Caffeic acid phenethyl ester (CAPE), a well proven and reported chemical constituent of propolis is chemically polyphenolic compound. CAPE has antitumor activity on various types of cancer cells with different mechanisms including inhibition of the NF-𝜅B and increased activity of caspase-3 or caspase-7. Formal normal cells CAPE has been reported as a harmless but it inhibits the growth of various types of cancer cells[3,4]. The potent antitumor nature of CAPE has been reported on cancer cells through NF-𝜅B inhibition [5,6]. But it is highly lipophilic in nature and thus it shows poor water solubility. CAPE is reported to get hydrolysis by an esterase plasma enzymes which leads to rapid clearance and short half-life. The overall effect gets results in lower bioavailability. Solubility parameter is a key factor which determines drug dissolution, absorption and bioavailability.It directly affects the biological effects. For improvement of drug solubility various approaches have been reported which includes solid dispersions, cyclodextrin complex formation or chemical modifications however there are various limitations associated with these approaches. Applications of nanotechnology in drug delivery are also established for solubility enhancement, drug targeting and to overcome other limitations associated with conventional approaches. Various biodegradable polymers have been reported in drug delivery applications to achieve improved solubility, drug targeting by enhanced permeability and retention and prolonging the biological activity[10,11]. Among those poly (ε-caprolactone) (PCL) is one of the biodegradable polymer known for pharmaceutical application in various drug delivery systems[12-14]. PCL has applicability in formulation development of pharmaceuticals because of its various advantages such as biocompatibility, biodegradability, high permeability and 6-hydroxycaproic acid is a biodegradation end product of PCL which is neutral and not disturb normal pH balance[15,16].
Design of experiments is an important approach implementing many formulation scientist to study effects of critical variables on the product. Nanoparticle formulations can be developed using DoE approach to study the interactions between formulation variables and reduce number of experiments. Factorial design method used to optimize the nanoparticle formulation [9, 7].
With aiming of anticancer drug delivery of CAPE some nanotechnology based attempts have been reported to overcome its limitations.However these studies have not attempted effects of formulation variables, detailed characterization of formulations, in vitroanticanceractivity and biosafety performance[1, 18-20].
Prompted by the above facts, the present study has been designed to develop CAPE loaded PCL nanoparticle formulation (denoted as CPL) to investigates its formulation characteristics and in vitro anticancer activity.
MATERIALS AND METHODS
CAPE (> 98%), PCL (poly-ε-caprolactone, mol weight 65,000 Da), Pluronic® F-68 (PF-68) and dialysis bag were purchased from Sigma-Aldrich, Bangalore, India). Sodium hydroxide, potassium dihydrogen phosphate and acetone GR were procured from Merck India. All other pharmaceutical grade chemical and reagents used in study.
Preparation of CAPE loaded polymeric nanoparticle formulation
The nanoparticle formulation batches were prepared by solvent displacement method reported by Bothiraja et al., 2011. Briefly, PCL and Pluronic® F-68 were first dissolved in 3 ml of acetone in which the CAPE was dissolved as per composition shown in Table 1. This phase (organic) was injected into water phase (10 ml) 10 ml/min with magnetic stirring at 2000 rpm for 1 h at 40°C. The polymer aggregates were removed by membrane filtration. The concentrations of PCL and Pluronic® F-68 (PF 68) were optimized to obtain stable suspension.
The particle size (PS) of developed formulations was measured with by laser diffraction technique and expressed in terms of d (0.9) μm value.
The CAPE concentration as drug content (DC) and encapsulation efficiency (EE) of the formulated batches were measured using previously developed and validated HPLC method.2 The nanoparticle solution was suitably diluted with alcohol prior to determination. Encapsulation efficiency was measured by by centrifugation at 12,000 rpm for 2 h at 40C followed by HPLC analysis.
DoE study was implemented for optimization of nanoparticle formulation. 32 (3 levels, 2 factors) factorial design was used, thus a total number of 9 batches were prepared. PS and EE values of formulated batches were measured. PS and EE were selected as dependent variables while the amount of PCL and amount of PF 68 were selected as independent variables.
Optimization by 32 factorial design
Trial batches were performed as preliminary experiments that helped in understanding the effects of variables on characteristics of polymeric nanoparticles. The amount of PCL (X1) and PF 68 (X2) concentration were identified as key factors in determining the characteristics of polymeric nanoparticles. Thus, a 32 factorial design was selected for optimization process while studying the effect of independent variables on PS and EE as responses.
DSC and FTIR study
DSC thermograms of vacuum evaporated samples of CAPE, PCL and CPL were recorded with 5 mg sample at heating rate of 10°C/min under a nitrogen atmosphere (flow rate 50 ml/min) using DSC 821e. KBr dispersion technique was used for FTIR study. FTIR spectra of CAPE, PCL and CPL were recorded on a FTIR spectrophotometer (JASCO FTIR-8400).
