Research Article

 

Codelivery of Nanosized Curcumin and Bioenhancer Using Acid Degradable Polymeric Nanoparticles Displayed Enhanced Anticancer Efficacy

 

Moorthi Chidambaram *, Kathiresan Krishnasamy

 

Department of Pharmacy, Annamalai University, Annamalai Nagar - 608 002, Chidambaram, Tamil Nadu, India.

 

* Corresponding author. E-mail: cmoorthitgodu@gmail.com

 

Received: Jan. 6, 2014; Accepted: Apr. 29, 2014; Published: June 15, 2014.

 

Citation: Moorthi Chidambaram and Kathiresan Krishnasamy. Codelivery  of Nanosized Curcumin  and Bioenhancer  using Acid Degradable Polymeric Nanoparticles Displayed Enhanced Anticancer Efficacy. Nano Biomed. Eng. 2014, 6(2), 47-59.

DOI: 10.5101/nbe.v6i2.p47-59.

 

Abstract

Curcumin, a functional food polyphenol reported to inhibit cancer cell proliferation, invasion, angiogenesis and metastasis through interaction with multiple molecular targets. However, the clinical usefulness of curcumin in the treatment of cancer is limited due to poor solubility in water at acidic and neutral pH, hydrolytic degradation in alkaline pH and metabolism in the liver and intestine, resulting in decreased or absence of therapeutic efficacy. Hence, the present study was aimed to overcome the limitations of curcumin in the treatment of cancer by codelivery of nanosized curcumin and bioenhancer using acid degradable polymeric nanoparticles. Modified nanoprecipitation method was used to prepare void, curcumin-piperine, curcumin-quercetin and curcumin-silibinin encapsulated polymeric nanoparticles. Prepared nanoformulations were evaluated for particle size, polydispersity index, zeta potential, surface morphology, drug content, encapsulation efficiency, drug loading, in- vitro release, stability at elevated storage conditions, toxicity on normal liver cells, anticancer activity on various cancer cell lines and on cancer induced rats. Prepared curcumin-bioenhancer encapsulated polymeric nanoparticles were (a) spherical in shape with size <100 nm and displayed excellent uniformity; (b) showed >95% release of curcumin and bioenhancers within 45 minutes in gastric fluid; (c) proved non-toxic to normal liver cells; (d) extremely stable at elevated storage conditions; and (e) demonstrated enhanced anticancer activity against various cancer cell lines and mammary cancer in rats than the pure curcumin. Study concludes that the prepared curcumin-bioenhancer encapsulated polymeric nanoformulations significantly overcome the limitations of curcumin in the treatment of cancer and synergistically enhance its anticancer activity. However, out of three polymeric nanoformulations, curcumin-silibinin polymeric nanoformulation showed superior anticancer activity.

 

Keywords: Curcumin; Piperine; Polymeric Nanoparticle; Quercetin; Silibinin

Introduction

Chemotherapy is the major remedial approach for the treatment of localized and metastasized cancer but suffers several limitations including poor aqueous solubility, severesy stemictoxicities, and drug resistance. Of all limitations, systemic toxicities pose a major concern [1, 2]. Hence, anticancer functional foods (i.e. biologically active compound) which are devoid of systemic toxicities at the therapeutic dose seem to be better alternative [3]. Curcumin (Fig. 1) is one such functional food, isolated from dried rhizome of turmeric (Curcuma longa Linn). [4, 5]. More than 3000 studies over last 60 years have revealed the multiple cellular targets of curcumin, which are responsible for various pharmacological ctivities including anticancer. Various cell based assays have reported that curcumin inhibit cancer cell proliferation, invasion, angiogenesis and metastasis through interaction with multiple molecular targets and also downregulates the expression of P-glycoprotein, multi-drug resistance protein and breast cancer resistance protein [6-9]. Moreover, curcumin has not shown any toxicity in humans up to 12000 mg/day and has been declared as ‘generally regarded as safe’ by United States Food and Drug Administration [10, 11]. However, the clinical efficacy of curcumin in the treatment of cancer is limited due to limited solubility, hydrolytic degradation, and metabolism via glucuronidation and sulfation leading to reduce the oral bioavailability resulting in decreased or absence of therapeutic efficacy of curcumin [12, 13]. Various pharmaceutical approaches have been tried to overcome the aqueous solubility of curcumin, which have shown significant improvement in oral bioavailability of curcumin. However, there were no reports of nanoparticulate drug delivery system that overcome the metabolism of curcumin. Bioenhancer such as piperine, quercetin and silibinin (Fig. 1) plays a significant role in preventing/minimizing the metabolism of curcumin in liver and intestine. Utilizing these bioenhancers along with curcumin as a dual drug loaded nanoparticles are hypothesized to overcome all the limitations of curcumin such as poor aqueous solubility, hydrolytic degradation, intestinal metabolism, hepatic metabolism and synergistically enhance the anticancer activity of curcumin [14-18]. Hence, the present study was aimed to overcome the limitations of curcumin in the treatment of cancer by codelivery of nanosized curcumin and bioenhancer using acid degradable polymeric nanoparticles.

