1. Enteric coated mucoadhesive bromelain nanoparticles comprising bromelain, chitosan, tripolyphosphate, lecithin and Eudragit polymer.

2. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein chitosan is present in an amount of 53.33 – 61.53 % w/w, bromelain is present in an amount of 7.69 – 20 % w/w, lecithin is present in an amount of 7.69 – 20 % w/w and Eudragit is present in an amount of 2-3 % w/v.

3. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein ratio of lecithin: bromelain is between 1:5 to 5:1.

4. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein ratio of chitosan: bromelain is 4:1 to 10:1.

5. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 3, wherein ratio of lecithin: bromelain is between 1:2 to 2:1.

6. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the Eudragit polymer is Eudragit L 100.

7. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a particle size of 200 to 800 nm.

8. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a zeta potential of -40 to 40 mV.

9. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a poly dispersibility index of 0.2- 0.7.

10. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the nanoparticle comprises bromelain, chitosan, tripolyphosphate, lecithin and Eudragit L-100.

11. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein chitosan is present in an amount of 57.14 % w/w, bromelain is present in an amount of 14.29 % w/w, tripolyphosphate is present in an amount of 14.29 % w/w, lecithin is present in an amount of 14.29 % w/w coated with Eudragit L-100.

12. A process for preparing enteric coated mucoadhesive bromelain nanoparticles as claimed in any of the preceding claims, said process comprising: (a) Mixing aqueous solution of chitosan with aqueous solution of bromelain and lecithin, (b) Adding a buffer to the mixture of step (a) to obtain a mixture having pH 5-6, (c) Adding aqueous solution of tripolyphosphate to the mixture of step (b) with continuous stirring for 1-3 h to obtain a translucent dispersion, (d) Sonicating the dispersion of step (c) for 10 - 40 minutes to obtain nanodispersion, followed by centrifuging the nanodispersion to obtain nanoparticles, (e) dispersing nanoparticles of step (d) in Eudragit L 100 coating solution (2.0 - 3.0 % w/v) under continuous stirring for 1 h to obtain dispersion, (f) centrifuging the dispersion of step (e) at 15,000 rpm for 30 min to obtain pellets of nanoparticles.

13. An anticancer pharmaceutical composition comprising bromelain nanoparticles as claimed in claim 1.

Description:Field of the invention:
The present invention relates to enteric coated mucoadhesive bromelain nanoparticles. In particular, the present invention relates to pH resistance enteric coated bromelain nanoparticles suitable for targeted delivery of bromelain to the colon. The present invention also provides a process for the preparation of enteric coated mucoadhesive bromelain nanoparticles. The enteric coated mucoadhesive nanoparticles of the present invention have enhanced absorption at the colon and are useful in the treatment of colon cancer.

Background of the invention;
Colon cancer (CC) is one of the most prevalent cancer with highest fatality rates globally. The incidence of colorectal cancer has substantially elevated 0.5 to 3% per year from 2011 to 2020, making it second most prevalent cancer in women and the third most common in men. A variety of genetic or epigenetic modifications leading to hyperproliferation of epithelial cells and development of polyps contribute to progress and metastasis of colon cancer by a number of different paths, such as chromosomal instability (CIN), serrated neoplasia and microsatellite instability (MSI). However, etiology of CC is complex and still unknown. Various anticancer agents like 5-fluorouracil, leucovorin, irinotecan and oxaliplatin, cetuximab, bevacizumab, encorafenib, binimetinib and masitinib, atezolizumab and pembrolizumab have been found effective in colon cancer management. However, higher cost and associated side effects limits their patient compliance. Therefore, an alternative complementary drug having anticancer potential with immunomodulation can be a drug of choice. Numerous studies have reported the therapeutic benefits of natural bioactive molecules in management of cancer. Bromelain, a cysteine protease enzyme derived from pineapple (Ananas comosus) stems is receiving a lot of interest because of its safety, excellent therapeutic efficacy and low cost. It has a number of biological properties, including anti-inflammatory, immunomodulatory, antioxidant, fibrinolytic, anti-thrombotic, wound healing and anti-cancer activity. Despite its several benefits as therapeutic moiety its low bioavailability, sensitivity to low pH and instability in GIT limits its oral use. Therefore, novel strategies utilizing application of nanotechnology, absorption enhancers, chemical modifications, colon-specific targeting are promising for improving therapeutic potential of protein/ peptide based drugs and defeating their associated problems. Colon exhibit supportive physiological domain for absorption of protein drugs due to its neutral pH and lower density of proteases. Several formulation approaches have been explored in development of colon-targeted drug delivery systems. These approaches involve the use of formulation components that interact with one or more aspects of gastrointestinal (GI) physiology, such as difference in pH across GI tract, presence of colonic microflora and enzymes to achieve colon targeting. However, hydrophilicity and high molecular weight of protein based drugs limits their penetration and absorption. Therefore, pH-responsive carrier system composed of Eudragit L 100 coated chitosan nanoparticles loaded with bromelain were fabricated in present study for targeted controlled delivery of drug to malignant colon cells and to enhance stability and therapeutic efficacy of drug.
Front Oncol. 2022; 12 disclose the anticancer properties of bromelain. It discusses the anticancer properties of Bromelain and its ability to synergize/ enhance the anticancer efficiency of the existing anticancer molecules.
Marketed product BromAc® (Mucpharm), combination of bromelain and acetylcysteine, has proved effective in the recurrent thoracic pseudomyxoma peritonei (PMP) treatment. According to the bromelain-based mucus-disrupting activity (see above), the mucolytic properties of BromAc® help the tumor dissolution when it was injected directly into mucinous disease.
Am J Cancer Res 2023;13(4):1522-1532 discusses the mucolysis and antitumour effects of BromAc® for several mucinous tumours. It discloses that it exhibits a synergistic potentiation of the effect of several cytotoxic agents on mucinous tumours in preclinical studies. Furthermore, it demonstrates locoregional safety and efficacy in animal and clinical studies. This literature review summarises the history of BromAc® for mucinous tumours, including its conception, preclinical development in vitro and in vivo, and clinical evidence.
Hence it is evident that Bromelain is emerging as a potent anticancer molecule alone. It also enhances the efficacy of existing anticancer molecules and hence can become a part of mainstream anticancer therapy.
Therefore, there is a need to develop novel bromelain formulations for effective use in anticancer therapy so that the drug is effective in lower concentration, has high availability at the active site. For oral therapy targeting colon, it is desired that the drug reaches the colon in therapeutic amount by safely bypassing the acidic environment of the stomach.

