1. Novel anti-thrombotic nanoparticles comprising a binary lipid matrix of a core and a shell, wherein - the core comprises of bromelain and lecithin, and - the shell comprises lipid matrix of stearic acid and tristearin, wherein said nanoparticles possess high entrapment efficiency, stability and improved drug loading capacity.

2. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said nanoparticle size is in the range of 120.56 ± 40.12 nm.

3. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said high entrapment efficiency is in the range of 86.32 ± 5.56 %.

4. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein zeta potential of the nanoparticles is in the range of -29.01±3.41 mV.

5. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said nanoparticles are analyzed till 6 months under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity and till 12 months under room temperature storage conditions of 25 ± 5 ºC at 60 ± 5 % relative humidity to retain colloidal property of the nanoparticle.

6. A process to prepare the novel anti-thrombotic nanoparticles as claimed in claim 1, said process comprises the steps of: (i) preparing an aqueous solution of bromelain and lecithin (W1), (ii) preparing an organic phase of stearic acid, tristearin and a surfactant dissolved in an organic solvent (O), (iii) emulsifying aqueous solution of step (i) (W1) with organic solvent of step (ii) (O) employing ultrasonic disruptor under predefined conditions to obtain a primary emulsion, (iv) preparing an aqueous solution of a synthetic polymer and a surfactant in predetermined ratio (W2), (v) emulsifying said primary emulsion of step (iii) with aqueous solution of step (iv) (W2) under probe sonication under predefined conditions to form a double emulsion (W1/O/W2), (vi) removing said organic solvent by continuous stirring of said double emulsion to obtain organic solvent free double emulsion, (vii) centrifuging said organic solvent free double emulsion at predefined conditions to obtain pellets, (viii) dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension, (ix) lyophilizing the said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain said antithrombotic nanoparticle.

7. The process as claimed in claim 6 wherein said organic solvent in step (ii) is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.

8. The process as claimed in claim 6 wherein said organic solvent is dichloromethane.

9. The process as claimed in claim 6 wherein said tristearin in step (ii) is added in a concentration upto 50 % w/w of total lipid phase content.

10. The process as claimed in claim 6 wherein said surfactant in step (ii) is Span 80 of 3 % w/v.

11. The process as claimed in claim 6 wherein said predefined conditions in step (iii) for emulsification by ultrasonic disruptor are disruption for 5 - 7min over an ice bath.

12. The process as claimed in claim 6 wherein said water soluble synthetic polymer in step (iv) is selected from the group comprising poly((meth)acrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.

13. The process as claimed in claim 6 wherein said surfactant in step (iv) is Tween 80.

14. The process as claimed in claim 12 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).

15. The process as claimed in claim 6 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant, preferably the surfactant is a non-ionic surfactant.

16. The process as claimed in claim 6 wherein said predetermined ratio of the synthetic polymer and the surfactant is 1% w/v : 0.5% w/v.

17. The process as claimed in claim 6 wherein said predefined conditions in step (v) are emulsifying the primary emulsion for 7.5 min over an ice bath.

18. The process as claimed in claim 6 wherein said predefined conditions in step (vii) are centrifugation at 30,000 rpm for 25 min at 4 ºC.

19. The process as claimed in claim 6 wherein said cryoprotectant is mannitol 10% w/v.

20. The process as claimed in claim 6 wherein said specific conditions for lyophilization are freezing to -80 °C for 5 h followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar.

21. A composition of nanoparticle formulation, said composition comprises: Ingredient Quantity (%) Bromelain 7-15% Lecithin 10-20% Stearic acid 24-75% Tristearin 0-50% Surfactant 0.25-.75% Volume of external phase 20-30 ml

22. The composition as claimed in claim 21 wherein said composition comprises: Ingredient Quantity (%) Bromelain 11.11% Lecithin 14.81% Stearic acid 37.04% Tristearin 37.04% Tween - 80 0.5% (w/v) Volume of external phase 20 ml

23. The composition as claimed in claim 21 wherein t90 of said nanoparticle formulation is 820±50 days.

24. The composition as claimed in claim 21 wherein said composition comprises said antithrombotic nanoparticles of bromelain with pharmaceutically acceptable excipients or surfactants or carriers or a combination thereof.

25. The composition as claimed in claim 21 wherein said composition is in dosage form suitable for oral drug delivery.

26. The composition as claimed in claim 25 wherein said dosage form is sustained release dosage form selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels.

The present invention relates to novel antithrombotic nanoparticles capable of being used in the treatment of thrombosis. The present invention also relates to a process of preparation of the novel antithrombotic nanoparticles and pharmaceutical compositions comprising thereof. More specifically, the present invention relates to bromelain nanoparticles capable of being used in the treatment of thrombosis.

BACKGROUND OF THE INVENTION
Thrombosis is a multifactorial disorder characterized by excessive clot formation inside the blood vessels due to imbalance of pro-coagulation and coagulation process. Clinically, thrombolytic drugs like streptokinase, tissue plasminogen activator (t-PA) and reteplase have been therapeutically used to dissolve the formed blood clots. However, in the constant endeavor of moving towards natural based therapies, dietary components of cysteine protease family are gaining immense attention for a variety of indications. Among them bromelain has earned growing acceptance as phytotherapeutic agent because of its higher efficacy and non-toxic nature.

Bromelain is a crude extract found in the tissues of species belonging to the Bromeliaceae family, in particular pineapple (Ananas comosus). It contains thiol endopeptidases and other components like, phosphatases, glucosidase, peroxidases, cellulases, glycoproteins and carbohydrates.

It has been widely utilized in traditional system of medicine due to its remarkable pleiotropic therapeutic effects as anti-oedematous, anti-inflammatory, anti-metastatic, immunomodulator, platelet aggregation inhibitor, fibrinolytic and antithrombotic agent. Available literature reports have provided evidence of reversible inhibition of platelet activation, platelet aggregation and thrombus formation by bromelain under in vitro as well as in vivo assays. Indirectly, bromelain enhances the time required for thrombin formation from prothrombin, activates plasminogen for production of plasmin and thereby inhibits the production of fibrin, and restrains the adhesion of human thrombocytes on endothelium cells, thereby contributing to inhibition of blood coagulation and reduction of the risk of thromboembolic diseases. In addition, bromelain downregulates COX-2 and PGE-2 expression and reduces cytokines like IL-1ß, IL-6 and TNF-a secretion from immune cells stimulated during inflammatory pathologies. The proteolytic activity of bromelain also controls inflammation by proteolytic degradation of advanced glycation end products receptor and cell surface markers regulating lymphocyte homing and migration to inflammatory site.

In spite of the widely reported therapeutic benefits, the clinical usage of bromelain is still limited, in part due to challenge associated with the development of its stable formulation. Because of its proteinaceous components, it is susceptible to degradation in the acidic environment of the stomach that may lead to the loss of its therapeutic efficacy. Mechanical instability, poor shelf-life, low bioavailability, higher probability of bromelain degradation, gastric instability and poor patient compliance due to high oral dose limits its therapeutic potential. The denaturation or aggregation of bromelain may also facilitate depletion of its therapeutic efficacy along with unpredictable prophylactic hypersensitivity or toxic reactions.

Therefore, there had been efforts to develop stable formulations of bromelain with delivery routes that can bypass harsh conditions of stomach and deliver bromelain efficiently at the desired site in therapeutically effective dose.