 

 

Fig. 1  Illustrate the chemical structure of (a) Curcumin, (b) Piperine, (c) Quercetin and (4) Silibinin.

 

Experimental section

Material

Curcumin, Piperine, Quercetin, Silibinin, Poloxamer 188, Potassium Bromide, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, Sulphorhodamine B, and 7,12-Dimethylbenz(a) anthracene were obtained from Sigma Aldrich, India. Dulbecco’s modified eagle medium, Fetal bovine serum and β-cyclodextrin were obtained from Himedia, India. Poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) polymer was obtained from Degussa, India. Methanol, Propanol and Tris Base were obtained from Merck, India. Ethanol was obtained from Brampton, Canada. Uranyl Acetate was obtained from Electron Microscopy Sciences, India.

 

Preparation of void and curcumin-bioenhancer encapsulated acid degradable polymeric nanoparticles

Void , curcumin-pipe rine (Cur-Pi ), curcumin - quercetin (Cur-Qu) and curcumin-silibinin (Cur- Si) encapsulated poly(butyl methacrylate-co-(2- dimethylaminoethyl) methacrylate-co-methyl methacrylate) nanoparticles were prepared using nanoprecipitation method with slight modifications [19-22]. Briefly, about 250 mg of poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate- co-methyl methacrylate) polymer with or without 50 mg of various curcumin-bioenhancer combinations [i.e. (25 mg curcumin and 25 mg of piperine for Cur-Pi); (25 mg curcumin and 25 mg of quercetin for Cur-Qu); and (25 mg of curcumin and 25 mg of silibinin for Cur-Si)] were dissolved in 16 mL of 60% ethanol, which was transferred at once into 500 mL beaker containing 49 mL of distilled water, 97 mg of Poloxamer 188 and 54 mg of β-cyclodextrin under mechanical stirring (RQT-124A, Remi) at 520 rpm. Polymeric nanoparticles were formed spontaneously but the stirring process was continued for 1 hour and 20 minutes to aid the size reduction and to evaporate the residual solvent. Prepared polymeric nanoformulations were stored at room temperature for one month to identify any aggregation and post-formulation degradation.

 

Particle size, polydispersity index and zeta potential analysis

Prepared void and curcumin-bioenhancer encapsulated polymeric nanoformulations were diluted appropriately using ultra pure water (Milli-Q Academic, Milli-Pore). Diluted samples were loaded separately in a disposable zeta cell and measured the average particle size, polydispersity index (PDI) and zeta potential using Zetasizer (ZEN3600, Malvern).

 

Particle surface morphology analysis

Prepared void and curcumin-bioenhancer encapsulated polymeric nanoformulations were dropped onto Formvar-coated copper grids and air dried. The samples were then negatively stained with 1% uranyl acetate for 10 minutes and air dried again. The samples were then imaged using transmission electron microscope (H-7500, Hitachi).

 

Drug content estimation

Prepared curcumin-bioenhancer encapsulated polymeric nanoformulations were mixed with equal volume of methanol and sonicated (Ultrasonic cleaner, Lark) for 5 minutes followed by filtration through 0.22 µm membrane. Samples were analyzed in triplicate using the developed high performance liquid chromatographic (HPLC) methods [23-25] and the values were expressed as WTotal.

 

Encapsulation efficiency and drug loading estimation

Prepared curcumin-bioenhancer encapsulated polymeric nanoformulations were centrifuged using a cooling centrifuge (C-24, Remi) for 45 minutes at 19,000 rpm at -20°C and the supernatant was separated. To 1 mL of supernatant, an equal volume of methanol was added and sonicated (Ultrasonic cleaner, Lark) for 5 minutes followed by filtration through 0.22 µm membrane. Samples were analyzed in triplicate using the developed HPLC methods [23-25] and expressed as Wfree. Encapsulation efficiency (EE) was calculated using formula EE (%) = [(Wtotal - Wfree) / (Wtotal)] X 100. Drug loading (DL) was calculated using formula DL (%) = [(Wtotal - Wfree) / (Wpolymer)] X 100.

