Selective isolation of hydrophobin SC3 by solid-phase extraction with polytetrafluoroethylene microparticles and subsequent mass spectrometric analysis.

Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

Poly(N-Isopropylacrylamide)-co-Acrylamide Hydrogels for the Controlled Release of Bromelain from Agroindustrial Residues of Ananas comosus.

This works reports the purification of bromelain extracted from Ananas comosus industrial residues by ethanol purification, its partial characterization from the crude extract as well as the ethanol purified enzyme, and its application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels. Bromelain was recovered within the 30-70 % ethanol fraction, which achieved a purification factor of 3.12-fold, and yielded more than 90 % of its initial activity. The resulting purified bromelain contained more than 360 U · mg(-1), with a maximum working temperature of 60 °C and pH of 8.0. Poly(N-isopropylacrylamide)-co-acrylamide hydrogels presented a swelling rate of 125 %, which was capable of loading 56 % of bromelain from the solution, and was able to release up to 91 % of the retained bromelain. Ethanol precipitation is suitable for bromelain recovery and application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels based on its processing time and the applied ethanol prices.

Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement.

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

Polymer-based alternative method to extract bromelain from pineapple peel waste.

Bromelain is a mixture of proteolytic enzymes present in all tissues of the pineapple (Ananas comosus Merr.), and it is known for its clinical therapeutic applications, food processing, and as a dietary supplement. The use of pineapple waste for bromelain extraction is interesting from both an environmental and a commercial point of view, because the protease has relevant clinical potential. We aimed to study the optimization of bromelain extraction from pineapple waste, using the aqueous two-phase system formed by polyethylene glycol (PEG) and poly(acrylic acid). In this work, bromelain partitioned preferentially to the top/PEG-rich phase and, in the best condition, achieved a yield of 335.27% with a purification factor of 25.78. The statistical analysis showed that all variables analyzed were significant to the process.

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate).

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.

A biophysical strategy to examine the impact of newly synthesized polymerizable ammonium-based ionic liquids on the structural stability and proteolytic activity of stem bromelain.

The present manuscript reports the synthesis and characterization of polymerizable ammonium-based ionic liquids (ILs) and explores their influence on structural stability of stem bromelain (BMN) by using various biophysical techniques. Thermal fluorescence results showed that N-ethyl-2-(methylacryloyloxy)-N,N-dimethylethan-1-ammonium bromide (IL2C) (at lower concentration (0.1 mg/mL)) is found to be increasing the thermal stability of BMN which can be evident from the transition temperature (Tm) for BMN in IL2C (68.51 °C) is higher than BMN in buffer (66.24 °C). Whereas, N-(2-(methacryloyloxy)ethyl)-N,N-dimethylpropan-1-ammonium bromide (IL3C) and N-(2-(methacryloyloxy)ethyl)-N,N-dimethylpentan-1-ammonium bromide (IL5C) are maintaining the Tm values very near to Tm of pure BMN. Though, N-(2-(methacryloyloxy)ethyl)-N,N-dimethylhexan-1-ammonium bromide (IL6C) is found to be destabilizing IL as it significantly decreased the Tm value of BMN at all concentrations. Additionally, consequence of ILs on the proteolytic activity of BMN has also performed for IL2C up to 5 mg/mL while IL3C and IL5C at 0.1 mg/mL and 0.5 mg/mL are enhancing the proteolytic activity of BMN. Later, molecular docking studies are also performed with AutoDock vina and results showed that all ILs have different binding sites on BMN however IL6C is observed to be binding to the catalytic site of the BMN, it turns out to be the most destabilizing IL.

Bromelain immobilization in cellulose triacetate nanofiber membranes from sugarcane bagasse by electrospinning technique.