Sharma et al RSC Adv., 2018, 8, 2541-2551 discloses enteric coated nano-formulation of bromelain by double emulsion solvent evaporation method to obtain gastro-resistant properties. Eudragit has been used as the coating polymer. It endorses that enteric formulation could improve the GIT stability and functional outcomes of bromelain in inflammation. It concludes that while bromelain loaded enteric nanoparticles can be considered as promising drug delivery system for improving therapeutic efficacy of bromelain via oral route, the scalability, safety and efficacy of formulation needs to be studied in clinics before its therapeutic use. However, the Eudragit coated nanoparticles exhibited poor sustained release due to burst release of drug, large particle size and less stability.

US11119861 discloses formulation of bromelain preparation by coating bromelain with organic network polymer constructed by cross linkage between organic acids and polysaccharides. Electron-beam irradiation is used to embed bromelain in the organic network polymer constructed through a crosslinking reaction between an organic acid and a polysaccharide.

CN103285383A discloses enteric coated bromelain tablets comprising polyethylene glycol. The document discloses the use of polyethylene glycol as stabilizing agent.

CN201610475396 discloses bromelain liposomes comprising bromelain, lecithin, cholesterol and pectin.

Although the literature is replete with various formulation strategy of bromelain so as to increase its stability, bioavailability and therapeutic efficacy, till date there is no clinical success. Also, the processes reported involve multiple steps and are cumbersome. Further the particles/ formulation obtained using such process also do not show any improvement in the loading of bromelain or its therapeutic efficacy.

Hence there is a need to develop stable bromelain formulations with increased therapeutic efficacy, bioavailability and stability using simple processes and advanced technologies such as nanotechnology.

Nanotechnology has gained attraction in the field of biopharmaceuticals for the development of novel therapies for proteins and enzymes to make their oral administration feasible. The main focus in development of thrombosis treatment via nanomedicine is to enhance selective drug permeability to inflamed tissues, prolong duration of action at lower effective dose and reduce dosing frequency. The fabrication of nanocarriers is primarily based on distinctive arrangement of synthetic, natural or biological constituents as their building blocks. Among the various nanocarrier systems, solid lipid nanoparticles (SLNs) exhibit good biocompatibility, improved pharmacokinetic profile, controlled drug release behavior and least toxicity under in vivo application with significant success in protein and peptide delivery at lower cost and ease of scalability. The encapsulation of proteins in lipid carrier also protects them against physical aggregation and in vivo enzymatic inactivation. This contributes to reduction in therapeutic effective dose and risk of dose dependent toxicities. As drug loading efficiency, stability and drug release response of SLNs are significantly affected by lipid’s crystallinity and its polymorphic nature, suitable selection of lipids to prepare binary lipid matrix with improved drug loading capacity and physical stability can overcome the drawback of individual lipid constituent. There have been no studies documented for fabrication of bromelain laden hybrid solid lipid nanoparticles (Br-HNPs).

OBJECTS OF THE INVENTION
In order to obviate the drawbacks in the existing state of the art, the main object of the present invention to provide novel antithrombotic nanoparticles capable of being used in the treatment of thrombosis.

Another object of the present invention is to provide novel antithrombotic nanoparticles of bromelain capable of being used in the treatment of thrombosis.

Yet another object of the invention is to provide novel antithrombotic nanoparticles of bromelain having enhanced bioavailability, therapeutic efficacy and stability.

Yet another object of the invention is to provide novel antithrombotic nanoparticles of bromelain having improved drug loading capacity.

Yet another object of the invention is to provide pharmaceutical compositions comprising the novel antithrombotic nanoparticles of bromelain.

Yet another object of the invention is to provide pharmaceutical compositions comprising the novel antithrombotic nanoparticles of bromelain showing sustained release of bromelain, enhanced bioavailability, therapeutic efficacy and stability.

Yet another object of the invention is to provide a process for the preparation of novel antithrombotic nanoparticles of bromelain having improved drug loading capacity and stability.

Yet another object of the invention is to provide a process for the preparation of novel antithrombotic nanoparticles of bromelain capable of being used in the treatment of thrombosis.

SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

The inventors of the present invention conducted strategic experimentation and have surprisingly found that bromelain when embedded in binary lipid matrix produces hybrid bromelain nanoparticles (Br-HNPs) which are stable and show sustained release. The present invention, therefore, provides cost-effective antithrombotic nanoparticles of bromelain, a process to prepare thereof and a composition thereof.

Accordingly, one of the aspects of the present invention is to provide novel antithrombotic nanoparticles capable of being used in the treatment of thrombosis. More specifically, the invention provides novel antithrombotic nanoparticles of bromelain having enhanced bioavailability, therapeutic efficacy, stability and improved drug loading capacity.

The antithrombotic nanoparticle comprises binary lipid matrix of a core and a shell, wherein the core comprises an antithrombotic agent and lecithin, and the shell comprises of stearic acid and tristearin.

In a preferred embodiment of the invention, the antithrombotic agent is bromelain.

In another preferred embodiment of the invention, the antithrombotic nanoparticles comprise bromelain, lecithin, stearic acid and tristearin with pharmaceutically acceptable surfactants or excipients or carriers or any combination thereof.

The antithrombotic nanoparticles have a particle size in the range of 50 nm to 300 nm. The antithrombotic nanoparticles have a zeta potential ranging from -35 mV to -10 mV. The antithrombotic nanoparticles show drug loading capacity of 80-95%.

In second aspect, the present invention provides a process for the preparation of antithrombotic nanoparticles by double emulsion solvent evaporation method.

In a non-limiting embodiment, the invention provides a process for the preparation of antithrombotic nanoparticles comprising bromelain, lecithin, stearic acid and tristearin, said process comprising the steps of:
a) preparing an aqueous solution of bromelain and lecithin (W1);
b) preparing an organic phase comprising stearic acid, tristearin and a pharmaceutically acceptable surfactant dissolved in an organic solvent (O);
c) emulsifying aqueous solution of step (a) and organic phase of step (b) to form a primary emulsion;
d) emulsifying the primary emulsion formed in step c) with a water soluble synthetic polymer and a surfactant to obtain double emulsion (W1/O/W2).

The organic solvent suitable for the process is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.

The water soluble synthetic polymer suitable for the process is selected from the group comprising poly((meth)acrylic acid and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose.

In a preferred embodiment the water soluble synthetic polymer is poly(vinyl alcohol).

The surfactant suitable for the process is selected from anionic, cationic and non-ionic surfactant. In a preferred embodiment the surfactant is a non-ionic surfactant.

In another preferred embodiment, the surfactant is Sorbitan monooleate (Span 80).

Third aspect of the present invention is to provide a pharmaceutical composition of the antithrombotic nanoparticle formulation of bromelain comprising bromelain nanoparticles with pharmaceutically acceptable excipients or surfactants or carriers or a combination thereof.

In one embodiment, the pharmaceutical composition is in the dosage form suitable for oral drug delivery.

In another embodiment the dosage form is selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels, etc.

In another embodiment the dosage form is a sustained release dosage form.