 

Fourier transform-infrared (FT-IR) spectral analysis

Prepared void and curcumin-bioenhancer encapsulated polymeric nanoformulations were loaded in FT-IR grade cell and scanned at moderate scanning speed between 4000-400 cm-1 using FT-IR Spectrometer (Nicolet iS5, Thermo Scientific). The generated signals were monitored and integrated using inbuilt Thermo Scientific OMNIC software.

 

In-vitro release kinetics study

Prepared curcumin-bioenhancer encapsulated polymeric nanoformulations (weight equivalent to 10 mg of drug) were introduced into 900 mL of simulated gastric fluid (pH 1.2). The medium was maintained at 37±0.5ºC at a rotation speed of 100 rpm. About 5 mL of samples were withdrawn at 5, 10, 20, 30, 45 and 60 minutes, which were replaced with an equal volume of temperature-equilibrated blank media. The samples were centrifuged using cooling centrifuge (C-24, Remi) for 5 minutes at 19,000 rpm at -20°C. The supernatant was separated and filtered using 0.22 µm membrane. The sample was analyzed using developed HPLC methods [23-25].

 

In-vitro toxicity of void polymeric nanoparticles

Prepared void polymeric nanoformulation was evaluated for cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on BRL3A rat liver cells (normal rat liver cells) [26, 27]. Briefly, prepared void polymeric nanoformulation and paclitaxel (positive control) was diluted with Dul be c c o’s m odi fi e d E a gl e m e di um (DME M) su p p l e m e n t e d wi t h 2 % i n a c t i v a t e d f e t a l b o v i n e serum (FBS) to obtain a stock solution of 5 mg/mL concentration, which was sterilized by filtration and finally centrifuged. Serial dilutions (1000, 500, 250, 125, 62.5 µg/mL) were made from the stock solution. About 0.1 mL of the diluted BRL3A cell suspension (approximately 10,000 cells) was added to each well of the 96-well plate. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µL of different concentrations of test drugs were added. The plates were then incubated at 37°C for 3 days in 5% CO2  atmosphere and microscopic examination was performed after every 24 hours interval. After 72 hours, the test drug solutions in the wells were discarded and 50 µL of MTT in phosphate buffered saline was added to each well. The plates were gently shaken and incubated for 3 hours at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 µL of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader (ELx800, Bio-Tek) at a wavelength of 540 nm. The percentage growth inhibition was calculated using formula GI (%) = [100 – (Mean optical density of test) / (Mean optical density of control)] X 100 and concentration of test drug needed to inhibit cell growth by 50% (CTC50) was generated from the dose-response curves. The experiments were performed in triplicate.

 

In-vivo acute and 28-day repeated dose oral toxicity study of curcumin-bioenhancer encapsulated polymeric nanoparticles