Cellulose triacetate (CTAB) synthesized by cellulose extracted from sugarcane bagasse, and commercial cellulose acetate (CA) were used to produce nanofiber membranes contained bromelain by electrospinning technique. About 1.3 g of cellulose acetate per gram of bagasse were obtained, and both CTAB and CA was characterized by analysis of Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC). The nanofiber membranes were produced by electrospinning process testing the following conditions: voltage 25 kV, flow rate 4 mL/h and distance 10 cm, using acetone/ dimethylformamide (DMF) (85:15 m/ m) to 15% cellulose triacetate (70% CA + 30% CTAB) or CA solutions. Scanning Electron Microscopy (SEM) was used to nanofiber membranes characterization. Bromelain was immobilized on the nanofiber membranes by crosslinking with glutaraldehyde and directly in the electrospinning step, the highest activity recovery was about 675% and in vitro controlled release tests were performed to semi-quantitatively evaluate the release of the enzyme bromelain thus demonstrating complete release process in 3 days.

Mussel-inspired polydopamine-assisted bromelain immobilization onto electrospun fibrous membrane for potential application as wound dressing.

There has been a recent increase in research interest regarding the development of wound dressings containing bioactive compounds capable of improving outcomes for complex healing needs. In the present study, we describe the generation of bromelain immobilized eletrospun poly(ε-caprolactone) (PCL) fibers (BrPDA-PCL fibers) using the dopamine-assisted co-deposition strategy. We wanted to combine the structural advantage of electrospun fiber and the activity of bromelain and PDA to develop functional wound dressings. We found that bromelain activity could be better stabilized when via its immobilization on electrospun fibers. The resultant BrPDA-PCL fibers exhibited promising properties including optimal mechanical stability, wettability, and rates of water vapor transmission. In addition, these BrPDA-PCL fibers were biocompatible, allowing for effective cellular adhesion and proliferation. The results of zone of inhibition testing further confirmed that these fibers achieved effective antibacterial activity against Escherichia coli and Staphylococcus aureus. When used in vivo, as compared with PCL fibers or control animals the BrPDA-PCL fibers enhanced wound healing rates while reducing associated inflammation. As such, these results indicate that these biocompatible BrPDA-PCL fibers exhibit desirable physicochemical properties making them ideal for use as a wound dressing to enhance the repair of full-thickness wounds to the skin.

How do biological stimuli modulate conformational changes of biomedical thermoresponsive polymer?

Continuing efforts to develop stimuli-responsive polymers (SRPs) as novel smart materials/biomaterials are anticipated to upgrade the quality life of humans. The details of the molecular, physico chemical and biophysical interactions between SRPs and proteins are not fully understood. Indeed, protein - polymer interactions play a major role in a wide range of biomedical/biomaterial applications. In this regard, we have demonstrated the influence of proteins (ß-lactoglobulin (BLG) and stem bromelain (BM) as biological stimuli) on the phase transition behavior of biomedical thermoresponsive poly(N-isopropylacrylamide) (PNIPAM). In order to predict these, we have used a set of biophysical techniques to unveil the influence of biological stimuli on the phase transition behavior of PNIPAM. Absorption spectroscopy, steady-state fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM) were operated at room temperature to examine the changes in absorbance, fluorescence intensity, molecular interactions and surface morphologies, respectively. Furthermore, temperature dependent fluorescence spectroscopy and dynamic light scattering (DLS) studies were also performed to analyze conformational changes, agglomeration behavior, particle size, coil to globule transition and phase behavior. The significant variations obtained in the phase transition temperature values, conformational changes and agglomeration behavior clearly reflects the different molecular interplay induced in presence of biological stimuli. The results demonstrated that the added proteins act as biological stimuli via preferential interactions between the amide group of the polymer and water molecules. The present study can be useful for the design and development of the next generation smart responsive materials/biomaterials.

Does macromolecular crowding compatible with enzyme stem bromelain structure and stability?