Object of the invention:
To obviate the drawbacks in the existing state of the art, it is an object of the present invention to provide enteric coated bromelain nanoparticles for the treatment of colon cancer.
It is another object of the present invention to provide enteric coated bromelain nanoparticles which can safely deliver bromelain at the active site on colon by bypassing the acidic environment of the stomach.
It is another object of the present invention to provide pH stable bromelain nanoparticles.
It is another object of the present invention to provide bromelain nanoparticles having enhanced absorption at the colon.
It is another object of the present invention to provide mucoadhesive bromelain nanoparticles having enhanced absorption at the colon.
It is another object of the present invention to provide enteric coated bromelain nanoparticles having enhanced entrapment efficiency.

Summary of the invention:
Accordingly, the present invention provides enteric coated bromelain nanoparticles comprising bromelain, chitosan, tripolyphosphate, lecithin and Eudragit polymer.
In one embodiment, the present invention provides enteric coated bromelain nanoparticles, wherein bromelain is present in an amount of 7.69 – 20 % w/w, chitosan is present in an amount of 53.33 – 61.53 % w/w, tripolyphosphate is present in an amount of 7.69 – 20 % w/w, lecithin is present in an amount of 7.69 – 20 % w/w and Eudragit is present in an amount of 2.0-3.0 % w/v.
In one embodiment, the present invention provides a process for preparing enteric coated bromelain nanaoparticle.
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 proceeded 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.

Brief description of drawings:
Figure 1: Fluorescence spectra of pure bromelain and bromelain released from optimized formulation incubated at distinct simulated gastrointestinal pH conditions respectively.
Figure 2: In vitro drug release profile of optimized formulation.
Figure 3: [A] Mucoadhesive nature of CNPs, Br-CNPs and Eu-Br-CNPs; Effect of mucin binding on colloidal properties of CNPs, Br-CNPs and Eu-Br-CNPs such as [B] particle size, [C] PDI and [D] zeta potential respectively.
Figure 4: % growth inhibition at various concentrations of bromelain, 5-FU and optimized formulation respectively.
Figure 5: Representation of cellular uptake of free dye and placebo-Eu-CNPs by fluorescence microscopy (500-600 nm filter) (20 X magnification).
Figure 6: [A] Plasma drug concentration v/s time profile and [B] Colon tissue drug concentration v/s time profiles of bromelain and optimized formulation respectively.
Figure 7: Effect of different treatment on (A) DAI and (B) Tumor burden in DMH+DSS induced colon cancer model respectively.
Figure 8: Effect of different treatments on (A) haemoglobin, (B) RBCs count, (C) lymphocyte, (D) neutrophil, (E) total leukocyte count, (F) platelet count, (G) SGPT (H) SGOT and (I) CEA level respectively.
Figure 9: The level of immunological markers (A) TNF-a, (B) IL-6, (C) IL-10 and (D) relative caspase-3 activity (?F X 103) in the serum of DMH+DSS, 5-FU, bromelain and optimized formulation treated wistar rats respectively.