Surface morphology and Zeta potential
Surface morphology of developed formulations was studied using Transmission electron microscopy (Zeiss EM 109) with carbon film covered copper grid technique. The zeta potential was measured with Zetasizer at a temperature of 25°C.
In vitro drug release study
in vitro drug release study was carried out in phosphate buffer (pH 7.4) as drug release medium with dialysis bag technique at 37 ± 0.5 °C with continuous magnetic stirring at 100 rpm. At predetermined time intervals sample was withdrawn maintaining sink condition. The samples were analyzed for CAPE content by developed and validated HPLC method. The percent cumulative release of CAPE was plotted against time. Pure CAPE release was analyzed in a similar manner.
In vitro cytotoxicity
In vitro cytotoxic activity of CAPE and CPL was carried out on MCF-7 and HT-9 cell line using sulforhodamine B (SRB) assay. The study performed as per the procedure described by Bothiraja et al, 2013 at Tata Memorial Center, Advanced Centre for Treatment Research and Education in Cancer (ACTREC) Mumbai, India. CPL was diluted in distilled water so as to obtain CAPE in the concentration ranges of 10, 20, 40 and 80μg/ml which were analyzed for cytotoxicity using SRB assay. Absorbance was measured on an ELISA plate reader at a wavelength of 540 nm with 640 nm reference wavelength[21, 22].
The hemolysisstudy on CAPE and CPL were performed using the method reported by love et al., 2012. Distilled water taken as 100% hemolysis control and NaCl solution as the non hemolysis control. CAPE and CPL samples corresponding to concentrations of 0.25, 0.5, 0.75, 1 and 1.5 mg/ml of formulations were taken for study. Absorbance of supernatants was determined at 453 nm with UV–VIS spectrophotometer.
CPL was transferred in glass vial and subjected to short term stability study at 4 °C in refrigerated condition. Optimized nanoparticles were analyzed for the change in particle size and encapsulation efficiency up to 180 days.
RESULTS AND DISCUSSION
Nanotechnology in drug delivery systems has proven for enhanced efficacy and safety of various drugs over the past few decades[24-26]. In this study, CAPE loaded polymeric nanoparticles have been investigated to improve its solubility, in vitro anticancer efficacy. The effect of the CAPE loaded polymeric nanoparticles composition on PS and EE was studied by DoE approach.
PS, DC and EE
The mean CPL nanoparticles size was obtained in the range of 187 + 2 to 220 + 5 and strongly affected by the selected variables. The DC and EE were obtained in range of 90.13–96.80 % and 64.37–74.80 %, respectively for CPL (Table 1). A good fit r2 for PS = 0.9284 and EE = 0.8134 for CPL was observed for the particle size and encapsulation efficiency the independent variables. Particle size affects in site specific drug delivery. In case very small particles also particle uptake by non targeted cells decreases and premature clearance occurs by the phagocytic system.
Optimization of CPL nanoparticles by 32 factorial design
Particle size and encapsulation efficiency are the key factors considered in nanoparticle formulation development. During the preliminary study, assessment of the concentrations of polymers and surfactants were done for obtaining non-sedimenting, non-aggregating nanoparticles. After preliminary studies, optimization was done by 32 factorial design to determine final proportions of polymer and surfactant (Table 1). CAPE content was kept constant for each batch. Batches were formulated as per 32 factorial design and responses shown in Table 1, Table 2 A&B. Multiple regression analysis performed with Design Expert® software and data summarized in Table 2 A&B. It was observed that positive coefficients of the terms X1 and X2 indicated a favorable effect on the mean particle size and EE with the PCL and PF68. The PCL had a linear effect on the PS and curvilinear effect on EE while PF 68 had linear effects on both PS and EE as seen in surface plot (Fig. 1).
Based on the results of the factorial design, the solution for optimum batch selection with highest desirability of 0.852 was obtained with F6 for CPL so F6 batch was selected as an optimized formulation and further evaluated for various parameters.
Zeta potential is essential parameter gives information about surface charges which has direct impact on colloidal stability. The CPL showed negative zeta potential –23.01 ± 0.45 mV that might be observed due to presence of PF 68 on surface. Desirable values of zeta potential obtained considering the colloidal stability of developed formulation.
Differential scanning calorimetry and Fourier transform-infrared spectroscopy (FTIR)
Study was performed for the free CAPE, PCL and CPL (Fig. 2A) in order to determine the molecular state of the CAPE. A sharp melting transition of free CAPE was observed at 127.48 °C with \H 38.01 J/g shows transit crystallinity. In CPL thermogram, the free CAPE peak was disappeared which indicates molecular dispersion of CAPE inside polymeric nanoparticles.