Animal experiments were performed as  per the protocol approved by the Institutional Animal Ethics Committee (160/1999/CPCSEA; Proposal Number 896). Healthy adult female Wistar albino rats (nulliparous; non-pregnant; weighing 250-350 gram) were housed in polypropylene cages layered with husk and maintained in a controlled room at a temperature (i.e. 22±3°C) and light (i.e. 12 hours light/dark cycle). Animals were allowed free access to water and standard pellet diet. In-vivo acute oral toxicity study was performed to evaluate the toxicities of single dose administration of prepared void and curcumin-bioenhancer encapsulated polymeric nanoformulations. Animals were randomly assigned to 5 groups, each containing 5 animals. Animals were withheld of food overnight prior to dosing and 3-4 hours after dosing but not the water. Following the period of fasting, animals were weighed and treated orally as follows (a) Animals in group 1 received water; (b) Animals in group 2 received void polymeric nanoformulation; (c) Animals in group 3 received curcumin-piperine encapsulated polymeric nanoformulation; (d) Animals in group 4 received curcumin-quercetin encapsulated polymeric nanoformulation; and (e) Animals in group 5 received curcumin-silibinin encapsulated polymeric nanoformulation. One animal in each group was gavaged once with respective doses (i.e. equivalent to 2000 mg/kg of body weight) and observed 48 hours for any death. Since there were no death, remaining four animals in each group were gavaged once with respective doses. Animals were observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours and daily thereafter, for a total of 14 days for clinical signs of toxicity or mortality. Daily observations include changes in skin, fur, eyes, mucus membrane (nasal), respiratory rate, circulatory signs (heart rate), autonomic effects (salivation, lacrimation, piloerection, urinary incontinence and defecation) and central nervous system (drowsiness, gait, tremors and convulsion). The 28-day repeated dose oral toxicity study was performed to evaluate the toxicities of continuous administration of the prepared void and curcumin- bioenhancer encapsulated polymeric nanoformulations. Animals were randomly assigned to 5 groups, each containing 5 animals. Animals were weighed and treated orally as follows (a) Animals in group 1 received water; (b) Animals in group 2 received void polymeric nanoformulation; (c) Animals in group 3 received curcumin-piperine encapsulated polymeric nanoformulation; (d) Animals in group 4 received curcumin-quercetin encapsulated polymeric nanoformulation; and (e) Animals in group 5 received curcumin-silibinin encapsulated polymeric nanoformulation. The animals were gavaged with respective doses (equivalent to 100 mg/kg of body wei ght ) for a peri od of 28 da ys. Ani m a l s we re observed periodically for changes in skin, fur, eyes, mucus membrane (nasal), respiratory rate, circulatory signs (heart rate), autonomic effects (salivation, lacrimation, perspiration, piloerection, urinary incontinence and defecation), central nervous system (drowsiness, gait, tremors and convulsion), body weight, food consumption and mortality for a total period of 28 days. At the end of the treatment period, the animals were bled from the retro orbital sinus for clinical pathology assessment, which included analysis of various haematology parameters and blood biochemistry parameters. Consequently the animals were sacrificed by cervical dislocation and necropsied to facilitate gross pathological examination of vital organs such as brain, liver, kidney and heart.

 

In-vitro anticancer activity of curcumin-bioenhancer encapsulated polymeric nanoparticles using MTT assay

Prepared curcumin-bioenhancer encapsulated polymeric nanoformulations were evaluated for anticancer activity using MTT assay on human breast adenocarcinoma (MCF-7) cells [26, 28]. Briefly, pure curcumin, prepared curcumin-bioenhancer e n c a p su l a t e d p o l ym e r i c n a n of o r m ul a t i o ns a n d paclitaxel (positive control) were diluted with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 5 mg/mL concentration, which was sterilized by filtration and finally centrifuged. Serial dilutions were made from the stock solution. About 0.1 mL of the MCF-7 cell suspension (approximately 10,000 cells) was added to each well of the 96-well plate. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µL of different concentrations of samples were added. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere and microscopic examination was performed after every 24 hours interval. After 72 hours, the samples solutions in the wells were discarded and 50 µL of MTT in phosphate buffered saline was added to each well. The plates were gently shaken and incubated for 3 hours at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 µL of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader (ELx800, Bio-Tek) at a wavelength of 540 nm. The percentage growth inhibition was calculated using formula GI (%) = [100 - (Mean optical density of test) / (Mean optical density of control)] X 100 and the concentration of sample needed to inhibit cell growth by 50% (CTC50) was generated from the dose-response curves. The experiments were performed in triplicate.

 

In-vitro anticancer activity of curcumin-bioenhancer encapsulated polymeric nanoparticles using SRB assay