Detailed description of the invention:
Bromelain, a key enzyme found in pineapple (Ananas comosus (L.) Merr.) has multiple beneficial effects for human health such as anti-inflammatory, immunomodulatory, antioxidant and anticarcinogenic. It is a promising candidate for anticancer therapy and hence research is being conducted for its enhanced delivery and therapeutic efficacy.
The present invention relates to advanced formulation of bromelain for targeted drug delivery. It relates to enteric coated bromelain nanoparticles. The enteric coated nanoparticles of the present invention (Eu-Br-CNPs) are resistant to the acidic gastric pH and hence can deliver bromelain to the colon.
In one embodiment the present invention provides bromelain nanoparticles comprising bromelain, chitosan, a cross-linker, one or more emulsifier and an enteric coating polymer.
In another embodiment, the present invention provides bromelain nanoparticles comprising bromelain in an amount of 7.69 – 20 % w/w, chitosan in an amount of 53.33 – 61.53 % w/w, cross-linker in an amount of 7.69 – 20 % w/w, one or more emulsifier in an amount of 7.69 – 20 % w/w and an anionic copolymer in an amount of 2.0-3.0 % w/v.
The cross linker is an inorganic compound selected from tripolyphosphate (TPP), sodium sulfate, or cyclodextrin (CD).
The emulsifier is selected from sucrose fatty acid esters, sorbitan fatty acid esters, glycerol fatty acid esters, or propylene glycol fatty acid esters, lecithin, phospholipids, glycolipids, carbohydrates and triglycerides or their mixtures.
The enteric coating copolymer is selected from methacrylate copolymers, hydroxypropyl methylcellulose acetate succinates and cellulose acetate phthalate and Eudragit.
Eudragit is selected from Eudragit®L 100-55, Eudragit®L 30 D-55, Eudragit®FL 30 D-55, Eudragit® L 100, Eudragit®L 12,5, Eudragit®S 100, Eudragit® S 12,5, Eudragit®FS 100 and Eudragit® FS 30 D.
In a particular embodiment, the present invention provides Bromelain nanoparticles comprising bromelain, chitosan, tripolyphosphate, lecithin and Eudragit L-100.
In the nanoparticles of the present invention, chitosan is present in an amount of 57.14 % w/w, bromelain is present in an amount of 14.29 % w/w, tripolyphosphate is present in an amount of 14.29 % w/w, lecithin is present in an amount of 14.29 % w/w coated with Eudragit.
In one embodiment, the ratio of lecithin: bromelain is between 1:5 to 5:1.
In one embodiment, the ratio of chitosan: bromelain is 4:1 to 10:1
In one embodiment the ratio of lecithin: bromelain is between 1:2 to 2:1.
In one embodiment, the Eudragit polymer is Eudragit L 100.
The Bromelain nanoparticles of the present invention have a particle size of 200 to 800 nm.
The Bromelain nanoparticles of the present invention have a Zeta potential of -40 to 40 mV.
The Bromelain nanoparticles of the present invention have a Entrapment efficiency of more than 95%.
The Bromelain nanoparticles of the present invention have poly dispersibility index of 0. 20 to 0.70.
In one embodiment, the present invention also provides a process for preparing enteric coated Bromelain nanoparticles (Eu-Br-CNPs).
The Enteric coated Bromelain nanoparticles (Eu-Br-CNPs) of the present invention can be prepared by modified ionic gelation technique. It is prepared by first preparing bromelain nanoparticles (Br-CNPs) and then coating it with an anionic copolymer suitable for enteric coating.
In one embodiment the present invention provides a process for preparing bromelain nanoparticles (Br-CNPs). An aqueous solution of bromelain and an emulsifier in buffer is prepared at pH 5-6. An aqueous solution of chitosan is also prepared in buffer at pH 5.5. The aqueous solution of chitosan is then mixed in aqueous solution of bromelain and emulsifier. An aqueous solution of cross linker is prepared in milli Q water. The aqueous solution of cross linker is then mixed with aqueous solution of bromelain, emulsifier and chitosan, with continuous stirring for 2 h at 1000 rpm at room temperature until translucent nanoparticulate dispersion developed. The translucent nanoparticulate dispersion is sonicated for 10 -40 minutes to obtain a nanodispersion. The nanodispersion is centrifuged at 15,000 rpm for 45 min at 4 ºC to obtain pellets of nanoparticles. The supernatant is collected and evaluated for bromelain encapsulation efficiency.
The pellets of nanoparticles are dispersed in enteric coating solution (2.0 - 3.0 % w/v) under continuous stirring for 1 h followed by centrifugation at 15,000 rpm for 30 min to obtain enteric coated pellets of the nanoparticles. The enteric coated pellets are dispersed in a cryoprotectant followed by lyophilization process under a working pressure of 0.07 bar at -80 °C for 48 h to obtain the enteric coated bromelain chitosan nanoparticles.
In one embodiment the emulsifier is lecithin, cross linker is tripolyphosphate (TPP) and the enteric coating is Eudragit.
The Cryoprotectant is selected from mannitol, ethylene glycol, propylene glycol, glycerol and DMSO.
In one embodiment the buffer is selected from tris-HCl buffer, phosphate buffer, acetate buffer, MES buffer.
In one embodiment the buffer is in particular the tris-HCl buffer.
In one embodiment the enteric coated Bromelain nanoparticles (Eu-Br-CNPs) are prepared by a process comprising:
(a) Mixing aqueous chitosan solution with aqueous solution of bromelain and lecithin,
(b) Adding a buffer to the resultant solution of step (a) obtain a mixture having pH 5-6,
(c) Adding aqueous solution of TPP to the mixture of step (b) with continuous stirring for 1-3 hr to obtain a translucent dispersion,
(d) Sonicating the dispersion of step (c) for 10 -40 minutes to obtain nanodispersion,
(e) Centrifuging the nano-dispersion to obtain nanoparticles (Br-CNPs),
(f) dispersing Br-CNPs dispersion in a coating solution of Eudragit with continuous stirring for 1 h followed by centrifuging at 10,000-20,000 rpm for 15-45 min to obtain pellets,
(h) dispersing the pellets in mannitol solution (10% w/v) followed by lyophilization process to obtain the enteric coated bromelain chitosan nanoparticles (Eu-Br-CNPs).