Fig. 2B shows FTIR spectra of drug, polymer and formulation. FT-IR spectrum of free CAPE showed specific bands obtained at 3482 cm−1 and 3333 cm−1 of –OH stretching and at 3061.44 cm -1 of C–H stretch. The strong and narrow peaks also observed at 1683 cm−1, 1602 cm−1, and 1184 cm−1 of C=O, C=C, and C–O. In case of PCL the specific bands at 2940 cm−1 cm−1 of C–H hydroxyl groups asymmetric stretching and 2860 of C-H hydroxyl groups symmetric stretching were observed. Also bands at 1722 cm-1 (C=O stretching vibrations of the ester carbonyl group), 1238 cm-1 (C-O-C asymmetric stretching) and 1160 cm-1 (C-O-C symmetric stretching) were observed. FTIR spectra of CPL, band intensity of CAPE at 3482 cm−1, 3333cm−1 and 1602cm−1 were significantly reduced gives confirmation of CAPE encapsulation.
Transmission electron microscopy (TEM) of nano-particles
Surface morphology of the CPL was studied TEM from which it can be observed that the nanoparticles were spherical in shape and freely dispersed (Fig. 3). Larger size nanoparticles were observed in TEM than that tested by laser diffraction that may be due to low melting points of PF 68 (~55 °C) and PCL (~60 °C), may undergoes the melting and expansion in TEM.
In vitro release study
Fig. 4 revealed that CAPE showed 92.39% release in 6 h. However, CAPE release from CPL showed initial burst release (19.00%) within the first 1 h and further sustained release up to 36 h.
Drug release from nanoparticle occurs through erosion, diffusion and degradation mechanisms. For developed system any or all three mechanisms may be contributed. The initial burst release may be because of drug adsorbed on the surface of the nanoparticles or dissolved in medium, while a sustained release could be caused by diffusion of the drug. For development of desired formulation, sustained release of entrapped drug from nanoparticles is an important parameter, as it maintains constant amount of drug persistently at site of action.
In vitro anticancer activity
The in vitro anticancer activity of CPL was performed on MCF-7 and HT-29 cells. The observations showed in Table 3, Fig. 5 indicated that CPL displayed superior anticancer activity than CAPE on MCF-7 cells. The TGI value for CPL was observed 27.20+0.010 μg/ml while for CAPE it was 46.00+0.020 μg/ml. In case of HT-29 cells lines Table 3, Fig. 6 indicated that CPL displayed superior anticancer activity than CAPE. The TGI for CPL was observed 36.00+0.010 μg/ml and for CAPE 47.20+0.015% μg/ml.
Results and microscopic images showed that in Fig. 5 A cells were appeared with high content and density which are of normal control group of MCF-7. In Fig. 5 B is of positive control group treated with Adriamycin, Fig. C & D were images of CAPE and CPL treated group respectively and appearing to be less dense and rounded.
Similarly Fig. 6 A cells were appeared with high content and density which are of normal control group of HT-29. In Fig. 6 B is of positive control group treated with Adriamycin, Fig. C & D were images of CAPE and CPL treated group respectively and appearing to be less dense and rounded.
The enhanced anticancer efficacy may be because higher cellular uptake of CPL with phagocytosis/ fusion which resulted in the EPR of the cell membrane to CAPE that allows sufficient drug concentration inside the cells. Therefore, CPL might serve as good carrier to improve in vitro anticancer activity of CAPE. The lower anticancer activity of free CAPE in solution may be because of P-glycoprotein pumps efflux. The GI50 value for CPL was observed less than 10 μg/ml indicates anticancer active on MCF-7 whereas it was 12 + 0.010 μg/ml for HT-29 cells. From the values obtain it can be concluded that the CPL showed better anticancer potential on MCF-7. Similarly on both cell lines CAPE showed better anticancer potential on MCF-7 breast cancer cell line. Also CPL showed enhanced cytotoxicity on both cell lines as compare to CAPE. The reason for enhanced cytotoxicity may be attributed to increased solubility, enhanced permeability and retention (EPR) effect because of reduction in particle size and developed formulation as a nanocarrier.
For in vivo application as a biomedicine it is necessary to evaluate the biosafety of developed formulation. The results of hemolysis study indicates that the hemolysis rate for up to 1.5 mg/ml concentration of CAPE and CPL were 2.5 and 2.6 % respectively which is within acceptable hemolysis rate and safe for internal use.
No significant deviations in EE and PS were observed as compared to initial values (P > 0.05) of freshly prepared nanoparticle formulation over the period of six months during stability study which indicates the developed formulation was physically stable for at least 6 months.
Nanoparticulate formulation composed of PCL and PF-68 was developed for delivery of anticancer bioactive, CAPE. The developed formulation displayed desirable particle size, encapsulation efficiency and sustain release with better stability. CPL demonstrated better anticancer efficacy as compared to CAPE. CPL was observed with better in vitro cyotoxicity in both MCF-7 and HT-29 than its free form, which may leads to reduction in dose. Developed formulation can be further investigated as a dosage form for CAPE to enhance therapeutic efficacy in cancer chemotherapy.
All Authors are thankful to Advanced Centre of Treatment Research and Education in Cancer (ACTREC), Mumbai, India for in vitro cytotoxicity study. This work was supported by the University Grants Commission, Government of India [grant numbers UGC-MRP, F.No.42-699/SR 2013].
CONFLICT OF INTEREST
The authors report no conflicts of interest in this work.