WO2019224709A1

WO2019224709A1 - Antibiotic delivery system

Info

Publication number: WO2019224709A1
Application number: PCT/IB2019/054179
Authority: WO
Inventors: Sounik Sarkar; Roshni THAPA; Jaydeep Bhattacharya
Original assignee: Jawaharlal Nehru University
Priority date: 2018-05-22
Filing date: 2019-05-21
Publication date: 2019-11-28

Abstract

The present invention relates to an antibiotic delivery system for combating microbial infection, comprising platelets loaded with antibiotics. The antibiotic loaded platelets are capable of killing microbes at very low antibiotic concentration. The present invention also provides a method of administration of the antibiotic delivery system for combating microbial infection and a device for mixing platelet and antibiotics to obtain platelets loaded with antibiotics.

Description

ANTIBIOTIC DELIVERY SYSTEM

FIELD OF INVENTION

The present invention relates to the development of a novel antibiotic (Ab) delivery system for combating microbial infection. In the present invention, platelet, the smallest blood component, is used as the antibiotic delivery system. The antibiotic loaded platelets are capable of killing microbes at very low Ab concentration. This invention can also be used to inhibit the growth and kill antibiotic resistance pathogenic strains. Moreover, the Ab loading in the platelets makes the Abs invisible to the microorganisms and does not generate any immunogenicity and further resistance.

BACKGROUND OF THE INVENTION

Till date, one of the most critical health challenges occurs due to the microbial pathogenesis worldwide. To combat this microbial explosion on public health, several ranges of antibiotics have been used on a regular basis. Still the microbes manage to invade the effect of antibiotic and become resistance. Sometimes bacteria show the intrinsic resistance property and some time the drug molecules encounter difficulties passing through the bacterial cell wall because of the high molecular weight and a large spatial structure ( Nicolosi et al, (2015) International journal of antimicrobial agents, 45(6), pp.622-626). This in turn compels the scientists to discover more antibiotic and designing newer delivery vehicle for the same. It is much more convenient to improve the existing delivery system rather than introduction of new antibiotics since inefficient delivery can lead to poor therapeutic effect of the administered drug. Over the last decade, innovative technological research has been done to change the pharmacokinetic profile of known antibiotics to reduce the clinical significance of acquired bacterial resistance. Several drug carriers have been developed for treating pathogens, including antibiotics loaded into liposomes and other lipid formulations, microspheres, polymeric carriers, fullerenes, dendrimers and nanoplexes (,, Salouti and Ahangari (2014) DOI: 10.5772/58423; Pinto -Alphandary et al, (2000) International journal of antimicrobial agents, 13(3), pp.155-168; Briones et al, (2008) Journal of Controlled Release, 125(3), rr.210-227; and Huh et al, (2011) Journal of controlled release, 156(2), pp.128-145) Among them most widely studied delivery system is liposomal systems. Another popular approach is association of antibiotics with colloidal nano-sized material. Though considered as a good delivery vehicle, liposomal system has some major drawbacks. Liposome mostly carries hydrophilic drugs rather than hydrophobic drugs. Release of hydrophobic drug from liposome faces some difficulties ( Foldvari el ah, (1993) Drug development and industrial pharmacy, 19(19), pp.2499-2517; and Nounou et ah, (2006) Acta Pharmaceutica, 56(3), pp.311-324). Moreover, elimination of liposome from blood is also a matter of concern (Torchilin (2005) Nature reviews Drug discovery, 4(2), p.l45). On the other hand, the metallic (or even polymeric) nanoparticle systems have limited biodegradability (Soppimath et ah, (2001) Journal of controlled release, 70(1-2), pp.l-20), toxicity and adverse immune-response (Le and Chen (2011) Small, 7(21), pp.2965-2980; and Ahamed et ah, (2010) Clinica chimica acta, 411(23-24), rr.1841-1848).

It is generally known in the art that platelets or antibiotics can be administered into a patient’s body by injection. EP1191962 provides a device for allowing the injection of all of antimitotic, antibiotics or blood platelets when administered by slow intravenous. The disclosure of EP1191962 further points out the drawabacks of any residual products that are stagnant in the evacuation tubing are often thrown with it, resulting in under-treatment of the patient and risks to the environment. EP1191962 aims at providing specialized device for slow intravenous chemotherapy infusions (IVL): antimitotic for the treatment of cancers, but also antibiotic, anesthetic and valuable blood products such as blood platelets.

US20180125893 provides a pure platelet-rich plasma (P-PRP) composition as an alternative to conventional antibiotic treatment of subclinical mastitis caused by Gram-positive bacteria in bovine including five live platelets and leukocytes, an anticoagulant, and an activating substance.