In one embodiment the eudragit is Eudragit L100.

Examples:
Example 1: Preparation of Enteric coated Bromelain nanoparticles (Eu-Br-CNPs)
Chitosan solution was mixed with aqueous solution of Bromelain and soya lecithin in tris-Hcl buffer. Subsequently, aqueous solution TPP (cross-linker) was added with continuous stirring for 2 h at 1000 rpm at room temperature until translucent nanoparticulate dispersion developed. The resulting dispersion was sonicated utilizing probe sonicator for 30 minutes (20 W power, 30 % amplitude with 20 sec on and 10 sec off cycle) to obtain nanodispersion followed by centrifugation at 15,000 rpm for 45 min at 4 ºC. Supernatant collected was evaluated for bromelain encapsulation efficiency. Pellet of nanoparticles (Br-CNPs) collected was dispersed in distilled water consisting mannitol (10 % w/v) as cryoprotectant and lyophilized under vacuum at working pressure of 0.07 bar at -80 °C for 48 h. Various batches of Br-CNPs were prepared to optimize various formulation variables such as amount of chitosan (53.33 – 61.53 % w/w), TPP concentration (7.69 – 20 % w/w), lecithin: drug (1:2 to 2:1), chitosan : drug (2.67:1 to 8:1) respectively as well as process variables such as stirring time (1- 3 h) respectively. Br-CNPs were dispersed in dispersion in Eudragit L 100 coating solution (2.0 - 3.0 % w/v) under continuous stirring for 1 h. Pellet of Eu-Br-CNPs collected by centrifuging the dispersion at 15,000 rpm for 30 min was dispersed in mannitol (10 % w/v) solution followed by lyophilization process under a working pressure of 0.07 bar at -80 °C for 48 h. Optimized formulation was stored in air tight container at 4 ºC and utilized for further studies.
Example 2: Conformational stability studies in simulated gastro-intestinal fluid
The fluorescence spectrum of pure bromelain and bromelain released from optimized formulation was compared to confirm the conformational integrity of tertiary structure of protein (Figure 1). The ?max of pure bromelain solution in distilled water was 343 nm with fluorescence intensity of 480.66±14.57 a.u. corresponding to emission chromophores such as tryptophan and tyrosine. However, slight shift in ?max to 370 nm was observed for pure bromelain incubated at gastric pH 1.2. The red shift in ?max with lower fluorescence intensity (272.66±13.50 a.u.) for pure bromelain might be due to conformational changes in the vicinity of tryptophan surface indicating denaturation of bromelain in acidic environment. Bromelain released from optimized formulation incubated at gastric pH 1.2, small intestine pH 6.8 and colon pH 7.4 showed ?max at 351 nm with fluorescence intensity 449.33±12.58 a.u., 452.33±15.30 a.u. and 471.66±13.05 a.u. respectively. The results indicated that pH conditions had no significant influence on aromatic chromophore of encapsulated bromelain. This too confirmed that vesicular system provided structural protection to bromelain in acidic milieu to maintain its tertiary structure as well as proteolytic activity. The superimposed fluorescence spectra of drug and optimized formulation characteristic of tertiary structure additionally supported adequacy of method and formulation as a carrier for bromelain (Figure 1).
Example 3: Dissolution studies
Initial drug release ~9 % was observed from Eu-Br-CNPs at pH 1.2 in 2 h (Figure 2). The diffusion of dissolution fluid from minor cracks formed over the surface of particles during lyophilization might have dissolved the drug adsorbed over the surface of nanoparticles as well as enabled the ionization of chitosan matrix to facilitate drug release. With progression of pH, optimized formulation facilitated prompt drug release ~40% at intestinal pH 6.8 followed by controlled extended release at colonic pH 7.4 over a period of 24 h (Figure 2). The ionization and dissolution of Eudragit L 100 coat might have attributed to rapid drug release during initial period whereas slow and prolonged drug release due to swelling of cross-linked chitosan matrix respectively. The drug release data remarkably presented targeted drug delivery to intestine in controlled sustained manner from optimized formulation.
The drug release data showed best fit to Higuchi model which can be confirmed by estimating regression coefficients (R2) of zero order (0.872), first order (0.733), Higuchi (0.961) and Korsmeyaer-Peppas (0.920) model. The numerical value of release exponent (n=0.81) i.e., 0.45 0.85 indicated that drug release was initially by diffusion followed by erosion.
Example 4: Mucoadhesive study