Non-limiting Example:
a) Preparation of bromelain nanoparticles
An aqueous solution of bromelain and lecithin was emulsified with organic phase employing ultrasonic disruptor over an ice bath. Stearic acid, tristearin and Span-80 dissolved in dichloromethane constituted the organic phase. Primary emulsion so formed was further emulsified with PVA and tween-80 solution under probe sonication over an ice bath. The resultant double emulsion (W1/O/W2) was stirred to remove organic solvent and centrifuged. The collected pellet was dispersed in milli-Q pure water with subsequent addition of cryoprotectant, and the resultant suspension was lyophilized to obtain the bromelain nanoparticles.
b) Characterization of bromelain nanoparticles: Morphological evaluation of bromelain nanoparticle in suspension was executed employing transmission electron microscopy.
The suspension is evaluated for key indicators, including but not limited to, particle size, poly dispersibility index (PDI), zeta potential, and entrapment efficiency.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1. (A) TEM images of optimized Br-HNPs formulation. (B) Diffractogram of optimized Br-HNPs formulation and various formulation components like lecithin, tristearin, stearic acid and bromelain respectively. (C) Thermogram of optimized Br-HNPs formulation, drug and formulation excipients like lecithin, tristearin and stearic acid respectively. (D) In vitro release profile of optimized Br-HNPs.
Figure 2. Effect of storage under cool (4 ± 2 ºC/ 65 ± 5 % RH) and ambient (25 ± 2 ºC/ 60 ± 5 % RH) conditions on the colloidal properties like (A) particle size, (B) PDI, (C) zeta potential of Br-HNPs; and stability of formulation by (D) percent cumulative drug release and (E) percentage bromelain activity remaining.
Figure 3. Effect of various treatments on (A) platelet aggregation, (B) thrombus tail length at 24 h and (C) redness of thrombotic tail of (a) diseased control (b) aspirin treated (c) bromelain (10 mg/Kg) treated and (d) optimized Br-HNPs (equivalent 10 mg/Kg) treated animals after 24 h of carrageenan injection.
Figure 4. Effect of aspirin, bromelain and optimized Br-HNPs treatment on hematological parameters (A) PT and APTT level, (B) leukocyte count, (C) platelet count, (D) pro-inflammatory cytokines (TNF-a and IL-1ß) level, (E) percentage of neutrophils, monocytes, PLCR and PCT and (F) MPV. Optimized formulation showed statistically significant (p<0.05) reduction in biomarkers with respect to disease control and bromelain respectively.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND ILLUSTRATIONS
The chemicals used in the invention were procured from Hi-media laboratory Pvt. Ltd, Mumbai, India and used as received. The reagents and solvents used in the invention are of analytical grade.

The present invention provides novel antithrombotic nanoparticles capable of being used in the treatment of thrombosis. More specifically, the invention provides novel antithrombotic nanoparticles of bromelain having enhanced bioavailability, therapeutic efficacy, stability and improved drug loading capacity. The antithrombotic nanoparticles of the present invention comprise a binary lipid matrix of a core and a shell, wherein the core comprises of bromelain and lecithin, and the shell comprises of lipid matrix, said lipid matrix comprises of stearic acid and tristearin in specific concentration.

The present invention also provides a process to prepare a novel anti-thrombotic nanoparticle of bromelain. An aqueous solution (W1) of bromelain and lecithin is emulsified with an organic phase (O) employing ultrasonic disruption under specific conditions to obtain primary emulsion. The organic phase (O) is prepared by dissolving stearic acid, tristearin and Span-80 in an organic solvent. The specific conditions are application of ultrasonic disruption for 5-7 min over an ice bath. The organic solvent is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof. The organic solvent is preferably dichloromethane.
Said primary emulsion is further emulsified with aqueous solution of a synthetic polymer and a surfactant (W2) under probe sonication under specific conditions to obtain secondary emulsion (W1/O/W2). Said specific conditions for probe sonication are sonication time of 7.5 min. over an ice bath. Said synthetic polymer is selected from the group comprising poly((meth)acrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose. Said surfactant is selected from anionic, cationic and non-ionic surfactant.

In a preferred embodiment the surfactant is a non-ionic surfactant. Said surfactant is preferably tween-80 (0.5 % w/v).

The secondary emulsion is continuously stirred overnight to remove organic solvent, thereby obtaining organic solvent free double emulsion. The organic solvent free double emulsion is centrifuged under pre-determined conditions to obtain pellets. Said pre-determined conditions are centrifugation at 30,000 rpm for 25 min at 4 ºC. The pellets are dispersed in milli-Q pure water with subsequent addition of a cryoprotectant to obtain a suspension. The cryoprotectant is preferably mannitol 10% w/v. Said suspension is lyophilized by freezing to -80 °C for 5 h, followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar using lyophilizer to obtain lyophilized bromelain laden hybrid solid lipid nanoparticles (Br-HNPs). The Br-HNPs is characterized and stored at 4 ºC till further use.

Characterization of Br-HNPs
Several Br-HNPs batches have been prepared by varying lipid concentration, sonication time, type and concentration of surfactant and external phase volume to optimize formulation parameters.

Proteolytic activity
Modified casein digestion method has been utilized to ascertain the proteolytic prospect of bromelain.
Particle size and zeta potential
Lyophilized Br-HNPs have been dispersed in milli Q water via sonication (30 s) prior to characterize particle size, polydispersibility index (PDI) and zeta potential determination utilizing Nanosizer.
Entrapment efficiency
Briefly, Br-HNPs (25 mg) have been lysed in dichloromethane (5 ml) by vortexing and centrifuged at 20,000 rpm for 20 min. The collected pellets have been dissolved in distilled water and assayed for protein content by lowery method using U.V./Vis spectrophotometer at 750 nm. Entrapment efficiency was calculated by formula