The above mentioned patent applications either aim at improving the conventional method of administering antibiotics viz. injection; or avoiding the administration of antibiotics at all to address the general problem associated with use of antibiotics viz. microbial resistance. Hence, there is a need in the art of developing a tool for delivering antibiotics in a patient’s body in a simple manner and in manner that the patient’s body does not develop resistance against such antibiotics; furthermore, improving the efficacy of platelets and antibiotics when employed alone. Thus, in the present invention, an altogether different delivery system for antibiotics has been introduced. The system does not have any biocompatibility issue as it is a component of body. Our choice of delivery system is platelet. Platelet is the smallest anucleated component of blood. They are cytoplasmic fragments derived from the megakaryocytes of the bone marrow (Machlus et ah, (2014) British journal of haematology, 165(2), pp.227-236; and Hamzeh-Cognasse et ah, (2015) Frontiers in immunology, 6, p.82).

In normal physiological condition, platelets play a vital role in hemostasis, a process by which bleeding is stopped at the site of interrupted endothelium. During vessel injury, circulating platelets attach with the exposed endothelium and get activated. After activation, they interact with each other and form platelet plug (primary hemostasis). This process in turn activate the coagulation cascade which results in fibrin deposition and formation of fibrin plug (secondary hemostasis) and thus prevent blood lose from vessel injuries.

Besides the obvious role in hemostasis, blood platelets have been recently shown to actively participate in immunity. Clinical investigation suggests that thrombocytopenia occurs during infection and blood platelets can able to bind infectious agents or engulf them (Garraud et ah, (2013) Critical care, 17(4), p.236). Previously, a new role of platelets in the field of drug delivery has been explored. They have been used as a carrier of drug for the cancer treatment and management. (Sarkar et ah, (2013) Pharmaceutical research, 30(11), pp.2785-2794). The present inventors have targeted‘platelet- pathogen interaction’ and used platelets as antibiotic carriers. This well interaction of platelets with pathogens especially bacteria inspire us to adopt the present concept of using platelets as antibiotic carrier. The concept has been verified using E. coli as model bacteria and chloramphenicol as model antibiotic.

Microbial pathogenesis, till date, is one of the most critical health challenges occurring worldwide. Several ranges of antibiotics have been developed to control microbial pathogenesis. Due to increasing rate of antibiotic resistance development, newer antibiotic or newer technology for antibiotic delivery is required. As introduction of new antibiotic is very difficult, it is much more convenient to improve the existing delivery system rather than introducing new antibiotics.

There is also development of many antibiotic resistant strains. As mentioned hereinabove that no new antibiotics have been discovered in recent years it is getting harder to combat the resistant strains. In the present invention the antibiotics have been delivered inside the platelets successfully. It not only prevents the generation of resistance but also kills the microorganism at very low concentration. Thus, the delivery system of the present invention has high industrial importance as the same antibiotics show increased efficacy once loaded inside the platelet. In general, the development of new and potent antibiotics and their availability in the market after necessary regulatory approval take long time,. On the contrary the delivery system of present invention can be used with existing antibiotics. The present invention also provides a portable device that allows the proper loading of the antibiotics inside the plate during the platelet transfusion.

The present invention provides for the first time a delivery system wherein the platelets are laoded with antibiotics to improve the efficacy of the antibiotics, decrease the chances of developing resistance against the said antibiotics and does not have any biocompatibility issues with the recipient’s body. The present invention opens a new era and provides a safer approach towards antibiotic delivery.

OBJECTS OF THE INVENTION

The present invention provides an antibiotic delivery system for combating microbial infection, comprising platelets loaded with antibiotics.

In another embodiment in antibiotic delivery system of the present invention the platelets are isolated from healthy human and/or animal donors.

In still another embodiment the present invention provides an antibiotic delivery system for combating microbial infection selected from infection caused by bacteria, viruses, protists, fungi or combination thereof.

In yet another embodiment the antibiotic delivery system of the present invention is in form of tablets, capsules, creams, ointments, aerosols and/or patches.

In another embodiment in the antibiotic delivery system of the present invention the platelets loaded with antibiotics comprises about 25 % of the loaded antibiotics.

In still another embodiment in the antibiotic delivery system of the present invention the antibiotics are selected from the group consisting water-soluble, water- insoluble (hydrophobic) antibiotics and/or combination thereof. In yet another embodiment the in antibiotic delivery system of the present invention the antibiotics are selected from the group consisting chloramphenicol, kanamycin, Oxytetracycline, Chlortetracycline, Tetracycline, Tiamulin, Gentocin, Lincomycin, Neomycin, Spectomycin, Sulfamethazine, Tylosin, Penicillin G Potassium, tetracycline hydrochloride, moxifloxacin hydrochloride, and ciprofloxacin hydrochloride and/or combination thereof.