The mucoadhesive strength of CNPs, Br-CNPs and Eu-Br-CNPs was determined by estimating their mucin binding efficiency (Figure 3 A). The positive zeta potential, bioadhesive nature of CS and nano size of CNPs might be responsible for high mucin binding. However, mucoadhesive strength of bromelain loaded-CNPs was low indicating that amount of chitosan accessible for interaction with mucin was less. In addition, increase in particle size with drug loading might have reduced the specific surface area for mucin adsorption correspondingly led to decrement in mucoadhesive strength. The anionic nature of Eudragit L 100 coat in Eu-Br-CNPs might have hindered the interaction of chitosan with mucin and reduced the mucoadhesive strength of Eu-Br-CNPs compared to Br-CNPs. In vitro results of mucoadhesion study indicated that Eu-Br-CNPs would exhibit significant mucoadhesive nature in colon after dissolution of Eudragit L 100 coat similar to Br-CNPs.
The increase in particle size and PDI of nanoparticles was observed on addition of nanoparticles in mucin which clearly indicated the interaction among them (Figure 3 B, C and D). In addition, decrease in zeta potential was observed in order of EU-Br-CNPs < Br-CNPs < CNPs demonstrated the binding of negatively charged sialic acid residues of mucin to surface of nanoparticles. Significant lowering of zeta potential was observed for CNPs and Br-CNPs compared to Eu-Br-CNPs with relatively higher increase in particle size and PDI confirming their higher mucoadhesive nature.
Example 5: Stability studies
The capacity of Eu-Br-CNPs to sustain colloidal properties, drug release behavior and bromelain activity was investigated under cool temperature (4±2 ºC/ 65±5% RH) and room temperature (25±5 ºC/ 60±5% RH) for 12 months while for 6 months at accelerated temperature storage conditions (40±2ºC/ 75±5 % RH). Simultaneously, effect of different storage conditions on pure drug was also determined. Pure drug showed significant decrease in active drug content under room and accelerated temperature storage conditions respectively compared to Eu-Br-CNPs. However, pure drug and formulation showed 96.85±0.79% and 97.79±0.46% active drug content under cool conditions respectively indicating that proteolytic activity of drug was protected at lower temperature. Bromelain followed first order degradation kinetics. Higher Kcal (8.43 x 10-4 days-1) and lower t90 (125 days) value for pure drug demonstrated faster degradation rate compared to Eu-Br-CNPs (Kcal 2.17 x 10-4 days-1, t90 482 days) under real time stability conditions. The calculated t90 (days) was ~3.85-fold higher for formulation than pure drug at room temperature manifesting significantly better stability of formulation. The visual changes in color of pure drug from off white to brown was observed at both room and accelerated temperature storage condition respectively whereas no color change was observed for Eu-Br-CNPs.
Example 6: Cytotoxicity study (Ex-vivo)
Therapeutic potential of bromelain and Eu-Br-CNPs for impeding proliferation of cancer cells was estimated against human HCT-116 colorectal cancer cells at different concentration using MTT assay. Both bromelain and optimized formulation exhibited dose dependent cytotoxic effects on HCT-116 cells. The IC50 value of bromelain, Eu-Br-CNPs and 5-FU was found to be 26.80, 14.71, and 19.10 ?g/ml respectively (Figure 4). The reduction in IC50 value confirmed the enhanced ability of optimized formulation to inhibit the cancer cell growth than pure drug. The enhanced cytotoxic effect of formulation over the same dose range as pure drug might be attributed to mucoadhesive properties, sustained drug release behavior, phagocytic uptake and membrane infiltration of nanoparticles (Figure 4).
Example 7: Cellular uptake of nanoparticles
HCT-116 cells seeded in 6-well plate (1 x 105 cells/well) were treated with the fluorescent dye (Rhodamine B) loaded chitosan nanoparticles for 6 h and the untreated cells served as control. Afterwards, cells were washed with buffer solution (pH 7.4), fixed with 4% w/v paraformaldehyde and subsequently labeled with DAPI dye and visualized under fluorescence microscope (FLoid cell imaging station, Life Technologies, USA) to determine the uptake of nanoparticles. The uptake of nanoparticles by HCT-116 cells was evaluated to predict the absorption of an orally administered drug. The results of study showed that cells treated with fluorescent Rhodamine B loaded chitosan nanoparticles emitted a strong fluorescence compared to free dye treated cells after 6 h confirming the significant internalization and confinement of nanoparticles inside the cell cytoplasm (Figure 5). This might be attributed to increased paracellular, transcellular and phagocytic uptake of nanosized carrier system along with mucoadhesion of nanoparticles to cell surface contributing to extend the period of exposure of cells to drug which provide an additional benefit for treatment of cancer.