Lyophilization parameters
The effect of lyophilization parameters like freezing temperature, freezing rate, secondary drying time on quality attributes of formulation like particle size and PDI have been utilized to determine their suitability. Freezing temperature of -40 °C and -80 °C for 5 h have been used for evaluation of temperature effect respectively while performing primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar respectively. Lyophilized samples have been analyzed for re-dispersibility by adding milli Q water (10 ml) followed by manual shaking for 1 min. Thereafter, samples have been analyzed for particle size and PDI after sonication in bath sonicator for 1 min.
In order to compare the effect of freezing rate, formulations have been either frozen at -80 °C on a precooled shelf for 5 h (“fast” freeze) or placed on a room temperature shelf, cooled to -80 °C at a rate of 0.5 °C/min, and then kept at -80 °C for an additional 2 h (“slow” freeze). The primary and secondary drying conditions have been maintained same as described above and analyzed similarly.
In addition, the effect of secondary drying time on quality of lyophilized product has been ascertained by freezing the samples at – 80 °C for 5 h followed by primary drying at -80 °C for 36 h at 0.07 mbar with subsequent drying at 20 °C and 0.07 mbar for 6 h and 8 h respectively.
Dilution study
Optimized Br-HNPs formulation has been diluted with milli-Q water in various ratios (1:50, 1:100 and 1:500) and effect on particle size, PDI and zeta potential has been observed utilizing Nanosizer.
Stability in gastrointestinal milieu
Lyophilized Br-HNPs (equivalent to 20 mg bromelain) has been placed in simulated gastric fluid pH 1.2 and simulated intestine fluid pH 5.0, 6.8 and 7.4 respectively at 37 ± 0.5°C with 100 shakes/min for 2 h in order to envisage their stability. Subsequently after incubation, samples have been analyzed for size, zeta potential, PDI and proteolytic activity respectively.
Dissolution studies
The drug release studies of optimized Br-HNPs have been performed employing modified dialysis bag diffusion method. Br-HNPs (equivalent to 25 mg bromelain) kept in dialysis bag has been immersed in pH progressive dissolution medium (HCl buffer pH 1.2 for 2h followed by phosphate buffer pH 7.4, 150 ml) stirred at 100 rpm and maintained at 37 ± 2ºC. Aliquots withdrawn at different time spans have been replaced with equal volume of dissolution media and analyzed spectrophotometrically.
Solid state characterization
Morphological evaluation of Br-HNPs suspension has been executed employing transmission electron microscopy. Appropriately diluted dispersion of optimized Br-HNPs (5-10µl) has been deposited on carbon coated copper grid and spattered by phosphotungstic acid (2 % w/v). Samples have been air dried and analyzed at 200 kV with different magnification.
Thermal behavior of bromelain, stearic acid, soya lecithin and optimized Br-HNPs has been evaluated using differential scanning calorimeter. Samples (5-10 mg) have been sealed in aluminum pans and heated at a rate of 10 ºC /min over a temperature range of 30 - 300 ºC, under nitrogen flow.
Diffraction pattern of bromelain, stearic acid, soya lecithin and optimized Br-HNPs have been obtained utilizing powder X ray diffractometer with CuKa radiation in the diffraction range of 2º to 80°, 2?. PXRD study has been conducted to characterize the crystallographic structures of all components and formulation along with alteration in physical state of bromelain in formulation.
Colloidal property retention study
Retention of colloidal property of bromelain nanoparticles in optimized Br-HNPs formulation has been ascertained according to ICH guideline for zone III and IV. Lyophilized formulation fastened in amber colored glass vials have been kept at 4 ± 2 ºC / 65± 5 % RH and 25 ± 2 ºC / 60 ± 5 % RH. The tendency of Br-HNPs to retain colloidal properties like average particle size, zeta potential and PDI was analyzed till 6 months under cool (4 ± 2 ºC/ 65 ± 5 % RH) and till 12 months under room temperature storage conditions (25 ± 5 ºC and 60 ± 5 % RH). Samples have been dispersed in puri?ed Milli-Q water before estimation for particle size, zeta potential and PDI.
Shelf life determination study
Samples stored under room temperature storage conditions were evaluated for percentage bromelain activity remaining after a suitable time interval of 0, 3, 6, 9 and 12 months respectively. Visual inspection of samples was also done after completion of stability study.

Optimization of formulation
In order to achieve higher drug loading, nanosize, good colloidal dispersibility and stability, various preliminary studies have been performed for selection of suitable lipid phase, surface active agent, critical formulation and process parameters.
The results showed that characteristic features like particle size, poly dispersibility index, zeta potential and entrapment efficiency have significantly affected by nature and composition of lipid. Stearic acid alone as lipid phase produced particles of bigger size (301.80 ± 31.52 nm) with poor entrapment efficiency (61.93 ± 5.12 %) (Table 1). Therefore, various batches have been prepared by varying stearic acid and tristearin concentration respectively. Increase in entrapment efficiency (61.93 ± 5.12 % to 86.32 ± 5.56 %) while decrease in polydispersity index (0.49 ± 0.02 to 0.21 ± 0.03) with change of zeta potential from -15.11 ± 3.01 mV to -29.01 ± 3.41 mV has been observed on increasing tristearin concentration upto 50 % w/w of total lipid phase content. However, additional rise in tristearin amount over 50 % w/w of lipid phase had no impact on drug loading capacity (Table 1). The remarkable reduction in particle size has been observed with tristearin addition due to its emulsifying property. Batches prepared on varying tristearin amount showed variable particle size due to difference in the emulsifying capacity of lipid phase in aqueous surfactant phase. The increment in tristearin content increased particle size (Table 1) which might be attributed by enhanced consistency of dispersed organic phase.
The remarkable change in particle size, zeta potential and PDI has been observed with different concentration of tween-80 and pluronic F-68 with PVA (1 % w/v) respectively in external phase. Among surfactants, tween-80 has facilitated the generation of smaller sized particles with higher entrapment efficiency compared to pluronic F-68 (Table 1). This indicated that tween-80 provides a more firm mechanical and thermodynamic barrier at interface to facilitate formation of uniform nanosized particles. Lowering of surfactant concentration (0.25 % w/w) produced large sized Br-HNPs (246.32 ± 33.27 nm) with lower entrapment efficiency (74.01 ± 3.87 %) whereas increased concentration (0.75 %) reduced both size (81.23 ± 28.54 nm) and entrapment efficiency (76.62 ± 4.64 %) (Table 1).
Elevation of sonication time from 5.0 to 7.5 min reduced size and PDI of Br-HNPs. However, further increment in sonication time during double emulsion formulation from 7.5 to 10.0 min had promptly made system inconsistent and expedited aggregation of lipid phase contributing to formation of higher sized particles with lower entrapment efficiency (Table 1). The distortion of the surfactant layer at the interface due to generation of higher energy in the system might had reduced drug loading and zeta potential.
The increase in external phase volume to 30 ml reduced the entrapment efficiency whereas increased the average particle size and PDI (Table 1) due to unavailability of sufficient shear force for the genesis of consistent micelles.
The numerical values of polydispersity index (PDI) of various batches ranging from 0.21 to 0.49 represented the degree of homogeneity of colloidal nanoparticulate formulations. Batches with PDI in middle of 0.10 – 0.30 showed consistency in their particle size while PDI values greater than 0.30 represented diversity of system (Table 1). It was observed that physical stability of batches prepared was regulated by surface charges estimated as zeta potential. Higher zeta potential near ±30 mV represented good dispersibility and colloidal stability of formulation due to electric repulsion between particles. Batches with higher tristearin concentration showed higher negative zeta potential compared to stearic acid due to possession of higher substituted carbon chains in tristearin (Table 1). Higher zeta potential of batches prepared with tween-80 (0.5 % w/v) and PVA (1 % w/v) also confirmed the surfactant suitability in reducing the interfacial tension during emulsification to make a stable colloidal system. However, yield of different batches varied from 61.86 ± 5.21 % to 77.47 ± 6.89 % independent of composition. Lower yield value during Br-HNPs production might be contributed by losses arising during sonication, centrifugation or lyophilization.
Depending upon the results obtained, Br4 batch having 120.56 ± 40.12 nm size and 86.32 ± 5.56 % entrapment efficiency with zeta potential of -29.01±3.41 mV was entitled for further explorations. During formulation of optimized Br-HNPs (Br4) batch, proteolytic activity of bromelain decreased from 932.15 ± 23.15 CDU to 769.06 ± 31.45 CDU. Around 18 % loss in bromelain activity observed during formulation might be contributed by organic solvent and stress experienced during sonication and lyophilization.
Table 1: Effect of varying formulation and processing variables on the properties of Br-HNPs.
Formu-lation code Lipid Sonication time (min) Surfactant addition during W1/O/W2 Volume of external phase (ml) Particle size (nm ± SD) Zeta potential (mV ± SD) Polydisp-ersity index (PDI ± SD) Entrapment efficiency (% ± SD)
Stearic acid (mg) Tristea-rin (mg) Tween-80 (% w/v) Pluronic F-68 (% w/v)
Br1 200 - 7.5 0.50 - 20 301.80 ± 31.52 -15.11 ± 3.01 0.49 ± 0.02 61.93 ± 5.12
Br 2 150 50 7.5 0.50 - 20 90.34 ± 21.61 -19.02 ± 2.82 0.37 ± 0.01 68.14 ± 4.54
Br 3 133.33 66.67 7.5 0.50 - 20 104.26 ± 24.91 -21.45 ± 3.14 0.33 ± 0.02 75.23 ± 4.74
Br 4 100 100 7.5 0.50 - 20 120.56 ± 40.12 -29.01 ± 3.41 0.21 ± 0.03 86.32 ± 5.56
Br 5 66.67 133.33 7.5 0.50 - 20 187.86 ± 29.68 -27.29 ± 4.51 0.27 ± 0.02 80.04 ± 3.51
Br 6 100 100 7.5 0.25 - 20 246.32 ± 33.27 -22.14 ± 4.25 0.52 ± 0.02 74.01 ± 3.87
Br 7 100 100 7.5 0.75 - 20 81.23 ± 28.54 -24.42 ± 3.56 0.22 ± 0.03 76.62 ± 4.64
Br 8 100 100 7.5 - 0.50 20 216.11 ± 39.31 -20.17 ± 3.49 0.39 ± 0.03 75.15 ± 3.85
Br 9 100 100 5.0 0.50 - 20 313.45 ± 34.68 -20.18 ± 4.84 0.48 ± 0.02 70.12 ± 4.24
Br 10 100 100 10.0 0.50 - 20 88.56 ± 24.87 -22.56 ± 3.69 0.24 ± 0.02 73.06 ± 6.72
Br 11 100 100 7.5 0.50 - 30 188.24 ± 27.74 -18.56 ± 3.08 0.29 ± 0.03 76.14 ± 4.37