In another embodiment in the antibiotic delivery system of the present invention hydrophobic antibiotics are encapsulated in lipid or polymer prior to loading on platelets.

In still another embodiment the antibiotic delivery system of the present invention is for treating Bronchitis, Chest colds, Common Cold, Ear Infection, Influenza (Flu), Sinus Infection (Sinusitis), Sore Throat, Urinary Tract Infection, viral infections, chicken pox, or Measles.

In yet another embodiment the antibiotic delivery system of the present invention is for combating microbial infection comprising mixing antibiotics with platelets in a platelets and drug mixing chamber.

In another embodiment the present invention provides a method of administration of an antibiotic delivery system for combating microbial infection, comprising platelets loaded with antibiotics wherein the delivery system is in liquid, and/or gel form.

In still another embodiment the in the method of administration of the antibiotic delivery system of the present invention the delivery system is in powdered and/or tablet form.

In yet another embodiment in the method of administration of the antibiotic delivery system of the present invention the delivery system is administered either by oral route, intravenous route, rectal, topical, sublingual, subcutaneous, buccal, nasal, intravaginal, and/or intradermal route.

In another embodiment the present invention provides a method of treating a recipient human and/or animal by administering the delivery system for combating microbial infection, comprising platelets loaded with antibiotics

In still another embodiment the present invention provides a device for mixing platelet and antibiotics to obtain platelets loaded with antibiotics comprising a component for providing platelets (1) to a magnetic bead stirrer (3); a component for providing antibiotics or encapsulated antibiotics in polymer or lipid (2) to a magnetic bead stirrer(3); and a magnetic bead stirrer (3) to receive antibiotics and platelets from components (1) and (2); wherein the device is controlled with a steeper motor and open source electronic platform based micro controller.

SUMMARY OF THE INVENTION

The present invention provides novel antibiotic delivery system, wherein the platelets isolated form healthy human donors are loaded with Ab. The delivery system shows enhanced efficacy, does not have any biocompatibility issues and reduce chances of strains developing resistance to the Ab loaded in the platelets.

In the present invention the inventors have used the property of platelets, a blood component to engulf small molecules even bacteria and other microbes and obtained a novel delivery system. The inventors of the present invention have utilized this property and loaded antibiotics inside platelet. The antibiotic loaded platelet was found to be stable when studied through experimental processes. It has been previously reported that platlets generally engulf 25% to 30% of the small molecules in vitro. As a case study the nonpathogenic bacterial strain E. coli as a model microorganism has been used in the present invention. The bactericidal efficacy of the antibiotics loaded platelets was much higher, and the loaded platelets were able to kill the bacteria in such a low concentration at which the free antibiotic is barely effective. Moreover, there are some solubility and stability issues with some of the antibiotics. These antibiotics were firstly loaded inside the FDA approved polymer or lipid vesicles and then they were loaded inside the platelet, whereas water soluble antibiotics were loaded inside the platelet directly. Surprisingly, both exhibited similar bactericidal efficacy. The most important part of this delivery system is the ability to kill even the drug resistance strains. The advantage of this delivery system is that the Ab is invisible to the microorganisms and thus it is not diploid any further resistance. The delivery system of the present invention is also devoid of any immunogenic responses as the delivery vehicle is a blood component. Moreover, the present invention also provides a portable, low cost fluidics device where the antibiotics are loaded into the platelets in a controlled manner during platelet transfusion.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

Figure 1: Aggregation profile of platelets in presence and absence of antibiotic with different agonists: The aggregation profile of the platelets in presence of ADP and collagen as agonists at different time interval (0, 2 and 4 hrs.). The percentage of aggregation of platelets in absence (Control ADP) and presence (Ab ADP) of chloramphenicol is provided (figure A). For collagen-induced aggregation the percentage of aggregation is provided in Figure B. Figure C provides the Arachidonic acid induced aggregation of platelets at 4 hours for control platelets and antibiotic loaded platelets respectively.

Figure 2: Confocal microscopic images of E. coli and platelets.

Figure 3: Z- section images of platelet and E. coli cells (stained with DAPI) after 1 hr. of incubation.

Figure 4: E. coli cell viability with different types of treatment.

Figure 5: Response of Kanamycin sensitive and resistant strain of E. coli towards kanamycin loaded platelet.