It is essential to explore the impact of macromolecular crowding on protein in order to understand the behavior of the protein in the biological system. In this context, the consequences of macromolecular crowding on stem bromelain (BM) are explored, which is important for its industrial application. Herein, the effect of dextran (D70) and polyethylene glycol (P12 and P20) on native BM structure are investigated. The effect of crowder molecules on BM are deduced by combining the results of absorption, circular dichroism, fluorescence and activity studies. Additionally, molecular level interactions are investigated with molecular docking studies. Our results display that BM acts differently in higher and lower concentrations of crowding agents. Furthermore, crowding leads to alteration of protein structure and activity as compared to the dilute solutions. We observed that D70, is a very effective thermal stabilizing agent, while P12 is moderately stabilizing, on the other hand, P20 is destabilizing the BM structure. In the present study, the overall effect of crowders was destabilizing and deactivating on enzyme. Therefore, this study provides yet another example where soft interaction is dominating over the excluded volume effect.

Amphiphilic bromelain-synthesized oligo-phenylalanine grafted with methoxypolyethylene glycol possessing stabilizing thermo-responsive emulsion properties.

A thermo-responsive amphiphile was developed from oligo-phenylalanine [oligo(Phe)]. The hydrophobic moiety of the amphiphile, oligo(Phe) was synthesized via reverse hydrolysis catalyzed by bromelain in dimethyl sulfoxide and dioxane solutions. The production of oligo(Phe) increased by 80.7% by screening suitable reaction conditions. The average degree of polymerization of oligo(Phe) was determined to be four by 1H NMR. By grafting with aldehyde-ended methoxypolyethylene glycol (mPEG), oligo(Phe) was converted to amphiphilic oligo(Phe)-mPEG. The surface tension of oligo(Phe)-mPEG solution increased with decreasing chain length of the mPEG moiety. Cytotoxicity studies showed oligo(Phe)-mPEGs are biocompatible. On varying temperature, a reversible phase transition of oligo(Phe)-mPEG solutions could be observed. N-octane-in-water emulsions and 0.5% beta-carotene containing squalene-in-water emulsions stabilized by oligo(Phe)-mPEGs occurred at 25 °C but de-emulsification took place at >40 °C. Emulsification could be restored once the separated mixture cooled and re-homogenized. The emulsification/de-emulsification cycling could be repeated many times. The time required for de-emulsification decreased with elevated temperature but increased with a reduced concentration of oligo(Phe)-mPEGs and a reduction in the chain length of the mPEG moiety.

Fabrication of a novel magnetic mesoporous molecularly imprinted polymer based on pericarpium granati-derived carrier for selective absorption of bromelain.

Bromelain, a cysteine endopeptidase enzyme of great commercial value, has been widely used in food, pharmaceutical, and cosmetic industries. Conventional methods for purification of bromelain are still limited by a low binding efficiency, time-consuming process, and expensive equipment. Therefore, for selective absorption of bromelain, we developed a facile and effective method to fabricate magnetic mesoporous molecularly imprinted polymers using pericarpium granati-derived carbon as the carrier for the first time. The characterizations of the imprinted polymers indicated that a polydopamine layer was coated on the surface of the carrier and the crystallinity of the carrier did not change. The obtained imprinted polymers exhibited favourable saturation magnetization, a high adsorption capacity of 135.96 mg g-1, a fast equilibrium time, and satisfactory reusability. The imprinted polymers were prepared by an eco-friendly method and exhibited rapid separation and good adsorption performance, thus making the method applicable to biomacromolecular separation, proteomic analysis, and biomedical research.

Effect of polyethylene glycols on the function and structure of thiol proteases.

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.

Thermosensitive polymers: synthesis, characterization, and delivery of proteins.

Three triblock copolymers based on the poly(lactide) or poly(lactide-co-glycolide) and poly(ethylene glycol) or poly(ethylene oxide) blocks were synthesized and characterized. The weight average molecular weight and number average molecular weight were determined by gel permeation chromatography and proton nuclear magnetic resonance spectroscopy, respectively. Fourier transform infrared spectroscopy was used to determine the completion of synthesis of polymers. Thermoreversible sol-gel transition temperature and concentration were determined by an inverted tube method. Two formulations each of three synthesized polymers containing 5% (w/v) of lysozyme or bromelain but differing in polymer concentrations (20-30%, w/v) were prepared and studied for in vitro release of the incorporated protein. In vitro biocompatibility of the delivery systems was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Biological activities of lysozyme and bromelain were determined by enzyme activity assays. Critical gelling concentrations were found in the range of 20-30% (w/v). In vitro biocompatibility study showed that all the formulations were biocompatible. Increasing the polymer concentration led to a decrease in burst release and extended the in vitro release of proteins. Furthermore, biological activities of lysozyme and bromelain in released samples were found to be significantly (p<0.05) greater in comparison to the control. Thus, the above thermosensitive polymers were able to deliver proteins in biologically active forms at a controlled rate for 2-8 weeks.

