected cells were washed twice with PBS and lysed with 200 μl lysis buffer containing 0.1% Triton X-100. Cell lysate was then plated in sterile agar plates (2% LB and 1.8% agar) in serial dilutions (maximum dilution 1:625) and incubated overnight at 37° C. for Salmonella enterica and for 48 h at 25° C. for Yersinia pseudotuberculosis. Following incubation, bacterial colonies were counted and multiplied by the appropriate dilution factor. The number of colonies from the cell lysate was then expressed as a percentage of the number of colonies from the initial amount of bacteria used for the infection (inoculum), referred to as the percentage of invasion (Equation 3). The conditions (namely, cell: bacteria ratio) which were shown to result in the highest percentage of invasion were selected for use in further studies employing liposome treatment of infected cells:

[00002]$.Math.\mathrm{Equation}.Math..Math.3..Math..Math.\mathrm{Percentage}.Math..Math.\mathrm{of}.Math..Math.\mathrm{Invasion}$$\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{Invasion}=\frac{N^{\mathrm{br}}.Math..Math.\mathrm{of}.Math..Math.\mathrm{colonies}.Math..Math.\mathrm{from}.Math..Math.\mathrm{cell}.Math..Math.\mathrm{lysate}}{N^{\mathrm{br}}.Math..Math.\mathrm{of}.Math..Math.\mathrm{colonies}.Math..Math.\mathrm{from}.Math..Math.\mathrm{inoculum}}100$

[0118] Infected cells were incubated with empty liposomes, invasin-functionalized empty liposomes, liposomes containing 50 μg/ml gentamicin and liposomes containing 50 μg/ml gentamicin functionalized with invasin, all of which were suspended in binding buffer. Cells containing intracellular Salmonella enterica were treated with liposome formulations for 1 h, while cells containing Yersinia pseudotuberculosis were treated with liposomes for either 1 h or 2 h. The analysis of liposomal treatment was carried out by calculating the percentage of invasion from each treatment condition according to (Equation 3). Then, the efficiency of treatment was assessed by measuring the percentage of decrease in invasion after normalizing the different treatments to the blank (un-treated).

Example 14

Overexpression and Purification of the Cell-Surface Exposed C-Terminal Domain of Invasin (InvA497) from Y. pseudotuberculosis

[0119] Two liters of E. coli BL21 expressing the His-tagged. C-terminal 497 amino acids of invasin (His.sub.6-Inv497) from Yersinia pseudotuberculosis were grown at 37° C. in Luria Bertani broth medium to an A.sub.600=0.4. The culture was shifted to 17° C. and grown to an A.sub.6oo=0.6. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 100 μM to induce the expression His.sub.6-InvA497. The cells were grown overnight at 17° C. The cell pellet was resuspended in 50 ml cold lysis buffer containing 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole (pH 8) plus protease inhibitor cocktail containing 5 mM phenylmethylsulfonyl fluoride, 10 mM pepstatin (Sigma, Steinheirn, Germany), 10 mM E64 protease inhibitor (Boehringer, Mannheim, Germany), 20 mM leupeptin (US Biochemical, Cleveland, Ohio, USA) and 10 mM chymostatin (Sigma, Steinheim, Germany). The cells were disrupted using a french press (2× at 1000 psi). The His.sub.6-InvA497 protein was purified by affinity chromatography with Ni-NTA Agarose (Qiagen), eluted in elution buffer containing 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazol (pH 8) and dialyzed twice against 10 mM Tris buffer pH 8 containing 300 mM NaCl. Protein concentrations were determined by the Bradford protein assay (Pierce, Rockford, Ill, USA).

Example 15

Covalent Attachment of InvA497 on the Liposomal Surface

[0120] Invasin conjugation to the liposomal surface was based on covalent immobilization of the N-terminal of the protein to the carboxylic groups on the liposomal surface which were first activated using EDC/NHS (EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma Aldrich, Steinheim,Germany; NHS: N-hydroxysuccinimide, 99% (Carbolution Chemical GmbH,Saarbrücken,Germany). A volume of 300 μl of 48 mM EDC/19 mM NHS in 100 mM MES buffer (pH 6) was incubated overnight with a 2 ml liposomal dispersion with shaking at room temperature, centrifuged (Rotina 420R; Hettich Zentrifugen, Tuttlingen, Germany) in Centrisart® tubes 300,000 MWCO (Sartorius, Goettingen, Germany) at 3270 g, 4° C. for 30 min to remove excess free reagent followed by three successive washing steps during which the MES buffer was gradually exchanged with PBS, pH 7.4. The volume was then completed to 2.5 ml with PBS. 300 μl of 1 mg/ml invasin in PBS was added and coating process was continued overnight in ice bath with shaking. This was followed by centrifugation and washing steps in Centrisart® tubes 300,000 MWCO to remove unbound invasin. Covalent attachment of BSA (Sigma Aldrich, Steinheim, Germany) on liposomes followed the same protocol and served as controls for cell adhesion experiments.