Figure 6: Comparison of Bactericidal efficacy of the hydrophobic antibiotics loaded in platelets.

Figure 7: Schematic representation of antibiotic delivery through platelets.

DETAILED DESCRIPTION OF THE INVENTION

It is known in the art that platelets can interact with pathogenic microorganisms inside body. In the present invention a novel antibiotic delivery system is provided by exploiting this property of plateletsd. This concept has been examined in vitro. The platelets were loaded with antibiotic and treated with antibiotic sensitive and resistant strain of E. coli (model organism) where free antibiotic served as control. It was found that the viability of E. coli cells become significantly lower when treated with antibiotic loaded platelets. This happens even with the antibiotic resistant strains. This increased efficacy of antibiotics loaded platelets may be due to the molecular confinement of the antibiotic inside the platelet which increases the localized concentration. So, when the bacteria are being engulfed they are killed. As the delivery agent is a blood component itself, there is no immunogenic response against it which is the major drawback of other delivery vehicles. Moreover, as the antibiotic will be hidden inside platelet, the antibiotic mediated drug resistance development will be minimized.

In this invention the main aim is to investigate the role of platelets as antibiotic delivery system. It is reported that platelet can uptake soluble drugs and small particulate matter from the surroundings (Deb et ah, (2011) Nanomedicine: Nanotechnology, Biology and Medicine, 7(4), pp.376-384; and White (2005) Platelets, 16(2), pp.121-131). The inventors of the present invention have exploited this property of platelets to load antibiotic in it. This is not exactly phagocytosis as the engulfed material remains intact inside the cells. So, the internalized materials (here chloramphenicol) retain its activity even when remaining inside the platelets.

Effect of antibiotic on platelet functionality

It is not recommended to use any biological material as a delivery vehicle if the drug to be delivered shows any adverse effect towards the vehicle. So, this was very important in the present case, to examine whether chloramphenicol (antibiotic) has any adverse effect on platelet functionality. This effect was investigated using platelet aggregometric study using different agonists at different time interval. For this purpose, ADP, collagen and AA were used as agonists. The experimental results of present invention suggest that the concentration of chloramphenicol applied for the experimental purpose does not have any adverse effect on platelet (Figure 1). The percentage of aggregation of Ab loaded platelet remain almost same with that of unloaded control over time for the ADP, collagen and AA associated pathways.

The concept of delivering antibiotic to the bacteria using platelet evolved from the fact that platelets express a variety of potential bacterial receptor and thus bacteria can bind to these receptors on platelets. The mechanism lying behind this interaction are primarily of three types, (1) indirect binding of a plasma protein, with bacteria, which serve as a ligand of platelet receptor (2) the binding of secreted bacterial products, particularly toxins, to platelets and lastly (3) direct binding of bacteria to platelet receptors (Hamzeh-Cognasse et al, (2015) Frontiers in immunology, 6, pp.82). Beside these, there is also evidence of another kind of interaction in which platelet engulf bacteria within it (Youssefian et al, (2002) Blood, 99(11), pp.4021-4029). The confocal microscopic study (Figure 2) suggests similar kind of interaction between platelet and E. coli. The E. coli cells were stained with the DNA binding fluorescent dye DAPI prior to the incubation with platelets so that they can be easily detected by the fluorescence property of DAPI. Figure 2 clearly indicates aggregate formation in presence of platelet (Figure 2 D-I), whereas bacterial population remains as separate entity in absence of platelets (Figure 2 A-2C). The striking aspect is that the size of the aggregates becomes bigger when E. coli cells were incubated with platelets for 2 hours (Figure 2 D-2F) in comparison with 1 hour of incubation (Figure 2 G-2I). This interaction is more clearly visible in the higher magnification images (Figure 2K and L). Not only the superficial interaction, Z sectioning of platelet- E. coli clump revealed that platelets also engulfed E. coli cells within it (Figure 3).