Lyophilization parameters optimization
Lyophilization of aqueous dispersions was performed to remove moisture and convert them into stable and easily re-dispersible dry state. Conventionally, lyophilization technique encompasses three stages i.e solidification (freezing), sublimation (primary drying) and desorption of thawed water (secondary drying). Therefore, effect of freezing temperature, freezing rate and secondary drying time was evaluated on the quality of finished product. Freezing temperature is inherently corelated with freezing rate since lower temperature (-80 °C) facilitates faster freezing rates. A coherent tendency of faster uniform re-dispersion and significantly smaller particle size (120.56 ± 40.12 nm) and PDI (0.21 ± 0.03) of Br-HNPs frozen at -80 °C was observed compared to collapsed appearance at -40 °C (185.36 ± 15.23 nm, 0.29 ± 0.02). Smaller particle size at lower temperature indicated that faster freezing facilitated formation of smaller ice crystals with higher specific surface area. Thus, samples were then frozen at optimal freezing temperature (-80 °C) using slow freeze condition for comparison. No significant change in particle size (139.26 ± 20.34 nm) was observed at slow freezing rate but PDI was significantly increased (0.28 ± 0.02). This might be contributed by relatively slow movement of freezing interface and hybrid nanoparticles might have enough time to form strong aggregates before freezing.

Secondary drying time had not showed any significant change in particle size and PDI for 6 h (135.63 ± 23.24 nm, 0.24 ± 0.02) and 8 h (120.56 ± 40.12 nm, 0.21 ± 0.03) respectively. However, secondary drying for 8 h was utilized during study due to smaller average particle size obtained as well as to ensure least residual moisture content of formulation.

CHARACTERISTICS OF OPTIMIZED NANOPARTICLE AND THE FORMULATION:
Solis state characteristics of Br-HNPs
TEM images of Br-HNPs confirmed the spherical shape with smooth surface morphology of particles along with their nanometric size range (< 150 nm) (Figure 1A).
Diffraction pattern of bromelain, stearic acid, tristearin, lecithin and optimized Br-HNPs was used to determine their degree of crystallinity (Figure 1B). Bromelain diffractogram displayed various intense peaks in the range of 12º to 24º (2O). Stearic acid exhibited semi crystalline characteristic with distinct intense peaks at diffraction angles 6.99°, 21.47° and 23.90° (2 O) while tristearin at 16.54°, 19.36°, 23.16° and 24.21° (2O) respectively. However, characteristic diffraction peaks of bromelain were not noticeable in diffractogram of formulation indicating that bromelain was dispersed in formulation at molecular level with reduced crystallinity.
DSC thermogram of bromelain, stearic acid, tristearin, lecithin and optimized Br-HNPs are shown in Figure 1C. Bromelain exhibited characteristic sharp endothermic peaks at 149.63 ºC and 224.57 ºC while broad endothermic peak was observed at 63.60 ºC and 72.11 ºC respectively for stearic acid and tristearin. However, Br-HNPs did not show any characteristic peak of drug indicating homogeneous dispersion of bromelain in formulation. This might be contributed by loss in crystal lattice energy of bromelain in molecular dispersion ensuing the formation of Br-HNPs. The results also confirmed the chemical compatibility of drug with formulation ingredients. Thus, both DSC and pXRD study revealed loss of crystallinity of bromelain and its homogeneous dispersion in Br-HNPs.
Dissolution studies
In vitro release profile of bromelain from optimized nanocarriers was explored in pH progressive media to estimate the preservation of content from acidity and an effective delivery in intestinal milieu. Around 15 % drug releases in 2 h was observed from Br-HNPs followed by gradual drug release ~85 % in 24 h (Figure 1D). Initial burst release might be contributed by presence of free drug on the surface of particles whereas extended drug release might be influenced by lipophilicity of lipid phase, interaction of drug with surfactant at interface, rigidity of barriers along with diffusion from dialysis membrane. Prolonged drug release behavior from optimized formulation assured that fabrication Br-HNPs might be a coherent perspective for sustained delivery of bromelain. The highest r2 value determined by regression analysis was considered as benchmark to select the best fit model for release data and elucidate release mechanism. Thus, Br-HNPs release data followed Korseymeyer Peppas model (r2 = 0.9776) and diffusion exponent (n) has value 0.64 indicating anomalous release behavior i.e. diffusion and dissolution-controlled release mechanism.
Dilution study
In dilution study, no significant change in particle size, PDI and zeta potential was observed on diluting the lyophilized formulation upto 1:500 times (Table 2). This indicated that surfactant layer maintained the rigidity at interface of emulsified particles and maintained the particles intact and stable.
Table 2: Effect of dilution on particle size, zeta potential and PDI of optimized formulation (Br4).
Dilution ratio Particle size (nm ± SD) Zeta potential (mV ± SD) Polydispersity index (PDI ± SD)
1:50 119.35 ± 31.23 -28.19 ± 4.24 0.21 ± 0.03
1:100 122.32 ± 28.80 -29.01 ± 3.22 0.22 ± 0.02
1:500 117.22 ± 33.71 -28.86 ± 3.51 0.21 ± 0.02