Example 16

Cell Cultures and Treatments

[0121] HEp-2 cells (CCL-23™; ATCC, Manassas, Va., USA) were cultivated in RPMI (Gibco by life technologies™, Paisley, UK) supplemented with 10% fetal calf serum (FCS) (Lonza, Cologne, Germany) and kept in culture for 2 months maximum after thawing. Caco-2 cells, clone C2Bbel (CRL-2102™; ATCC, Manassas, Va., USA) were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 1% non-essential amino acids (PAA cell culture company, Pasching, Austria) and used at passages 58-72.

Example 17

Cell Viability Assay: ATP (Vialight® Plus) Assay

[0122] This assay kit (Lonza) measures cytoplasmic adenosine triphosphate (ATP) to assess the functional integrity of living cells. This bioluminescent assay utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin. The emitted light intensity is linearly related to ATP concentration. A volume of 100 μl of a series of 0.12-1.92 μM liposomal dispersions was tested on confluent HEp-2 cells, and confluent and 50% confluent Caco-2 cells seeded in 96-well plates (Greiner Bio One, Frickenhausen, Germany). Invasin-coated and uncoated liposomes were tested in parallel. Cells were incubated with the liposomes for 4-8h 50 μl/well of cell lysis reagent was added to extract ATP from the cells. A volume of 100 μl of each of the cell lysate and ATP monitoring reagent Plus8 were incubated in 96-well white walled luminometer plate (Optiplate™-96; Perkin Elmer Inc., Waltham, Mass., USA) for 2 min at room temperature in the dark. Bioluminescence was measured (Tecan Deutschland GmbH, Crailsheim, Germany). Cells grown in culture medium only were considered as high control (100% cell viability) and others incubated with Triton X-100 (2% w/v) were used as low control (0% cell viability). Percentage cell viability was calculated based on five replicates as follows:

[00003]$\%.Math..Math.\mathrm{Cell}.Math..Math.\mathrm{viability}=\frac{{\mathrm{Lum}}_{\exp }-{\mathrm{Lum}}_{\mathrm{low}.Math..Math.\mathrm{control}}}{{\mathrm{Lum}}_{\mathrm{high}.Math..Math.\mathrm{control}}-{\mathrm{Lum}}_{\mathrm{low}.Math..Math.\mathrm{control}}}*100$

[0123] In parallel, ATP controls in concentrations of 1.5 and 0.015 μM were prepared. 50 μl of each of the control and the liposomes was incubated together with 100 μl ATP monitoring reagent Plus® and bioluminescence was measured to check for wavelength interference in absence of cells.

Example 18

Cell Adhesion Experiments

[0124] One day before adhesion experiments, HEp-2 cells were seeded in 8 well μ-slides (ibidi cell infocus, Martinsried, Germany) at a density of 1×10.sup.4 cells/well. In parallel, constitutively GFP-expressing Yersinia pseudotuberculosis were grown in LB broth prior to infection. Two types of liposomes at a concentration of 4×10.sup.4 liposomes/ml were tested: liposomes to which invasin was covalently coupled (invas-cov) versus liposomes to which invasin was physically adsorbed by incubation of the liposomes with 1 mg/ml invasin at 37° C. for 2 h (invasphys). For control liposomes BSA was covalently attached (BSA-cov) or physically adsorbed (BSA-phys). Three sets of experiments were performed in parallel: The first set resembles late infection stage where cells were infected with bacteria (50 μl of 1×10.sup.6 bacteria per well) 30 min before addition of 50 μl/well of test liposomal dispersion. In the second set, resembling early infection stage, both bacteria and liposomes were applied together. For the third set (control), the liposomes were applied to cells without any pretreatment; i.e. representing the healthy state. Cells were washed three times with PBS and incubated in binding buffer (RPMI 1640 medium supplemented with 20 mM HEPES (pH 7) and 0.4% BSA) before addition of bacteria and liposomes. Cells were incubated for 1 h after liposomal application, after which the medium was removed and cells were washed three times with PBS. This was followed by cell fixation using 4% paraformaldehyde in PBS for 10 min, blocking and cell permeabilization with blocking buffer (5% goat serum, 0,1% Triton X-100 in 1× PBS) for 60 min and nuclei staining by DAPI mounting medium (Roth, Karlsruhe, Germany). Cell adhesion was examined using fluorescence microscopy (Zeiss Axioskope; Zeiss, Jena, Germany) followed by image analysis by ImageJ (http://rsbweb.nih.gov/ij/). Image analysis was based on a previously established method in which the number of pixels due to liposomal fluorescence was calculated. The number of liposomes was estimated based on the area of a single diffraction-limited fluorescent spot; π(r.sub.xy).sup.2, 0.359 μm.sup.2 in this study based on λ (emission wavelength for rhodamine)=564 nm and NA (numerical aperture of the optical lens)=1.1.