Example 8: Pharmacokinetics studies (In vivo)
Figure 6 A represent plasma concentration- time profile of pure drug and optimized Eu-Br-CNPs following oral administration in wistar rats respectively. The considerable increase in Cmax (3.09-fold) and Tmax (4-fold) was observed after single dose administration of Eu-Br-CNPs compared to pure bromelain. The prevention of premature leakage of drug in upper GIT and protection provide by polymeric carrier system from gastric milieu might have contributed to attain higher Cmax. Long Tmax indicated that Eudragit coated chitosan nanoparticles effectively delivered the drug in intestinal region and absorbed thereof. Furthermore, slower degradation of chitosan by colonic enzymes might be contributing prolonged absorption of drug. In addition, improved MRT (2.26- fold) and half-life (2.59-fold) of Eu-Br-CNPs respectively indicated prolonged duration of drug absorption due to controlled drug release behavior of Eu-Br-CNPs contributing to decreased drug clearance upon encapsulation. The significant improvement in AUC0–24h of Eu-Br-CNPs (~3.99-fold) as compared to bromelain via oral route might be contributed by paracellular and transcellular uptake respectively as well as prevention of pre-systemic elimination of drug from gastrointestinal tract by avoiding denaturation in gastric milieu and proteolytic degradation. The results confirmed the improved pharmacokinetic profile of bromelain via Eu-Br-CNPs carrier system.
Example 9: Colon distribution study
High Cmax of encapsulated bromelain in colonic tissue (3.23-fold) as compare to pure bromelain indicated the localized drug delivery in colonic site form coated chitosan nanoparticles. This might be contributed by nanosize of polymeric system facilitating higher uptake of formulation and their mucoadhesive nature. Additionally, it also provides suitable platform for localized delivery in colon. Moreover, improved half-life (2.00- fold) and MRT (1.73-fold) of formulation too confirmed sustained release behavior of nanocarrier system. The improved AUC0–24h of Eu-Br-CNPs (~6.49-fold) in colonic region compared to bromelain confirmed the localization of optimized formulation in colon. The colon tissue kinetics of optimized formulation confirmed more effective drug delivery to colon by generating a homogenous drug reservoir in colonic region which could elicit prolonged drug delivery at tumor site (Figure 6B).
Pharmacodynamic studies
Example 10: Disease activity index
The body weight loss, stool consistency and occult blood loss were the perceptible variables estimated for determination of disease activity index (DAI). The severity of chronic intestinal inflammation was directly related to the value of DAI score. The reduction in body weight and stool consistency along with enhanced occult blood loss increased the DAI score (Figure 7A). DMH + DSS treatment led to noticeable reduction in average body weight, loosening of fecal matter and severe occult blood loss. The results confirmed significant increment in DAI score of experimental control in comparison to naïve control. However, animals treated with drug, standard drug and optimized formulation showed a significant improvement in average body weight gain, stool consistency and occult blood loss after 13 weeks of treatment compared to control group respectively. No significant variation in DAI score was observed in standard drug and formulation treated group respectively. However, formulation showed significant lowering of DAI score compared to pure drug (p<0.05). The ability of formulation to reduce DAI score might be contributed to its stability in gastric milieu, sustained drug delivery nature, paracellular uptake of nanoparticles as well as improved therapeutic value of bromelain. Additionally, tissue distribution study also confirmed the localized delivery of formulation and achieved higher amount of drug at the site of action which might have significantly reduced the DAI score and confirmed effectiveness of Eu-Br-CNPs.
Example 11: Tumor burden
Rectal swelling, aberrant crypt foci formation, tumor formation and tumor multiplicity was observed in DMH-DSS treated animals. DMH-DSS treatment had significantly elevated the formation of multiple lesions in proximal, middle and distal region of colon compared to naïve control group. Lesions were counted and tabulated. Pure drug, 5-FU and optimized formulation significantly reduced the tumor development and progression in DMH+DSS induced colorectal cancer associated with colitis (Figure 7 B)..
Example 12: Hematological profile