Prepared curcumin-bioenhancer encapsulated polymeric nanoformulations were evaluated for anticancer activity using Sulforhodamine B (SRB) assay on human ovarian cancer cell (Ovkar-3), human leukaemia cell (HL60), human hepatoma cell (HEPG2), human cervix cancer cell (HeLa), human colon cancer cell (Colo205) and human lung cancer cell (A549) [29]. Briefly, pure curcumin, prepared curcumin- bioenhancer encapsulated polymeric nanoformulations and Adriamycin (i.e. Doxorubicin, a positive control) was diluted with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 5 mg/ mL concentration, which was sterilized by filtration and finally centrifuged. Serial dilutions (10, 20, 40, 80 µg/mL) were made from the stock solution. About 0.1 mL of the diluted cell suspension (approximately 10,000 cells) was added to each well of the 96-well plate. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 µL of different concentrations of test drugs were added. The plates were then incubated at 37°C for 3 days in 5% CO2 atmosphere and microscopic examination was performed after every 24 hours interval. The cells were fixed using ice-cold trichloroacetic acid (TCA) for 1 hour at 4°C and then washed using distilled water to remove excess TCA and allowed to dry in the air. After 72 hours, the drug solutions in the wells were discarded and 50 µL of SRB solution was added to each well and allowed to stain at room temperature for 30 minutes. The plate was washed with 1% v/v acetic acid to remove unbound dye and allowed to dry in the air. About 100 µL of 10 mM unbuffered Tris Base (pH 10.5) was added to each well and the plates were gently shaken for 5 minutes on a shaker platform to extract the bound SRB. The absorbance was measured using a microplate reader (ELx800, Bio-Tek) at a wavelength of 492 nm. Parameters such as GI50 (Concentration of the drug that produces 50% inhibition of the cells), TGI (Concentration of the drug that produces total inhibition of the cells) and LC50 (Concentration of the drug that kills 50% of the cells) were calculated. The experiments were performed in triplicate.

 

In-vivo anticancer activity of curcumin-bioenhancer encapsulated polymeric nanoparticles

Prepared curcumin-bioenhancer encapsulated polymeric nanoparticles were evaluated for anticancer activity using cancer induced rat. The animal experiment was performed as per the protocol approved by the Institutional Animal Ethics Committee (160/1999/CPCSEA; Proposal Number 896). Briefly, healthy adult female Sprague dawley rats (120-200 gram body weight) were procured and randomly assigned to 6 groups, each containing 6 animals in polypropylene cages layered with husk and maintained in a controlled room at a temperature (22±3°C) and light (12 hours light/dark cycle). Animals were allowed free access to water and standard pellet diet. The 7,12-Dimethylbenz(a)anthracene (DMBA) solution was freshly prepared at a dose of 25 mg/kg in 0.75 ml of sunflower oil and 0.25 ml of physiological saline. Animals (except animals in control group) received a single dose of 25 mg/kg of DMBA through intraperitoneal route and were allowed to develop tumor, which was identified by palpitation and tumor induced animals were selected for 60 days treatment through oral route. Animals in group 1 and 2 received no other treatment, animals in group 3 received pure curcumin (100 mg/kg of body weight), animals in group 4 received curcumin-piperine encapsulated polymeric nanoformulation (equivalent to 100 mg/kg of body weight), animals in group 5 received curcumin- quercetin encapsulated polymeric nanoformulation (equivalent to 100 mg/kg of body weight); and animals in group 6 received curcumin-silibinin encapsulated polymeric nanoformulation (equivalent to 100 mg/ kg of body weight). Anticancer efficacy of the test samples were assessed using tumerogenesis parameter such as body weight, tumor burden and tumor volume.

 

Statistical analysis

Statistical analysis was performed using SPSS 18.0 (SPSS, Inc., Chicago, IL) statistical package. Data were expressed as mean±standard deviation. One way analysis of variance (ANOVA) followed by Duncan multiple comparison method was used to correlate the difference between the variables. Data were considered statistically significant if P value was < 0.001, < 0.01 and < 0.05.

 

Stability study

Prepared void and curcumin-bioenhancer encapsulated polymeric nanoparticles were packed in a clear glass vial and stored at 40±2°C and 75±5% relative humidity in a stability chamber (SS134, Shivani Scientific) for 6 months. At the end of storage period, samples were analysed for average particles size, polydispersity index, zeta potential and drug content. The experiments were performed in triplicate.

 

Result and Discussion

Preparation of void and curcumin-bioenhancer encapsulated polymeric nanoparticles

Void and curcumin-bioenhancer encapsulated polymeric nanoparticles were prepared using modified nanoprecipitation method. Prepared polymeric nanoformulations were stored at room temperature for one month but there were no visual aggregation and post-formulation degradation.

