Chemicals and materials
Piperlongumine was purchased from Indofine Chemical Company (Hillsborough, NJ, USA). Curcumin and quercetin were purchased from the SRL (India). Leaves of the Ficus benghalensis were obtained from the Punjab university campus, Chandigarh (India). Dextran sulphate sodium salt (DSS) and DMH were purchased from the MP Biomedicals (Solon, OH, USA). Poly-D,L-lactide (PLA, mw=75000–120000) was purchased from the Sigma Aldrich and HPLC grade acetonitrile and water purchased from the SRL (India).
Five to six week old male BALB/c weighing 20–25 g were procured from Central Animal House, Panjab University, Chandigarh, India. All experimental protocols were first approved by Institutional Ethics Committee (PU/IAEC/S/14/47) and conducted according to the guidelines of Indian National Science Academy for the use and care of experimental animals. The animals were housed in ventilated polypropylene cages and acclimatized for one week in the animal room before the commencement of the study. The animals were fed on standard mouse chow pellet diet supplied by Aashirwad industries, Punjab, India and water ad libitum.
Preparation of leaf extracts
For the formation of leaf extracts (LEs), leaf materials were crushed with the help of mortar and pestle into fine powder and 5.0 g material was dissolved in the 100 mL distilled water and heated at 60°C. After cooling the remaining 50 mL material was centrifuged (10000 rpm, 20 min, 4°C) and filtered through a 0.22 µm filter to get finally freshly prepared LEs for the synthesis PLA NPs. The extracts were stored at 4 °C for further use.
Synthesis of LEs-mediated QPC-PLA NPs
Poly-D,L-lactide (mw=75 000–120000), was used for the synthesis of polymeric NPs using solvent evaporation method. Fifty five milligram PLA was dissolved in 2 mL dichloromethane (DCM) with 6 mg each of three molecules curcumin (C), quercetin (Q) and piperlongumine (P) and allowed to sonicate for 40 s. After sonication 4 mL of LEs was added and again sonicated for 40 s to form emulsion and finally diluted to 80 mL using distilled water. DCM was evaporated using rota-vapour for 20 min and synthesized loaded NPs were centrifuged at 4°C for 15 min. Similar conditions were followed for the synthesis of blank NPs. After separating the NPs using centrifuge, NPs were re-dissolved in the distilled water (4 mL). The freshly prepared NPs were twicely washed and ready for the characterization. The blank and molecules loaded LEs mediated NPs were named as LEs-PLA NPs and LEs-QPC-PLA NPs respectively.
Coating of sugar and its evaluation on the surface of NPs
For the encapsulation of sugar on the surface of NPs, the blank and loaded NPs were incubated overnight with the dextrose sugar solution (2 mg/mL). Anthrone test method was used for the estimation of sugar on the surface of LEs-S-PLA NPs and LEs-S-QPC-PLA NPs. The sugar coated LEs-S-PLA NPs and LEs-S-QPC-PLA NPs were centrifuged and incubated with the Anthrone for sugar estimation and scanned at 620 nm using spectrophotometer. The sugar concentration was calculated on the surface of LEs-S-PLA NPs and LEs-S-QPC-PLA NPs using formula (1).
OD = optical density
Morphological Characterization of blank and loaded NPs
High resolution transmission electron microscopy (HRTEM, FEI, Netherlands) was used for shape and size measurement of LEs-PLA NPs and LEs-QPC-PLA NPs. Negative stain phosphotungstic acid was used to coat the surface of NPs and allow to dry at room temperature on copper grid. The images were obtained with a Tecnai, Twin 200 kV TEM (FEI, Netherlands) operated at 200 kV at desired magnification.
Evaluation of encapsulation of curcumin, piperlongumine and quercetin in loaded NPs
Supernatant (10 µL, after separating out NPs) was evaluated for the encapsulation and loading of three molecules (Q, P and C) using HPLC (Waters, USA) instrument having auto sampler (Auto-2707) and UV-Visible detector (PDA 2998) using analytical C-18 column (Waters, USA, 4.6 × 250 mm). HPLC method was validated using solvent acetonitrile (0.1% TFA) and water (50:50) at 370 nm, 325 nm and 420 nm wavelength for Q, P and C respectively. The wavelength is selected based on the existing method. The limits of detection (LOD) and limit of quantitation (LOQ) for Q, P and C was evaluated. The formula 2 and formula 3 were used to calculate the encapsulation efficiency and loading of molecules respectively.
In vitro release of quercetin, piperlongumine and curcumin in PBS buffer from loaded NPs
From three formulation we have selected only freshly prepared and lyophilized 5 mg LEs-QPC-PLA NPs. LEs-QPC-PLA NPs were incubated in 10 mL 0.1 M phosphate buffer/saline at physiological pH (pH 7.4). To study the in vitro release kinetics, LEs-QPC-PLA NPs were continuously stirred by a magnetic stirrer at 37°C. Q, P and C containing released sample were collected (1 mL) at 0, 4, 8, 12 and 24 h, lyophilized and again dissolved in acetonitrile (1 mL). The dissolved sample were centrifuged at 10000 rpm for 20 min at 4°C. The amount of Q, P and C released (%) at any time ‘t’ was calculated using the formula 4:
Development and standardization of colon cancer model on animal male BALB/c mice
Male BALB/c mice were procured from central animal house, Panjab University Chandigarh, India. All the experimental procedures were first approved from ethical committee and conducted according to the guidelines of Indian National Science Academy, New Delhi. The animals were treated with single intra-peritoneal dose of DMH (20 mg/kg body weight). After one week, DSS (3% w/v) was given in drinking water for one week followed by normal drinking water for two weeks. The animals were subjected to three such DSS cycles. The animal model was standardised for the development of colon cancer and observed through the histological section and staining under microscope (Nikon Eclipse 80i).
Eight group of animal (n=5) including control (DMH-DSS), pure molecules (QPC), LEs-PLA NPs (blank), LEs-S-PLA NPs (blank), LEs-QPC-PLA NPs (loaded) and LEs-S-QPC-PLA NPs (loaded) were used for the anticancer study. The dose of curcumin was selected as standard (40 mg/kg) dose from the already published paper. And we have reduced the dose half to set 20 mg/kg due to three molecules available in the loaded formulation. Dose were (Pure molecules, blank and loaded NPs) given in saline (200 µL for each animals) via oral route for 15 days at alternate days. Only saline was given to the controller group (DMH-DSS) at alternate date for the same period (15 days).
Detection of quercetin, piperlongumine and curcumin in different organs using HPLC
After the completion of dose, animals were sacrifice and liver, kidney, colon, serum were analysed for the presence of quercetin, piperlongumine and curcumin by HPLC. Each organ (20 mg) were crushed in acetonitrile (1 mL) with mortar and pestle, filtered and 10 µL of each was injected into the HPLC with help of auto sampler. The presence of the Q, P and C was recorded and analysed.
Anticancer activity of pure molecules, blank and loaded NPs using histopathology in colon, liver, kidney and serum
The animals were sacrificed by cervical dislocation after completion of dose and then their colon, liver, kidney and serum were collected for further study. Buffer washed organs were preserved in the buffered formalin (10%) and fixed into the wax blocks for sectioning (4-5 μm). The fixed sections were stained with standard haematoxylin and eosin (H&E) staining using standard protocol. The well stained oven dried section slides were observed under microscope (Nikon 80i) and evaluated for the presence of adenoma, hyperplasia and cancer in situ.