Multiple polysaccharide-drug complex-loaded liposomes: A unique strategy in drug loading and cancer targeting.

In the present study, a unique strategy was developed to develop nanocarriers containing multiple therapeutics with controlled release characteristics. In this study, we demonstrated the synthesis of dextran sulfate-doxorubicin (DS-DOX) and alginate-cisplatin (AL-CIS) polymer-drug complexes to produce a transferrin ligand-conjugated liposome. The targeted nanoparticles (TL-DDAC) were nano-sized and spherical. The targeted liposome exhibited a specific receptor-mediated endocytic uptake in cancer cells. The enhanced cellular uptake of TL-DDAC resulted in a significantly better anticancer effect in resistant and sensitive breast cancer cells compared to that of the free drugs. Specifically, DOX and CIS at a molar ratio of 1:1 exhibited better therapeutic performance compared to that of other combinations. The combination of an anthracycline-based topoisomerase II inhibitor (DOX) and a platinum compound (CIS) resulted in significantly higher cell apoptosis (early and late) in both types of cancer cells. In conclusion, treatment with DS-DOX and AL-CIS based combination liposomes modified with transferrin (TL-DDAC) was an effective cancer treatment strategy. Further investigation in clinically relevant animal models is warranted to prove the therapeutic efficacy of this unique strategy.

Development of an Economical Method to Reduce the Extractable Latex Protein Levels in Finished Dipped Rubber Products.

Natural rubber latex (NRL) allergy is caused by the extractable latex proteins in dipped rubber products. It is a major concern for the consumers who are sensitive to the allergenic extractable proteins (EP) in products such as NRL gloves. Objective of this research was to develop an economical method to reduce the EP in finished dipped NRL products. In order to reduce the EP levels, two natural proteases, bromelain from pineapple and papain from papaya, were extracted and partially purified using (NH4)2SO4. According to the newly developed method, different glove samples were treated with a 5% solution of each partially purified enzyme, for 2 hours at 60°C. Residual amounts of in treated samples were quantified using the modified Lowry assay (ASTM D5712-10). Bromelain displayed a 54 (±11)% reduction of the EP from the dipped rubber products, whereas it was 58 (±8)% with papain. These results clearly indicate that the selected natural proteases, bromelain, and papain contribute significantly towards the reduction of the total EP in finished NRL products. Application of bromelain enzyme for the aforementioned purpose has not been reported up to date, whereas papain has been used to treat raw NRL towards reducing the EP.

Does 1-Allyl-3-methylimidazolium chloride Act as a Biocompatible Solvent for Stem Bromelain?

The broader scope of ILs in chemical sciences particularly in pharmaceutical, bioanalytical and many more applications is increasing day by day. Hitherto, a very less amount of research is available in the depiction of conformational stability, activity, and thermal stability of enzymes in the presence of ILs. In the present study, the perturbation in the structure, stability, and activity of stem bromelain (BM) has been observed in the presence of 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using various techniques. This is the first report in which the influence of [Amim][Cl] has been studied on the enzyme BM. Fluorescence spectroscopy has been utilized to map out the changes in the environment around tryptophan (Trp) residues of BM and also to discuss the variations in the thermal stability of BM as an outcome of its interaction with the IL at different concentrations. Further, the work delineates the denaturing effect of high concentration of IL on enzyme structure and activity. It dictates the fact that low concentrations (0.01-0.10 M) of [Amim][Cl] are only changing the structural arrangement of the protein without having harsh consequences on its activity and stability. However, high concentrations of IL proved to be totally devastating for both activity and stability of BM. The observed decrease in the stability of BM at high concentration may be due to the combined effect of cation and anion interactions with the protein residues. The present work is successful in dictating the probable mechanism of interaction between BM and [Amim][Cl]. These results can prove to be fruitful in the studies of enzymes in aqueous IL systems since the used IL is thermally stable and nonvolatile in nature thereby providing a pathway of alteration in the activity of enzymes in potentially green systems.