The remarkable changes in hematological profile of DMH+DSS intoxicated animals were observed compared to naïve animals. DMH+DSS intoxication induced significant decrement in level of hemoglobin and RBCs while augmented level of leukocyte count, lymphocytes, platelets and neutrophils respectively (Figure 8). The elevated levels of oxidative stress after DMH administration might have promoted oxidative degradation of erythrocytes which facilitated release of their content in blood, reduced RBC count and interfered with erythrocyte function. In addition available literature has reported that DMH deplete hemoglobin level by inhibiting RBC production and knocking down erythrocyte in hemopoietic organs. These events upsurge the immune response with increment in total leukocytes count associated with the inflammatory conditions due to induction of colon cancer. Increased platelet count too ensured the infiltration of leukocytes to inflammatory site of colon (Figure 8).
The progress toward the normal level of hematological values was observed following the course of therapy with pure drug, 5-FU and optimized formulation respectively. Optimized formulation significantly improved hemoglobin and RBCs count with reduction in the level of neutrophils, total leukocyte count, lymphocytes and platelets compared to 5-FU and pure drug group (p<0.05).
Subsequently, elevation in serum SGOT and SGPT level was observed in DMH intoxicated animals (Figure 8). Elevated SGOT and SGPT level indicated alteration in transport function and membrane permeability of hepatocytes along with leakage of these cytoplasmic enzymes from hepatocytes into blood stream confirming hepatotoxicity due to DMH and its active metabolite synthesized in liver. However, bromelain treatment reduced SGOT and SGPT level indicating its hepatoprotective potential. The remarkable restoration of SGOT and SGPT levels to normal was observed with Eu-Br-CNPs treatment confirming the healing of hepatic cells by reducing the free radical mediated oxidative stress.
The notable rise in expression of CEA protein, a colorectal cancer-specific marker level in serum was observed in DMH-DSS group in comparison to naïve control (p<0.05) indicating cancerous status of tissue. Usually, serum CEA concentration is sparse in a healthy individual but significantly elevate with development of cancer. DMH in association with DSS generates a cascade of event by disrupting the antioxidant balance of colon tissues followed by inflammation, mutation and genesis of tumor in tissues. The developed microtumors elevated the CEA content which was significantly diminished by bromelain and optimized formulation treatment due to anti-oxidant, anti-inflammatory and anti-cancer properties of bromelain respectively (Figure 8).
The protection provided by nanocarrier system to bromelain from gastric milieu and proteolytic enzymes enhanced paracellular and transcelluar uptake from inflamed colon cells and sustained drug release behavior of Eu-Br-CNPs had significantly potentiated hepatoprotective, anti-oxidant, anti-inflammatory and anti-cancer properties of bromelain respectively compared to pure drug (Figure 8).
Example 13: Immunological estimation
DMH+DSS intoxiaction significantly elevated the level of inflammatory markers i.e. TNF-a, IL-6 due to sensitization of receptors in macrophages. In addition, reduced level of anti-inflammatory cytokine IL-10 and caspase-3 activity in DMH+DSS control group confirmed the inflammatory condition in control group. Since IL-10 is an immunoregulatory Th2 cell cytokine inhibiting the production of inflammatory mediators by Th1 cells. Contrarily, treatment groups such as pure bromelain, 5-FU and formulation down-regulated the enhanced expression of TNF-a and IL-6 respectively (p<0.05) (Figure 9 A and B). Bromelain's inhibitory effect on release of various inflammatory mediators like TNF-a and IL-6 by inhibiting NF-kB overexpression as well as improving expression of Nrf2 pathway has contributed to its anti-inflammatory activity. Significant elevation in IL-10 level was observed in bromelain, 5-FU and optimized formulation treated groups (p<0.05). The results indicated that bromelain promoted IL-10 production by promoting Th2 cells (Figure 9 C). Furthermore, IL-10 harmonizes the pro-inflammatory response by down-regulating major histocompatibility complex class II expression, oxygen radical production, NO synthesis, and intercellular adhesion molecule (ICAM)-1 expression. These properties make IL-10 a critical factor in regulation of magnitude of inflammatory response and in prevention of an overwhelming response to inflammation.