Example 19

Cell Uptake Assays

[0125] Cells were seeded in 24-well imaging plates (Zell-Kontakt, Norten-Hardenberg, Germany) to 70-80% confluency for HEp-2 cells and 50%-90% confluency for Caco-2 cells. Liposomes, at a concentration of 1.92 μM, were used for cell uptake experiments. Liposomes were first centrifuged at 20000 g, at 4° C. for 30 min and redispersed in biological medium. Cells were washed with PBS after removing the biological medium and liposomes (500 ul/well) were incubated with the cells for 1,4 or 5 h. In order to assess the uptake mechanism of invasin-coated liposomes (invas-cov) into HEp-2 cells, experiments were conducted at 37° C. or 4° C. for 4 h. In addition, the following inhibitors in RPMI supplemented with 20 mM HEPES buffer (pH 7) and 0.4% BSA were incubated with the cells for 1 h at 37° C.: anti-integrin β.sub.1-antibody, 1:100 dilution (clone P4C10; Sigma Aldrich, Schnelldorf, Germany), 1 μM NPC-15437 dihydrochloride (Sigma Aldrich, Schnelldorf, Germany) and 25 μM Akt inhibitor VIII (Calbiochem; EMD Chemicals Inc., San Diego,Calif., USA). At the end of 1 h incubation, inhibitors were removed and cells were washed with PBS before incubation for further 4 h with liposomes. At the end, the biological media were removed and the cells were washed with PBS. Cell membrane was stained by 6.25 μg/ml fluorescein wheat germ agglutinin (Flu-WGA) (Vector Laboratories, Inc., Burlingame, Calif., USA). Cells were fixed with 4% formaldehyde. Nuclei were stained with DAPI (6.66 ng/ml) (Sigma Aldrich, Schnelldorf, Germany). Plates were protected from light and stored at 4° C. till further imaging. At least three replicates were performed. Uncoated liposomes were used as a control.

Example 20

Confocal-Multiphoton Laser Scanning Microscopy and Image Analysis

[0126] Fluorescence imaging was performed using an inverted confocal/two photon microscope (ZEISS LSM 510 MTA, Carl Zeiss, Jena, Germany). The objective used was a water immersion lens 40+ (NA=1.1). Wavelengths of 543 nm, 488 nm and 720 nm were used for excitation of rhodamine-labelled liposomes, fluorescein-labeled cell membrane and DAPI-labeled nuclei, respectively. Z-stacks of the skin samples were taken with steps every 0.8 μm. Each optical scan is of a size of 0.22 μM×0.22 μM. The gain settings were adjusted for each measurement individually. For each captured z-stack optical layers encompassing only taken up liposomes were chosen and z-projection image of the red channel (red fluorescence due to liposomes) was developed using ImageJ. Number of pixels was computed and converted into weighed number of liposomes as described earlier.

Example 21

Live Cell-Imaging

[0127] HEp-2 cells were seeded in 8-well μ-slide chambers and used when 70-80% confluent. Liposomes were first redispersed in biological medium and sterilized upon filtration through 0.2 μm membrane filter. Cells were washed with the biological medium, stained with Flu-WGA for 5 min at 37° C. and washed again before application of liposomes. The whole setup was transferred into a special incubation chamber of the confocal microscope with a constant temperature of 37° C. and 5% CO2 to maintain cell viability throughout the experiment. An area of interest was selected and imaging using the same optical settings was performed as indicated above, except for the size of the optical image; 0.27×0.27 μm.sup.2. Z-stack was sequestered at different time intervals (1, 2, 3 and 4 h). The thickness of the optical layer was kept as 0.8 μm. ImageJ was then used to develop a z-projection image of the red channel of the optical layers in the sequestered z-stack followed by pixel analysis. The weighted number of liposomes was plotted versus time to determine cell-uptake kinetics of invasin-coated (invascov) versus uncoated liposomes. To get more information on the first hour kinetics, cell uptake experiments at 10 and 30 min for both liposomes were conducted in parallel using the same set-up, however the cells were fixed afterward and imaged similarly.