The main objective of the study was to develop an invisible and highly biocompatible mode of delivery system that can effectively kill bacteria. There are several factors that led to choosing of platelets as the delivery vehicle. The factors were: firstly, being a blood component, they are non-immunogenic. Secondly, a well- defined interaction of platelets with microorganisms may be expected. Thirdly, platelets can uptake a substantial amount of drug (25% to 30% of applied drug concentration) (Sarkar et al, (2013) Pharmaceutical research, 30(11), pp.2785-2794) and upon aggregation, they can release the same. Figure 4 provides that when platelets were loaded with Chloramphenicol (Ab) and treated with E. coli, the presence of viable bacteria (in terms of Colony Forming Units (CFU)) at 30 min is 45.8% and become almost zero after 2 hours. Whereas, in case of free drug although an initial lagging of growth was observed but the bacteria gets a boom after 1 hr. and reached to 552% after 2 hours. Final dose of the free Ab was calculated according to previous work of the inventors of present invention where the use of platelets as vehicle of anti- cancer drug was reported for first time (Sarkar et al, (2013) Pharmaceutical research, 30(11), pp.2785-2794). The present invention provides employing platelets as antibiotic delivery vehicle. E. coli cells show a typical growth pattern when treated with only platelets. Percentage of viable count of the bacterial cells keeps decreasing till 1 hr. (74% and 50% after 30 min and 60 min respectively) and then it starts increasing to 88% after 2 hours (Figure 4). Formation of platelet- bacterial aggregates might be the probable reason of initial growth arrest. Untreated E. coli cells show the normal growth, used as the control here. The most striking aspect of this method is the lower requirement of antibiotic (Ab). It is clear that the dose (nanogram level) at which virtually there is no appreciable killing of bacteria by the free Ab, leads to significant killing when loaded in the platelet (Figure 4).

This is further extendable to prevent antibiotic resistance in bacteria. When examined with Kanamycin (Ab) resistant E. coli cells, it was clearly seen that kanamycin loaded platelets exert anti-bacterial efficacy towards both Kanamycin sensitive and resistant species (Figure 5).

In this regard, a small portable device has been designed that allows the proper loading of the antibiotics inside platelet.

Thus, the experimental results provide that platelet can be used as antibiotic delivery vehicle. The proposed method can be used to combat the bacterial infection where direct interaction between platelet- bacteria takes place and can be further extended to treatment of viral infection as there is well documented interaction observed between platelet and virus (Youssefian et al., (2002) Blood, 99(11), pp.402l- 4029).

Platelet isolation and antibiotic loading is done by collecting blood from healthy donors by venipuncture into plastic tubes containing a suitable buffer. The collected blood was then centrifuged at about 150 to 250 g and preferably at about 200 g for about 10 to 14 min and preferably for about 12 min to isolate platelet rich plasma (PRP). Platelet poor plasma (PPP) was obtained by centrifuging the blood at about 1000 rpm to 1400 rpm and preferably at about 1200 g for about 8 to 12 min and preferable for about 10 min. The PPP served as the blank for aggregometric study. All in vitro experiments were done within about 2 to 6 hours and preferably within about 4 hours of drawing the blood from the donors.

Antibiotic of choice was loaded into the platelets by means of diffusion method. Antibiotic (final applied concentration of about 6.25 pg / ml) was incubated with PRP at about 35 to 39 °C and preferably for about 37 °C for about half to one and half hour and preferably for about 1 hour. To discard the excess antibiotic remaining into the PRP solution, apyrase (final concentration 0.2 U/mL) was added and centrifuged at about 1500 to 2500 rpm and preferably at about 2000 rpm in a swing out centrifugation machine. The excess drug containing supernatant was discarded carefully and the pellet was dissolved in PBS. The antibiotic-loaded platelet was washed for another two to four times or preferably for two times following the same procedure. After final washing, the pellet was re-suspended in LB broth keeping platelet count of about 1,50, 000/ml.

Platelet aggregation study was done to detect the effect of applied Ab on platelets. For this purpose, Adenosine di phosphate (ADP) (final concentration 10 mM), collagen (final concentration 4 pg / ml) and arachidonic acid (AA) (final concentration 500 pg/ ml) were used as agonists. To the PRP isolated by the same procedure as stated earlier the Ab (final concentration of about 6 to 7 or preferably about 6.25 pg/ ml) was added and incubated at about 35 °C to 39 °C and preferably at about 37 °C. Another portion of PRP was kept as control. At different time interval PRP was taken from both control and test; and functionality of platelet was measured by ADP, Collagen and AA. The aggregation experiment was run for about 2 to 6 minutes and preferably for about 4 minutes with a predetermined stirring rate of about 800 to 1200 rpm or preferably at about 1000 rpm at about 35 °C to 39 °C or preferably at about 37°C. This experiment was repeated for 2 to 4 times or preferably for 3 times.

Thus, the delivery system of the present invention has high industrial importance as the same antibiotics show increased efficacy once loaded inside the platelet.