Stability study in gastrointestinal milieu
Optimized formulation incubated in simulated gastric environment pH 1.2 showed increase in particle size (2.54 ± 0.26 µm) and PDI (0.64 ± 0.02) along with drop of zeta potential (-7.13 ± 1.01 mV). Although no notable change in particle size (132.23 ± 18.57 nm), PDI (0.24 ± 0.02) and zeta potential (-25.32 ± 3.41 mV) was observed in simulated intestinal fluid pH 7.4. This indicated that simulated gastric milieu facilitated aggregation of nanoparticles. Yet the size of aggregates was less than 0.5 mm which confirmed no significant delay in gastric emptying. Optimized formulation retained 88.04 ± 5.14 %, 92.23 ± 4.25 %, 95.66 ± 3.24 % and 97.26 ± 4.28 % proteolytic activity after 2 h incubation period under simulated gastric pH 1.2 and intestinal pH 5.0, 6.8 and 7.4 respectively (Table 3). On the contrary, pure drug retained only 31.26 ± 6.23% proteolytic activity under simulated gastric pH 1.2. Thus, Br-HNPs for oral delivery would be competent enough to maintain structural robustness of bromelain during transit through stomach.
Table 3 : Effect of pH on the stability of formulation
Parameter Particle size Zeta potential (mV±SD) Polydispersity index (PDI) Proteolytic activity remaining
pH 1.2 2.54 ± 0.26 µm -7.13 ± 1.01 0.64 ± 0.02 88.04 ± 5.14 %
pH 5.0 1.09 ± 0.56 µm -15.36 ± 3.58 0.51 ± 0.04 92.23 ± 4.25 %
pH 6.8 209.14 ± 20.36 nm 21.89 ± 4.12 0.26 ± 0.03 95.66 ± 3.24 %
pH 7.4 132.23 ± 18.57 nm -25.32 ± 3.41 0.24 ± 0.02 97.26 ± 4.28 %
Colloidal property retention
The tendency of Br-HNPs to retain average particle size, zeta potential and PDI was analyzed till 6 months under cool (4 ± 2 ºC/ 65 ± 5 % RH) and till 12 months at room temperature storage conditions (25 ± 5 ºC and 60 ± 5 % RH). The results demonstrated no marked change in any of the parameters assessed under cool and ambient conditions respectively after 6 months of storage (Figure 2). Stability studies performed manifested that formulation and process variables to prepare optimized Br-HNPs had been congruously optimized which contributed better retention of colloidal property of Br-HNPs at room temperature (Table 4).

Table 4: Colloidal property stability data of optimized Br-HNPs formulation under cool (4 ± 2 ºC/ 65 ± 5 % RH) and ambient conditions (25 ± 2 ºC/ 60 ± 5 % RH) of storage.
Duration (Month) Storage condition Particle size (nm ± SD) Zeta potential (mV ± SD) PDI
0 - 120.56 ± 40.12 -29.01 ± 3.41 0.21 ± 0.03
3 4 ± 2 ºC/ 65 ± 5 % RH 123.41 ± 28.22 -28.23 ± 3.24 0.20 ± 0.04
25 ± 2 °C/ 60 ± 5% RH 129.32 ± 30.24 -25.57 ± 5.41 0.23 ± 0.04
6 4 ± 2 ºC/ 65 ± 5 % RH 126.01 ± 33.74 -27.19 ± 3.27 0.22 ± 0.03
25 ± 2 °C/ 60 ± 5% RH 181.25 ± 25.27 -21.02 ± 3.34 0.302 ± 0.04
9 25 ± 2 °C/ 60 ± 5% RH 190.06 ± 21.71 -21.01 ± 3.56 0.362 ± 0.03
12 25 ± 2 °C/ 60 ± 5% RH 211.25 ± 22.52 -19.65 ± 3.68 0.368 ± 0.04

Shelf life (Stability study)
Bromelain followed first order degradation kinetics. The calculated t90 of the formulation at real time stability conditions was 820±50 days. Numerical values of Kcal/t90 suggested that optimized formulations would be able to assure almost twenty-seven-month shelf life (t90) for the product. Stability studies performed manifested that formulation and process variables to prepare optimized Br-HNPs had been congruously optimized which contributed better stability of Br-HNPs at room temperature. The results demonstrated no marked change in any of the parameters assessed under ambient condition after 12 months of storage.
Table 5: Effect of storage on entrapment efficiency, drug release and percentage bromelain activity remaining of optimized Br-HNPs formulation
Duration (Month) Storage condition Entrapment efficiency (% ± SD) Cumulative percentage release (% ± SD) Bromelain activity remaining (% ± SD)
0 - 86.32 ± 5.56 86.56 ± 8.32 99.96 ± 3.65
3 4 ± 2 ºC/ 65 ± 5 % RH 85.83 ± 4.31 84.04 ± 5.97 99.31 ± 4.65
25 ± 2 °C/ 60 ± 5% RH 81.31 ± 5.47 83.87 ± 3.17 99.68 ± 4.15
6 4 ± 2 ºC/ 65 ± 5 % RH 83.87 ± 4.87 83.68 ± 4.19 98.01 ± 4.28
25 ± 2 °C/ 60 ± 5% RH 78.23 ± 3.04 81.32 ± 3.02 98.38 ± 5.14
9 25 ± 2 °C/ 60 ± 5% RH 78.23 ± 3.04 76.32 ± 3.15 97.25 ± 4.24
12 25 ± 2 °C/ 60 ± 5% RH 78.23 ± 3.04 74.18 ± 3.29 95.66 ± 5.47

In vivo evaluation of optimized formulation
Ex vivo platelet aggregation assay
The antiplatelet efficacy of bromelain and optimized Br-HNPs has been evaluated by performing ex vivo platelet aggregation assay. Twenty-eight wistar rats have been arbitrarily divided within seven groups (n=4). Group I, II, III, IV, V, VI and VII have been treated prophylactically orally with normal saline (control), aspirin (20 mg/Kg), bromelain solution (10mg/ Kg), bromelain solution (20 mg/Kg) and optimized formulation (equivalent to 5mg/Kg, 10 mg/Kg and 20 mg/Kg bromelain respectively) respectively for 7 days. Rats of respective group have been anesthetized with ketamine hydrochloride (60 mg/Kg) and xylazine hydrochloride (10 mg/Kg) intraperitoneally to collect blood via cardiac puncture. The accrued whole citrated blood has been centrifuged at 1000 rpm for 10 min at 37 °C to prepare platelet rich plasma. Rat’s platelet rich plasma has been adjusted to 3 X 108 platelets/ml using platelet poor plasma. Platelet rich plasma has been centrifuged at 3500 rpm for 10 min at 37 °C to obtain platelet poor plasma. Platelet aggregation has been induced in platelet rich plasma by thrombin (0.1 U/ml) addition followed by measuring absorbance of samples at 540 nm for 10 min. The percentage platelet aggregation has been determined by following equation.
Platelet aggregation (%) = [(R1- R2)/ (R1- P)] X 100
Where R1 is absorbance of platelet rich plasma before addition of thrombin; R2 is absorbance of platelet rich plasma after 10 min of addition of thrombin; and P is absorbance of platelet poor plasma.

Tail bleeding time
Tail transection bleeding time of animals (n=4) receiving normal saline, aspirin (20 mg/Kg), bromelain solution (10 mg/Kg) and optimized formulation (equivalent 10 mg/Kg bromelain) respectively prophylactically for 7 days has been determined. Animals have been anesthetized before measuring bleeding time by transecting tail 2 mm from their tips. Bleeding time has been described as time needed for blood flow to cease and determined after 2 h of administration of respective treatment.
Thrombolytic activity
Carrageenan-induced rat tail thrombosis model with slight modification have been utilized to measure in vivo thrombolytic potential of bromelain and optimized formulation. A total of 16 wistar rats having tail length exceeding 13 cm have been arbitrarily categorized into 4 groups (n=4). First group served as control and has been treated with normal saline orally while second, third and fourth group received aspirin (20 mg/Kg), oral bromelain solution (10 mg/ Kg) and optimized Br-HNPs (equivalent to 10 mg/Kg bromelain) respectively prophylactically for 7 days. Rat tails have been ligated and k-carrageenan (2 mg/Kg) was injected in dorsal rat tail vein after 30 min of last treatment respectively. Ligation has been unfastened after 10 min of injection. The redness and length of infarcted region of rat tail has been measured after 24 h of carrageenan injection. The percentage thrombus formation and actual thrombus dissolved after 24 h has been calculated by formula