Nanoparticles decorated with proteolytic enzymes, a promising strategy to overcome the mucus barrier.

The intestinal mucus gel layer represents a stumbling block for drug adsorption. This study is aimed to formulate a nanoparticulate system able to overcome this barrier by cleaving locally the glycoprotein substructures of the mucus. Mucolytic enzymes such as papain (PAP) and bromelain (BRO) were covalently conjugated to poly(acrylic acid) (PAA). Nanoparticles (NPs) were then formulated via ionic gelation method and characterized by particle size, zeta potential, enzyme content and enzymatic activity. The NPs permeation quantified by rotating tube studies was correlated with changes in the mucus gel layer structure determined by pulsed-gradient-spin-echo NMR (PGSE-NMR), small-angle neutron scattering (SANS) and spin-echo SANS (SESANS). PAP and BRO functionalized NPs had an average size in the range of 250 and 285 nm and a zeta potential that ranged between -6 and -5 mV. The enzyme content was 242 µg enzyme/mg for PAP modified NPs and 253 µg enzyme/mg for BRO modified NPs. The maintained enzymatic activity was 43% for PAP decorated NPs and 76% for BRO decorated NPs. The rotating tube technique revealed a better performance of BRO decorated NPs compared to PAA decorated NPs, with a 4.8-fold higher concentration of NPs in the inner slice of mucus. Addition of 0.5 wt% of enzyme functionalized NPs to 5 wt% intestinal mucin led to c.a. 2-fold increase in the mobility of the mucin as measured by PGSE-NMR indicative of a significant break-up of the structure of the mucin. SANS and SESANS measurements further revealed a change in structure of the intestinal mucus induced by the incorporation of the functionalized NPs mostly occurring at a length scale longer than 0.5 µm. Accordingly, BRO decorated NPs show higher potential than PAP functionalized NPs as mucus permeating drug delivery systems.

Bromelain nanoparticles protect against 7,12-dimethylbenz[a]anthracene induced skin carcinogenesis in mouse model.

Conventional cancer chemotherapy leads to severe side effects, which limits its use. Nanoparticles (NPs) based delivery systems offer an effective alternative. Several evidences highlight the importance of Bromelain (BL), a proteolytic enzyme, as an anti-tumor agent which however has been limited due to the requirement of high doses at the tumor site. Therefore, we illustrate the development of BL loaded poly (lactic-co-glycolic acid) NPs that show enhanced anti-tumor effects compared to free BL. The formulated NPs with a mean particle size of 130.4 ± 8.81 nm exhibited sustained release of BL. Subsequent investigation revealed enhanced anti-tumor ability of NPs in 2-stage skin tumorigenesis mice model. Reduction in average number of tumors (∼ 2.3 folds), delay in tumorigenesis (∼ 2 weeks), percent tumorigenesis (∼ 4 folds), and percent mortality rate as well as a reduction in the average tumor volume (∼ 2.5 folds) in mice as compared to free BL were observed. The NPs were found to be superior in exerting chemopreventive effects over chemotherapeutic effects at 10 fold reduced dose than free BL, validated by the enhanced ability of NPs (∼ 1.8 folds) to protect the DNA from induced damage. The effects were also supported by histopathological evaluations. NPs were also capable of modulating the expression of pro-apoptotic (P53, Bax) and anti-apoptotic (Bcl2) proteins. Therefore, our findings demonstrate that developed NPs formulation could be used to improve the efficacy of chemotherapy by exerting chemo-preventive effects against induced carcinogenesis at lower dosages.