Experimental details of the invention

Methods

1. Platelet isolation & antibiotic loading

Blood was collected from healthy donors by venipuncture into plastic tubes containing sodium citrate buffer. The collected blood was then centrifuged at about 200 g for about 12 min to isolate platelet rich plasma (PRP). Platelet poor plasma (PPP) was obtained by centrifuging the blood at about 1200 g for about 10 min. The PPP served as the blank for aggregometric study. All in vitro experiments were done within 4 hours of drawing the blood from the donors. Chloramphenicol antibiotic was loaded into the platelets by means of diffusion method. In short, Chloramphenicol (final applied concentration of about 6.25 pg / ml) was incubated with PRP at about 37 °C for about 1 hour. To discard the excess antibiotic remaining into the PRP solution, apyrase (final concentration 0.2 U/mL) was added and centrifuged at about 2000 rpm in a swing out centrifugation machine. The excess drug containing supernatant was discarded carefully and the pellet was dissolved in PBS. The antibiotic-loaded platelet was washed for another two times following the same procedure. After final washing, the pellet was re-suspended in LB broth keeping platelet count of about 1,50, 000/ml.

The hydrophobic antibiotic was loaded into the Polymer nanoparticles by nano precipitation technique. The hydrophobic antibiotic was solubilized in a volatile organic solvent containing hydrophobic polymer (Poly-caprolactone). The aqueous phase for polymer nanoparticles synthesis was 1% Pluronic F127 (w/v). The particles size of the antibiotic loaded nanoparticles was found to be around 200 nm. The antibiotic loaded nanoparticles were incubated with PRP and were taken up by the platelets. Any other FDA approved polymers like PLGA can also be used for the same purpose. The hydrophobic antibiotics can be encapsulated inside lipid vesicles too where POPC, DPPC etc. can be used as antibiotic carrier. All the nanoformulations are taken up by the platelets. On performing the killing assay, the bactericidal activity of polymer encapsulated/ lipid encapsulated antibiotic loaded platelet showed similar enhanced efficacy as it was found in case of hydrophilic antibiotic loaded platelet.

2. Platelet Aggregometry

Platelet aggregation study was done to detect the effect of applied Chloramphenicol (Ab) on platelets. For this purpose, Adenosine di phosphate (ADP) (final concentration 10 mM), collagen (final concentration 4 pg / ml) and arachidonic acid (AA) (final concentration 500 pg/ ml) were used as agonists. All the reagents were purchased from Chrono-log and Chrono-log aggregometer (model no 700) Chrono-log, Havertown, USA, was used for platelet functioning test. PRP was isolated by the same procedure as stated earlier in step 1. Then Chloramphenicol (Ab) (final concentration 6.25 pg/ ml) was added to the PRP and incubated at about 37°C. Another portion of PRP was kept as control. At different time interval (0, 2 and 4 hr) PRP was taken from both control and test; and functionality of platelet was measured by ADP, Collagen and AA. The aggregation experiment was run for about 4 minutes with a predetermined stirring rate of about 1000 rpm at about 37 °C. This experiment had been repeated for about 3 times.

Figure 1 demonstrates the aggregation profile of platelets in presence and absence of antiobiotic with different agonists viz. Adenosine di phosphate (ADP), collagen and Arachidonic acid (AA). The aggregation profile of the platelets in presence of ADP and collagen as agonists at different time interval (0, 2 and 4 hrs.) is shown in Figure 1A and 1B. The percentage of aggregation of platelets in absence (Control ADP) and presence (Ab ADP) of chloramphenicol (Ab) was about 74%, 71%, 70% and 72%, 73% 70% respectively at 0, 2 and 4 hr when ADP was used (Figure 1A). For collagen-induced aggregation the percentage of aggregation was about 74%, 74%, 72% and 73%, 74%, 70% respectively in absence (Control Col) and presence (Ab Col) of the antibiotic for the same time intervals (Figure IB). Figure 1C is the Arachidonic acid (AA) induced aggregation of platelets at about 4 hours where percentage of aggregation was about 74% and 75% for control platelets and antibiotic loaded platelets respectively.

3. E. coli culture and killing assay

Gram -ve bacteria E. coli ATCC 25922 was used for the study. The bacterial cells were grown overnight in LB media until mid-logarithm phase was achieved. The optical density of the culture was measured and adjusted to about 10⁶ CFU/ml. The bacterial culture was then exposed to various treatment groups including antibiotic solution, control platelets and platelets loaded with antibiotic and further incubated at about 37 °C. The aliquots were withdrawn immediately, and at time interval of about 30 minutes, 60 minutes and 120 minutes and placed on the agar plates. After overnight incubation, the colonies were counted manually and bactericidal activity was calculated (Shireen et ah, (2009) Peptides, 30(9), pp.1627-1635).

For treatment of kanamycin resistant strain, the same protocol mentioned above was followed. After about 2 hrs of incubation with platelets and/or Kanamycin (Ab) loaded platelet, cells were plated (Figure 5).