Moreover, caspase-3 is important molecule in apoptotic pathways playing key role in cancer development and progression. The reduced caspase-3 activity particularly down-regulate the apoptosis process in DMH+DSS control group (Figure 9 D). While the improved caspase-3 activities in pure drug and optimized formulation treated groups are a sign of apoptosis and a positive indicator of their efficacy in cancer treatment (Figure 9 D). The unique characteristics of Eu-Br-CNPs i.e., nanosize promoting paracellular, transcellular and phagocytic uptake, site specific colonic delivery, sustained and controlled drug release behavior at colonic tumor site reduced inflammation associated carcinogenic changes in colon due to improved anti-inflammatory and anti-oxidant potential of bromelain. Thus, it can be concluded that enteric nano-formulation of bromelain (Eu-Br-CNPs) significantly hampered the cascade of events induced by DMH+DSS by inhibiting cytokine storm and molecular markers throughout the onset and progression of malignancy (Figure 9).
Benefits of the invention
• Eu-Br-CNPs showed pH responsive mucoadhesive behavior. pH responsiveness provided by Eudragit L 100 coat imparted colon targeting potential to Eu-Br-CNPs. In addition, presence of crosslinked chitosan core provided prolonged residence time of nanoparticles in colon due to its mucoadhesive nature.
• Present invention provides thorough information of optimization of formulation and three dimensional stability of bromelain incorporated in Eu-Br-CNPs.
• Eu-Br-CNPs exhibited enhanced biocompatibility of nanocarrier system due to presence of natural chitosan polymeric core. The Eudragit coating further stabilizes the nanoparticles, protecting the payload from degradation, and improving therapeutic outcomes.
• The results of present invention correlate well the formulation optimization (via physicochemical optimization of their particle size, PDI, zeta potential and entrapment efficiency), in vitro gastric stability, ex-vivo cellular uptake and anti-cancer potential with its pharmacokinetic, tissue distribution and pharmacodynamic profile.
• The stability in gastric milieu, colon targeted prolonged drug release behavior, higher intracellular uptake, higher bioavailability and availability of drug in colon tissues as well as reduction of development of tumor burden, disease activity index, cancer specific haematological and immunological markers confirmed the therapeutic potential of Eu-Br-CNPs.
, Claims:We claim
1. Enteric coated mucoadhesive bromelain nanoparticles comprising bromelain, chitosan, tripolyphosphate, lecithin and Eudragit polymer.
2. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein chitosan is present in an amount of 53.33 – 61.53 % w/w, bromelain is present in an amount of 7.69 – 20 % w/w, lecithin is present in an amount of 7.69 – 20 % w/w and Eudragit is present in an amount of 2-3 % w/v.
3. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein ratio of lecithin: bromelain is between 1:5 to 5:1.
4. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein ratio of chitosan: bromelain is 4:1 to 10:1.
5. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 3, wherein ratio of lecithin: bromelain is between 1:2 to 2:1.
6. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the Eudragit polymer is Eudragit L 100.
7. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a particle size of 200 to 800 nm.
8. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a zeta potential of -40 to 40 mV.
9. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the said nanoparticles have a poly dispersibility index of 0.2- 0.7.
10. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein the nanoparticle comprises bromelain, chitosan, tripolyphosphate, lecithin and Eudragit L-100.
11. The enteric coated mucoadhesive bromelain nanoparticles as claimed in claim 1, wherein chitosan is present in an amount of 57.14 % w/w, bromelain is present in an amount of 14.29 % w/w, tripolyphosphate is present in an amount of 14.29 % w/w, lecithin is present in an amount of 14.29 % w/w coated with Eudragit L-100.
12. A process for preparing enteric coated mucoadhesive bromelain nanoparticles as claimed in any of the preceding claims, said process comprising:
(a) Mixing aqueous solution of chitosan with aqueous solution of bromelain and lecithin,
(b) Adding a buffer to the mixture of step (a) to obtain a mixture having pH 5-6,
(c) Adding aqueous solution of tripolyphosphate to the mixture of step (b) with continuous stirring for 1-3 h to obtain a translucent dispersion,
(d) Sonicating the dispersion of step (c) for 10 - 40 minutes to obtain nanodispersion, followed by centrifuging the nanodispersion to obtain nanoparticles,
(e) dispersing nanoparticles of step (d) in Eudragit L 100 coating solution (2.0 - 3.0 % w/v) under continuous stirring for 1 h to obtain dispersion,
(f) centrifuging the dispersion of step (e) at 15,000 rpm for 30 min to obtain pellets of nanoparticles.
13. An anticancer pharmaceutical composition comprising bromelain nanoparticles as claimed in claim 1.