 

Particles size, polydispersity index and zeta potential analysis

Void polymeric nanoparticles have shown an average particle size of 64.42 nm with PDI of 0.234 and zeta potential of 29.3 mV. However, encapsulation of Cur-Pi and Cur-Qu has increased the average particle size (86.87 nm and 71.03 nm), increased the polydispersity index (0.239 and 0.281) and altered the zeta potential (22.2 mV and 38.8 mV). Curcumin and piperine possess cationic charge on its structure and after encapsulation these phytochemicals are accommodated in the polymeric matrix and expected to repel each which would have increased the size. Similarly, curcumin possess strong cationic charge and quercetin possess week anionic charge on its structure and after encapsulation these phytochemicals are accommodated in the polym eric matrix and expected to attract each other. Hence, curcumin- quercetin combination has produced lesser particle size than the curcumin-piperine combination. Whereas, encapsulation of Cur-Si has decreased the average particle size (46.95 nm), decreased the zeta potential (26.6 mV) and decreased the PDI (0.142). Curcumin possess strong cationic charge and silibinin possess strong anionic charge on its structure and after encapsulation these phytochemicals are accommodated in the polymeric matrix and expected to attract each other strongly, which might have caused significant size reduction than void, curcumin-quercetin & curcumin-piperine combination. Particles size spectrum of prepared curcumin-bioenhancer encapsulated polymeric nanoformulations was displayed in Fig. 2. Prepared polymeric nanoparticles (Void, Cur-Pi, Cur- Qu, Cur-Si) displayed significant size reduction with excellent uniformity (i.e. <100 nm and <0.282 PDI) in comparison to pure curcumin (i.e. 38.617 µm and 0.551 PDI).

 

Particle surface morphology analysis

Prepared void and curcumin-bioenhancer encapsulated polymeric nanoparticles were spherical in shape. Hence, curcumin and bioenhancers encapsulated in the polymer matrix will also be in spherical shape and expected to enhance the basic function, release of from the polymer matrix, transport of in the body and internalization by many folds than the pure curcumin and bioenhancers.

 

Drug content, encapsulation efficiency and drug loading estimation

The results of drug content, encapsulation efficiency and drug loading were summarized in Table 1. Drug content in all curcumin-bioenhancer encapsulated polymeric nanoformulations was within ±2% from the actual quantities, which shows that there was no significant post-formulation drug degradation or drug loss. However, encapsulation efficiency of the drug in all curcumin-bioenhancer encapsulated polymeric nanoformulations was greater than 74% but less than 98.5, which shows that major volume of drugs were encapsulated in the polymer matrix and only negligible quantity of curcumin and bioenhancer were available as a free drug. Whereas, drug loading in all curcumin- bioenhancer encapsulated polymeric nanoformulations was greater than 7% but less than 9.9%, which shows that the polymer added to the formulation was optimal.

 

FT-IR spectral analysis

Void polymeric nanoparticles showed characteristic absorption bands at (a) 2924 cm-1 (CH3 stretch, dimethylamino groups); (b) 1737 cm-1 (C=O stretch); (c) 1479, 1467, 1451 and 1383 cm-1 (CHX stretch); (d) 1119 cm-1 (C-N stretch); and (e) 1020 cm-1 (CH3 rocking). The obtained spectrum was comparable with poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) spectrum. Hence, formation of poly(butyl methacrylate-co- (2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) nanoparticles was confirmed. Similarly, Cur-Pi, Cur-Qu and Cur-Si encapsulated polymeric nanoparticles also demons trated characteristic absorption bands between (a) 2923 to 2924 cm-1 (CH3 stretch, dimethylamino groups); (b) 1736 to 1737 cm-1 (C=O stretch);  (c) 1478 to 1479, 1461 to 1468, 1450 to 1451 and 1350 to 1383 cm-1 (CHX stretch); (d) 1106 to 1121 cm-1 (C-N stretch); and (e) 1020 to 1030 cm-1 (CH3 rocking), which confirm the formation of poly(butyl methacrylate-co-(2-dimethylamino-ethyl) methacrylate-co-methyl methacrylate) nanoparticles and encapsulation of curcumin and bioenhancers in the polymeric nanoparticles. FT-IR spectrum of Cur-Pi encapsulated polymeric nanoparticles were displayed in Fig. 3.

 

In-vitro release kinetics study

Prepared curcumin-bioenhancer encapsulated polymeric nanoparticles showed >40% release of drugs within 5 minutes, >60% release of drugs within 10 minutes, >85% release of drugs within 20 minutes, >92% release of drugs within 30 minutes and >95% release of drugs within 45 minutes (Table 2 and Fig. 4). Released nanosized curcumin and bioenhancers displayed enhanced aqueous solubility in gastric fluid. Hence, the movement of undissolved curcumin to the intestine will be prevented; thereby hydrolytic degradation of curcumin in the intestine will also be prevented.

 

In-vitro toxicity of void polymeric nanoformulation