Figure 5 and Table 1 clearly provide the comparative analysis of the response of Kanamycin (Ab) sensitive and resistant strain of E. coli towards kanamycin loaded platelet (Plt-Ab). The upper panel of the image represents plates of kanamycin sensitive strain of E. coli treated with free Kanamycin (Kan treated), Kanamycin loaded platelet (Platelet-Kan treated) and free platelet. Untreated cells of E. coli strain served as the control. Lower panel represents plates of the Kanamycin resistant strain of E. coli treated with free Kanamycin and platelet loaded with Kanamycin.

Figure 5 explicitly provides the comparative analysis of efficacy between Kan loaded platelets, platelets and only Ab. Both the Ab-sensitive and Ab-resistant strains of E. coli showed highsusceptibility to platelets loaded with Ab (Plt-Ab), as compared to other controls. This makes it evident that the platelets loaded with Ab are also effective (in addition to Ab sensitive strains) against those strains which are or have grown resistance towards the known Ab.

Figure 6 provides the bactericidal activity of the hydrophobic antibiotics which are insoluble in water and cannot be loaded to the platelets. Prior to loading they were encapsulated in FDA approved polymer or lipid vesicles. The polymer encapsulated/ lipid encapsulated antibiotic loaded platelet showed similar enhanced efficacy as it was found in case of hydrophilic antibiotic loaded platelet. The antibiotic control here shows the activity of the antibiotics when dissolved in organic solvents such as ethanol/ DMSO.

Confocal Microscopy:

Interaction of platelets with bacteria was studied by confocal microscope. In short, E. coli was cultured as stated earlier. Platelet was prepared following the same procedure. After preparation of platelets, E. coli cells were stained by DAPI (concentrate 0.5 pg/ml) following PBS washing to remove excess stain and was mixed with platelet ( E . coli and platelet ratio is 1:2) and incubated in a bacterial incubator at about 37 °C with a stirring speed of about 200 rpm for about 1 hour. Next, the platelet- E. coli mixture was fixed with about 4% para- formaldehyde. Then in a grease-free glass slide, 10 pl of the suspension was taken and a cover slip was placed on the slide and edges were sealed. The slides were then seen under a laser confocal microscope of Olympus models no IX 81 FV 1,000 with 60x objective lenses.

Figure 2 provides Confocal microscopic images of E. coli and platelets. Figure (2A-2C) demonstrates the bright field, fluorescent and corresponding over lapping images of E. coli cells respectively. Figures (2D-2F) represents the respective bright field, fluorescent and corresponding over lapping images of E. coli in presence of platelets after 1 hr. of incubation. Figures (2G-2I) shows bright field, fluorescent and corresponding over lapping images of E. coli in presence of platelets after 2 hr. of incubation. Figure (2J) is the bright field image of platelets. Figures (2A-2J) were captured at 60X magnification. Figures (2K-2L) represent the higher magnification (400X) images of platelet and bacterial cell interaction in bright field and its fluorescent overlapping mode respectively.

Furthermore, Figure 3 provides Z- section images of platelet and E. coli cells (stained with DAPI) after 1 hr. of incubation.

4. Fabrication of the device (Figure 7)

Drug mixed platelets can be delivered to the patients (4) in a simple manner by the device as depicted in Figure 7. The platelets (1) and Antibiotic or polymer/ lipid encapsulated antibiotic (2) were injected separately in a magnetic bead for stirring (3) in a Platelet antibiotic mixing device. Once the stirring was done the platelets loaded with antibiotics can be injected into patients (4). The platelets antibiotic mixing device and the magnetic bead stirrer (3) are further controlled with a steeper motor (not shown in Figure) and open source electronic platform (for example aurdino) based micro controller throughout the mixing.

It was found that about 25 % of the Antibiotic (Ab) was loaded successfully on the platelets. For this purpose, if for example about 6.25 pg/ml chloramphenicol (Ab) was incubated with platelet solution (150000/ml) about 1 of 25th portion of the drug was taken up by the platelets. So, the bulk concentration of Ab taken up by platelets was about 250 ng/ml.

Figure 4 provides E. coli cell viability with different types of treatment. Figure (4A) represents the percentage of viability of E. coli cells when treated with free Chloramphenicol (Ab control), platelet (Plt control) and chloramphenicol loaded platelet (Plt-Ab) in a time dependent manner. E. coli cells without any treatment serves as the control. Figure (4B) demonstrates the bar diagram with error bar of the viability percentage of E. coli cells of individual treatment group at 0, 30, 60 and 120 min respectively. The results of Figure 4 clearly demonstrate the success of the delivery system of the present invention. The platelets loaded with Ab (Plt-Ab) showed a sharp decrease in the percent viability, leading to total annihilation of the target microorganism within about 2 hours of the treatment. Figure 4 clearly provides comparative analysis of the delivery system of the present invention with antibiotic and platelet controls used separately on same target microorganisms. The data of Table 1 further substantiates that not only the delivery system of the present invention is more effective than the individual components; the delivery system works synergistically to provide better results than the individual components.

Table 1: The following Table depicts the % viability of the bacterial culture when exposed to various treatment groups (Ab control; Plt control; Plt-Ab) at different time intervals.

Although the present invention has been described herein with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Furthermore, precise and systematic quantitative experimental measurements on all above aspects are currently being made. Work is still underway on this invention. It will be obvious to those skilled in the art to make various changes, modifications and alterations to the invention described herein. To the extent that these various changes, modifications and alterations do not depart from the scope of the present invention, they are intended to be encompassed therein.

Advantages of the present invention:

The delivery system of present invention is effective against microorganisms, even when loaded with very small amount or concentrationn of Ab. The delivery system of present invention is also effective against microorganisms which are or have turned into resistant strains to the Ab loaded in the platelets.

The delivery system of the present invention does not allow the microorganisms which are sensitive to the said Ab, to develop into a resistant strain.

The delivery system of the present invention provides a fast and effective way of killing microorganisms.

The delivery system of the present invention is a very simple and easy method without employing any expensive technology or equipment(s).

The delivery system of the present invention does not pose any biocompatibility issues in the recipient’s body.

Claims

We claim:

1. An antibiotic delivery system for combating microbial infection, comprising platelets loaded with antibiotics.

2. The antibiotic delivery system as claimed in claim 1, wherein the platelets are isolated from healthy human and/or animal donors.

3. The antibiotic delivery system as claimed in claim 1, wherein the microbial infection is selected from infection caused by bacteria, viruses, protists, fungi or combination thereof.

4. The antibiotic delivery system as claimed in preceding claims wherein the delivery system is in form of tablets, capsules, creams, ointments, aerosols and/or patches.

5. The antibiotic delivery system as claimed in claim 1, wherein the platelets loaded with antibiotics comprises about 25 % of the loaded antibiotics.

6. The antibiotic delivery system as claimed in claim 1, wherein the antibiotics are selected from the group consisting water-soluble, water- insoluble antibiotics (hydrophobic) and/or combination thereof.

7. The antibiotic delivery system as claimed in claim 8, wherein the antibiotics are selected from the group consisting chloramphenicol, kanamycin, Oxytetracycline, Chlortetracycline, Tetracycline, Tiamulin, Gentocin, Lincomycin, Neomycin, Spectomycin, Sulfamethazine, Tylosin, Penicillin G Potassium, tetracycline hydrochloride, moxifloxacin hydrochloride, and ciprofloxacin hydrochloride and/or combination thereof.

8. The antibiotic delivery system as claimed in claim 1, wherein the hydrophobic antibiotics are encapsulated in lipid or polymer prior to loading on platelets.

9. The antibiotic delivery system as claimed in preceding claims wherein the delivery system is for treating Bronchitis, Chest colds, Common Cold, Ear Infection, Influenza (Flu), Sinus Infection (Sinusitis), Sore Throat, Urinary Tract Infection, viral infections, chicken pox, or Measles.

10. A method of preparing a delivery system for combating microbial infection comprising mixing antibiotics with platelets in a platelets and drug mixing chamber.

11. A method of administration of an antibiotic delivery system for combating microbial infection, comprising platelets loaded with antibiotics wherein the delivery system is in liquid, and/or gel form.

12. A method of administration of the delivery system as claimed in the claim

11, wherein the delivery system is in powdered and/or tablet form.

13. A method of administration of the delivery system as claimed in the claim

11 or 12, wherein the delivery system is administered either by oral route, intravenous route, rectal, topical, sublingual, subcutaneous, buccal, nasal, intravaginal, and/or intradermal route.

14. A method of treating a recipient human and/or animal by administering the delivery system as claimed in any preceding claims.

15. A device for mixing platelet and antibiotics to obtain platelets loaded with antibiotics comprising a component for providing platelets (1) to a magnetic bead stirrer (3); a component for providing antibiotics or encapsulated antibiotics in polymer or lipid (2) to a magnetic bead stirrer(3); and a magnetic bead stirrer (3) to receive antibiotics and platelets from components (1) and (2); wherein the device is controlled with a steeper motor and open source electronic platform based micro controller.