Thrombus dissolved (TD, %) = 100- TF %
Evaluation of hematological parameters
After 24 h of carrageenan injection animals have been anaesthetized and blood samples have been collected via cardiac puncture. The collected whole blood has been evaluated for leukocyte count, monocyte percentage, neutrophils percentage, platelet count, platelet–larger cell ratio (PLCR), mean platelet volume (MPV) and procalcitonin (PCT) level to determine therapeutic potential of treatments. Blood cell counting has been carried out utilizing Automated Hematology Analyzer. Serum separated from whole blood has been collected and stored at -80 ºC until used to measure TNF-a and IL-1ß level using commercial assay kits as per the manufacturer’s guidelines. Coagulation assay has been performed within 3 h of blood collection.
Coagulation assay
The collected whole citrated blood after 24 h of carrageenan injection from animals of respective group has been centrifuged to prepare platelet poor plasma. So obtained platelet poor plasma (150 µl) of treated and control groups respectively have been used to measure prothrombin time (PT) and activated partial thromboplastin time (APTT). PT and APTT of samples have been illustrated in seconds only.
Statistical Analysis
Results have been illustrated as mean ± SD. One-way ANOVA with Bonferroni's multiple comparison test has been used to analyze the statistical significance of results utilizing GraphPad Prism software version 5.00, USA. The variation in results have been considered statistically significant at 95% confidence interval (p < 0.05).

In vivo performance
Platelet aggregation
Ex vivo platelet aggregation induced by thrombin was significantly inhibited with oral treatment with aspirin, bromelain (10 mg/ Kg) and Br-HNPs (equivalent to 10 mg/ Kg bromelain) to 26.19 ± 3.23 %, 11.33 ± 2.36 % and 25.59 ± 2.14 % respectively (Figure 3A). Bromelain’s ability to suppress thrombin induced COX-2 expression and PGE2 production facilitating activation of ERK1/2 and p38 MAPK might have contributed to inhibition of platelet aggregation. However, poor platelet aggregation inhibitory effect of bromelain might be contributed by its inactivation in acidic gastric milieu and degradation of bromelain by proteases in gastrointestinal tract. The results of study confirmed that Br-HNPs possessed improved antiplatelet activity which might be the outcome of enhanced in vivo stability, their sustained drug release behavior and lymphatic uptake of Br-HNPs by M-cells of Peyer’s patches. Thus, optimized formulation could serve as an alternative antiplatelet therapy for prevention of thrombotic complications associated with platelet adhesion, activation and aggregation amplified by thrombin, an endogenous agonist.
However, oral administration of bromelain (20 mg/ Kg) and Br-HNPs (equivalent to 20 mg/ Kg bromelain) inhibited platelet aggregation to 15.87 ± 2.24 % and 28.12 ± 2.56 % respectively. Increase in dose for pure drug enhanced the platelet aggregation inhibitory effect significantly but increase in dose of Br-HNPs had not shown any significant improvement in antiplatelet activity. Sustained release behavior of optimized formulation prevented increase in antiplatelet activity. However, lower dose 5mg/Kg showed only 10.36 ± 2.08 % antiplatelet activity. This indicated that lower dose was not sufficient to exhibit sufficient therapeutic effect. Therapeutic effect obtained with Br-HNPs was almost 2.2-fold higher than pure drug at same dose (10 mg/Kg).
The increased or continuous bleeding is the most typical untoward outcome related with antiplatelet therapies in clinics due to imbalance of procoagulation and anticoagulation properties. Thus, tail bleeding time was determined to predict the relative safety of pure drug and optimized formulation at antithrombotic dose. Tail bleeding time of healthy rats was 323.15 ± 65.23 s which had significantly increased to 736.94 ± 71.23 s in aspirin treated animals. In contrast, bromelain and Br-HNPs treated animals did not significantly extended bleeding time i.e. 370.23 ± 59.36 s and 358.39 ± 62.23 s respectively. Br-HNPs exhibited platelet aggregation inhibitory effect comparable to aspirin along with significantly lower bleeding time. Sustained drug release from Br-HNPs had not caused unrestrained bleeding due to bromelain’s ability to reversibly inhibit platelet activation and platelet aggregation. Thus, it can be inferred that optimized formulation is relatively safe at effective antithrombotic dose.
Thrombolytic potential
During present study, k-carrageenan, a potent thrombogen was used to induce local in?ammation and endothelial cell injury facilitating release of in?ammatory mediators which triggers the genesis of thrombosis by inducing agglutination of blood cells. Tail redness corresponding to thrombus formation was measured as principle major of the study which increased to maximum after 24 h. Tail thrombosis with 100 % frequency was attained by carrageenan injection in control group indicating break in the homeostasis. The diseased control group showed thrombus formation in 89.92 ± 4.36% of total tail length. While the animals on oral therapy of aspirin, bromelain and Br-HNPs showed the reduced frequency of thrombosis i.e. 26.72 ± 2.69 %, 59.36 ± 5.36 % and 22.40 ± 2.18 % of total tail length respectively (Figure 3B). The anti-inflammatory potential of bromelain has contributed to reduction in thrombus formation by suppressing local inflammation, endothelial cell injury and platelet aggregation induced by carrageenan. Since inflammation and coagulation are intensely related processes with substantial consequences on each other. The remarkable reduction (p<0.001) in infarcted tail length and redness after Br-HNPs treatment compared to bromelain was observed (Figure 3C). Br-HNPs treatment facilitated 1.9-fold higher dissolution of thrombus compared to pure drug. Protection to bromelain offered by Br-HNPs in gastric milieu, extended drug release pattern and lymphatic uptake of nanoparticulate formulation by M-cells of Peyer’s patches were the major contrivances contributing to improved thrombolytic potential of Br-HNPs.
Change in blood coagulation pro?le of rats
Thrombosis is a multifactorial disorder associated with breakdown of homeostasis between blood cells (including platelets), plasma proteins, coagulation factors, inflammatory factors and the endothelial lining within the lumen of arteries and veins. Thus, to evaluate the effect of bromelain and Br-HNPs on coagulation process in carrageenan induced tail thrombosis model, PT and APTT level was measured. PT and APTT represents time required to form clot via different coagulation pathways. Level of PT and APTT exhibited by animals of the healthy control group were 14.33 ± 0.51 s and 35.83 ± 2.91 s, respectively during the study. However, carrageenan challenge dropped the PT (7.20 ± 0.23 s) and APTT (19.60 ± 0.90 s) significantly. Significant lower PT and APTT in diseased control group confirmed coagulation disorder and thrombotic disease compared to healthy group. On the contrary, oral dosing of aspirin (20 mg/Kg), pure drug and Br-HNPs at a maintenance dose of 10 mg/kg/day equivalent to bromelain for 7 days maintained the high PT and APTT levels after thrombotic insult (Figure 4). The remarkably high level of PT and APTT (p<0.05) observed in bromelain and Br-HNPs treated animals confirmed elevation in activation time of extrinsic and intrinsic routes of coagulation process due to a slow fibrin clot formation. The results also justify the anti-thrombotic potential of bromelain. Statistically significant (p<0.05) higher PT and APTT level among Br-HNPs treated group compared to pure drug could be attributed to characteristic features imparted by carrier system on stability and prolonged availability of drug in blood. The results confirmed Br-HNPs potential analogous to aspirin in antagonizing thrombotic effect of carrageenan and efficacy in preventing hypercoagulation states.
Hematology
The increase in leukocyte count, monocyte percentage and neutrophils percentage confirmed the ability of kappa-carrageenan to cause acute and systemic inflammation in diseased control group. Pre-treatment with aspirin, pure drug and Br-HNPs guarded against carrageenan fostered elevated levels of leukocyte count, monocyte percentage, neutrophils percentage, TNF-a and IL1-ß level respectively (Figure 4). Inhibitory effect of bromelain on inflammatory cytokines secretion by hindering diverse molecular pathways including NFKB overexpression along with improvement of expression of Nrf2 pathway has bestowed its anti-inflammatory potential. In addition, results of study showed that treatment with both drug and Br-HNPs exhibited positive platelet count reduction which impeded thrombosis formation and alleviation of blood viscosity (Figure 4). Significantly (p<0.05) higher reduction of platelet count and inflammatory markers with Br-HNPs might be contributed by higher in vivo stability attributed by lipid-based carrier system, controlled drug delivery and their probable lymphatic drainage due to nanosize.
Platelet indices typically platelet–larger cell ratio (PLCR) and mean platelet volume (MPV) are regarded as the quality indicator of hemostasis and thrombosis. The elevated level of PLCR and MPV in diseased control group confirmed the thrombotic events induced by carrageenan injection (Figure 4). PLCR recognizes the largest-sized fraction of platelets and their potential proportion correlates more firmly to thrombotic events. Since large sized platelets are highly active enzymatically and metabolically, lodging greater number of granules, inducing greater production of thromboxane A2 and expressing more surface proteins. Furthermore, platelet activation induces morphologic changes in platelets and increases MPV. Oral treatments with pure drug as well as Br-HNPs reduced PLCR and MPV respectively and progression of thromboembolic events by inhibiting platelet activation and aggregation.
Elevation of procalcitonin (PCT) level in diseased control group compared to healthy group also acknowledged the inflammatory retaliation for carrageenan injection (Figure 4). Oral treatment with both pure drug and Br-HNPs decreased PLCR, MPV and PCT level confirming anti-inflammatory activity of bromelain. Although decrease in MPV in animals treated with pure drug and Br-HNPs was not statistically significant. A reverse shift of MPV, PCT and PLCR to values similar to those of healthy subjects correlated well with increased stability of bromelain in Br-HNPs. Thus, the results inferred the effectiveness of bromelain’s nano-formulation as antithrombotic therapy by inhibition of neutrophil infiltration and reduction of cytokine storm, PCT level, platelet count, PLCR, MPV and thrombus formation.
The present invention provides suitable oral delivery vehicle for bromelain in the form of Br-HNPs. Optimized formulation exhibits higher stability following oral administration and increased anti-thrombotic activity in comparison to free drug. ,CLAIMS:We Claim:
1. Novel anti-thrombotic nanoparticles comprising a binary lipid matrix of a core and a shell, wherein
- the core comprises of bromelain and lecithin, and
- the shell comprises lipid matrix of stearic acid and tristearin,
wherein said nanoparticles possess high entrapment efficiency, stability and improved drug loading capacity.
2. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said nanoparticle size is in the range of 120.56 ± 40.12 nm.
3. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said high entrapment efficiency is in the range of 86.32 ± 5.56 %.
4. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein zeta potential of the nanoparticles is in the range of -29.01±3.41 mV.
5. The novel anti-thrombotic nanoparticles as claimed in claim 1 wherein said nanoparticles are analyzed till 6 months under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity and till 12 months under room temperature storage conditions of 25 ± 5 ºC at 60 ± 5 % relative humidity to retain colloidal property of the nanoparticle.
6. A process to prepare the novel anti-thrombotic nanoparticles as claimed in claim 1, said process comprises the steps of:
(i) preparing an aqueous solution of bromelain and lecithin (W1),
(ii) preparing an organic phase of stearic acid, tristearin and a surfactant dissolved in an organic solvent (O),
(iii) emulsifying aqueous solution of step (i) (W1) with organic solvent of step (ii) (O) employing ultrasonic disruptor under predefined conditions to obtain a primary emulsion,
(iv) preparing an aqueous solution of a synthetic polymer and a surfactant in predetermined ratio (W2),
(v) emulsifying said primary emulsion of step (iii) with aqueous solution of step (iv) (W2) under probe sonication under predefined conditions to form a double emulsion (W1/O/W2),
(vi) removing said organic solvent by continuous stirring of said double emulsion to obtain organic solvent free double emulsion,
(vii) centrifuging said organic solvent free double emulsion at predefined conditions to obtain pellets,
(viii) dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension,
(ix) lyophilizing the said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain said antithrombotic nanoparticle.
7. The process as claimed in claim 6 wherein said organic solvent in step (ii) is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.
8. The process as claimed in claim 6 wherein said organic solvent is dichloromethane.
9. The process as claimed in claim 6 wherein said tristearin in step (ii) is added in a concentration upto 50 % w/w of total lipid phase content.
10. The process as claimed in claim 6 wherein said surfactant in step (ii) is Span 80 of 3 % w/v.
11. The process as claimed in claim 6 wherein said predefined conditions in step (iii) for emulsification by ultrasonic disruptor are disruption for 5 - 7min over an ice bath.
12. The process as claimed in claim 6 wherein said water soluble synthetic polymer in step (iv) is selected from the group comprising poly((meth)acrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.
13. The process as claimed in claim 6 wherein said surfactant in step (iv) is Tween 80.
14. The process as claimed in claim 12 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).
15. The process as claimed in claim 6 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant, preferably the surfactant is a non-ionic surfactant.
16. The process as claimed in claim 6 wherein said predetermined ratio of the synthetic polymer and the surfactant is 1% w/v : 0.5% w/v.
17. The process as claimed in claim 6 wherein said predefined conditions in step (v) are emulsifying the primary emulsion for 7.5 min over an ice bath.
18. The process as claimed in claim 6 wherein said predefined conditions in step (vii) are centrifugation at 30,000 rpm for 25 min at 4 ºC.
19. The process as claimed in claim 6 wherein said cryoprotectant is mannitol 10% w/v.
20. The process as claimed in claim 6 wherein said specific conditions for lyophilization are freezing to -80 °C for 5 h followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar.
21. A composition of nanoparticle formulation, said composition comprises:
Ingredient Quantity (%)
Bromelain 7-15%
Lecithin 10-20%
Stearic acid 24-75%
Tristearin 0-50%
Surfactant 0.25-.75%
Volume of external phase 20-30 ml
22. The composition as claimed in claim 21 wherein said composition comprises:
Ingredient Quantity (%)
Bromelain 11.11%
Lecithin 14.81%
Stearic acid 37.04%
Tristearin 37.04%
Tween - 80 0.5% (w/v)
Volume of external phase 20 ml
23. The composition as claimed in claim 21 wherein t90 of said nanoparticle formulation is 820±50 days.
24. The composition as claimed in claim 21 wherein said composition comprises said antithrombotic nanoparticles of bromelain with pharmaceutically acceptable excipients or surfactants or carriers or a combination thereof.
25. The composition as claimed in claim 21 wherein said composition is in dosage form suitable for oral drug delivery.
26. The composition as claimed in claim 25 wherein said dosage form is sustained release dosage form selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels.