Folic acid conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel for site specific delivery of hydrophobic drugs to cancer cells
© Pillai et al.; licensee BioMed Central Ltd. 2014
Received: 19 March 2014
Accepted: 4 July 2014
Published: 15 July 2014
The hydrogel based system is found to be rarely reported for the delivery of hydrophobic drug due to the incompatibility of hydrophilicity of the polymer network and the hydrophobicity of drug. This problem can be solved by preparing semi-interpenetrating network of cross-linked polymer for tuning the hydrophilicity so as to entrap the hydrophobic drugs. The current study is to develop a folic acid conjugated cross-linked pH sensitive, biocompatible polymeric hydrogel to achieve a site specific drug delivery. For that, we have synthesized a folic acid conjugated PEG cross-linked acrylic polymer (FA-CLAP) hydrogel and investigated its loading and release of curcumin. The formed polymer hydrogel was then conjugated with folic acid for the site specific delivery of curcumin to cancer cells and then further characterized and conducted the cell uptake and cytotoxicity studies on human cervical cancer cell lines (HeLa).
In this study, we synthesized folic acid conjugated cross-linked acrylic hydrogel for the delivery of hydrophobic drugs to the cancer site. Poly (ethyleneglycol) (PEG) diacrylate cross-linked acrylic polymer (PAA) was prepared via inverse emulsion polymerization technique and later conjugated it with folic acid (FA-CLAP). Hydrophobic drug curcumin is entrapped into it and investigated the entrapment efficiency. Characterization of synthesized hydogel was done by using Fourier Transform-Infrared spectroscopy (FT-IR), Transmission Electron Microscopy (TEM), Differential Scanning Calorimetry (DSC). Polymerization and folate conjugation was confirmed by FT-IR spectroscopy. The release kinetics of drug from the entrapped form was studied which showed initial burst release followed by sustained release due to swelling and increased cross-linking. In vitro cytotoxicity and cell uptake studies were conducted in human cervical cancer (HeLa) cell lines.
Results showed that curcumin entrapped folate conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel showed higher cellular uptake than the non folate conjugated form. So this can be suggested as a better delivery system for site specific release of hydrophobic cancer drugs.
Hydrogels are polymeric networks having three-dimensional configuration capable of imbibing high amounts of water or biological fluids. Their water absorbing property is mainly attributed to the presence of hydrophilic groups such as –OH, −CONH–, −CONH 2 –, and –SO 3 H in the polymers. Due to the contribution of these groups and domains in the network, the polymer is thus hydrated to different degrees, depending on the aqueous environment and polymer composition . These ionizable functional groups present in it affect its permeability mechanical stability and biocompatibility to a greater extends . Along with that these structures have some common physical properties resembling that of the living tissues, which is attributed to their high water content, soft and rubbery consistency, and low interfacial tension with water or biological fluids [3–5]. The high water content make it soft and wet just like a biological material mimicking the extracellular matrix similar to macromolecular based compound in the human body [6, 7]. Hydrogel based drug delivery is a type of controlled delivery system were the gel swell resulting in release of drug from the polymer in a controlled manner. As water penetrate through the polymer chain the glass temperature of the polymer decreases and make the hydrogel rubbery . These hydrogels have highly porous structure which helps incorporation of drug into it. The high water content and high porosity help them easy release of the drug within certain hours to days. The porosity of hydrogel can be tuned to the required size of the drug by the addition of cross linker to it. And thus it help in the controlled release of drug [9, 10]. The polymer used for the preparation of hydrogel is of natural, synthetic and semi-synthetic origin. Even though natural polymers have good bioactive properties  they are found to have low mechanical strength so we make use of synthetic polymers because of their good mechanical strength and well-defined structure which can be modified to improve the biocompatibility and biodegradability [12, 13].
Polyacrylic acid based polymers are one of such ideal candidates for the synthesis of hydrogel system for the controlled drug delivery because of the swelling behavior in aqueous environment . It is a type of pH sensitive polymer [15, 16] which shows swelling at higher pH due to the presence of ionizable carboxyl groups in it and can release the drug at neutral pH. The main drawback associated with PAA based drug delivery system is rapid release of drug from it which can be controlled by cross-linking. The cross-linking helps in slow drug release due to small mesh size which can be advantageous for controlled drug delivery applications. Cross-linking also improves the physical properties of the hydrogel including mechanical strength, degradability and diffusivity of drugs from the system. The solubility of polymer in the aqueous environment can also be prevented using cross-linking [17, 18].
Curcumin, a naturally occurring yellow coloured polyphenol obtained from the rhizome of the perennial herb Curcuma longa, is found to have potent anticancer properties . It inhibit proliferation and induces apoptosis in various cancer cell lines isolated from malignancies like leukemia, breast, lung, prostate and colon tumors [20–23]. Studies were also done in various tumerogenetic models [24–29] and some clinical trials were also done in patients which confirmed the potential of curcumin as a tool for cancer therapy. But its clinical application becomes limited due to poor water solubility, minimal systemic bioavailability, degradation in alkaline pH and photo degradation. So the therapeutic efficacy of curcumin can be increased by incorporating curcumin in a biocompatible polymer which enhances the solubility in aqueous solution and extends the release.
One of the main problems associated with cancer therapy is its unwanted side effect towards normal cells along with the cancer cells. An active targeting strategy can improve the therapeutic efficacy of drugs and reduces the side effects [30–34]. For that we have to modify the polymer nanoparticles with certain ligands that have its specific receptor on cancer cell surface. Folate receptor has been extensively investigated for targeting various tumor cells since it is normally expressed in various types of cancer cells [35–38]. So the cross-linked polymeric hydrogel nanoparticles which were structurally modified with folic acid can help in easy targeting and up taking of drugs by the cancer cells.
The hydrogel based system is found to be rarely reported for the delivery of hydrophobic drug due to the incompatibility of hydrophilicity of the polymer network and the hydrophobicity of drug. This problem can be solved by preparing semi-interpenetrating network of cross-linked polymer for tuning the hydrophilicity so as to entrap the hydrophobic drugs. The current study is to develop a folic acid conjugated cross-linked pH sensitive biocompatible polymeric hydrogel to achieve a site specific drug delivery. For that, we have synthesized a folic acid conjugated PEG cross-linked acrylic polymer (FA-CLAP) hydrogel and investigated its loading and release of curcumin. Here we used inverse emulsion polymerization technique proposed by Vanderhoff et al. for the polymerization of acrylic acid where an aqueous solution of hydrophilic monomer acrylic acid is dispersed in a continuous lipophilic phase with the aid of surfactants to promote the formation of water in oil (W/O) emulsion . The formed polymer hydrogel was then conjugated with folic acid for the site specific delivery of curcumin to cancer cells and then further characterized and conducted the cell uptake and cytotoxicity studies on human cervical cancer cell lines (HeLa).
Results and discussion
Synthesis and characterization of FA-CLAP hydrogel
The DSC of FA-CLAP hydrogel shows endothermic transition peak at 115°C which can be due to the loss of loose and bound water in the hydrogel . The gel appeared to be thermally stable up to 200°C. Curcumin was loaded to the cross-linked acrylic polymer through physical adsorption by post loading method. And the DSC of curcumin loaded FA-CLAP hydrogel shows an endothermic peak at 85°C. Cross-linking using PEG diacrylate provides hydrophobicity to the acrylic hydrogel which enhances the uptake of curcumin, a hydrophobic drug. Swelling of the polymer occurs at a pH above the pKa of the carboxyl group of acrylic acid. Swelling increases with COO- groups and decreases with increasing cross-links.
In-vitro drug release
Cell uptake studies
Curcumin entrapped FA-CLAP show better cellular uptake compared to free curcumin
Folic acid-conjugated PAA (FA-CLAP) nanocurcumin induce cytotoxicity in HeLa cells
Acridine orange (AO)/ethidium bromide (EB) staining
Conjugation of Folic acid on PAA nanocurcumin slightly enhance the apoptotic effect induced by the latter in HeLa cells
Curcumin, which has many medicinal properties mainly, lacks its activity due to its low water solubility. So through this work the delivery of hydrophobic drug curcumin is done by incorporating the drug into cross-linked hydrogel matrix and the cell uptake which was another problem was further enhanced by introducing folic acid into the system. It was found that folic acid conjugated cross-linked hydrogel polymer (FA-CLAP) loaded with curcumin showed better cellular uptake compared to the non-folate hydrogel particles. So this can be used as a better system for the site specific delivery of hydrophobic drugs.
Acrylic acid (Mw ~ 72), cross-linker poly (ethylene glycol) diacrylate (Mw ~ 238,), Curcumin, Ammonium persulphate (APS), Span 80 (Sorbitan monooleate), Tween 80 (Poly (ethyleneglycol) sorbitan monooleate), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), ethylenediamine and folic acid were purchased from Sigma-Aldrich, Germany. Acridine orange and ethidium bromide were purchased from Sigma Aldrich. DMEM was purchased from Life Technologies (Grand Island, NY, USA). MTT was purchased from Calbiochem, Germany and Propidium Iodide from Calbiochem, USA. All other reagents and chemicals were of analytical grade or above, and used without further purification.
Preparation of folic acid conjugated cross-linked polymeric nanoparticles
Preparation of cross-linked acrylic polymer
The cross-linked acrylic polymer (1%) was prepared by inverse emulsion polymerization technique. Emulsification was done by dispersing aqueous phase consisting of 10% acrylic acid, 5% sodium hydroxide and 15% water with continuous lipophilic phase consisting of liquid paraffin (68%), emulsifiers (2%), Span 80 and Tween 80 (75:25 ratio). For cross-linking, 1% of PEG diacrylate was added to the mixture followed by the addition of initiator, ammonium persulfate (APS). The temperature for polymerization was at 60°C for 6 hours. The cross-linked polymeric particles were isolated by centrifugation (10,000 rpm) for 30 min. The isolated polymeric particles were washed several times with hexane and stored for further structural modifications.
Conjugation of folic acid to the prepared cross-linked polymeric particles (FA-CLAP)
For the conjugation of folic acid to the prepared cross-linked polymeric particles, first the cross-linked polymeric particle (850 mg. 1.2 eq) has to be treated with ethylenediamine (3.2 ml, 0.65 eq) through carbodiimide chemistry for the availability of free amine group for the binding of activated folic acid to it. Simultaneously folic acid (220 mg, 0.5 eq) has to be ester activated then it is allowed to react with ethylenediamine conjugated cross-linked acrylic hydrogel. A measured amount of this ethylenediamine conjugated cross-linked polymeric particles (550 mg) was dissolved in DMSO. The reaction mixture is kept overnight stirring, for the completion of folic acid conjugation; cross-linked polymeric particles were isolated by centrifugation (10,000 rpm) for 30 min. The isolated folic acid conjugated cross-linked polymeric hydrogel (FA-CLAP) was washed several times and then freeze dried to remove solvent and water. The freeze dried product was stored in vacuum. The polymerization of acrylic acid with PEG diacrylate cross-linking and folic acid conjugation was characterized using DSC and FT-IR spectroscopy. The amount of folic acid conjugated was also estimated (see Additional file 1).
Loading of curcumin in folic acid conjugated cross-linked polymeric nanoparticles (FA-CLAP) was done by post-polymerization method. 100 mg of the lyophilized powder was dispersed in 10 mL distilled water. Curcumin was dissolved in chloroform and the drug solution in chloroform was added to the polymeric solution with constant vortexing and sonication. The curcumin loaded FA-CLAP hydrogel was then lyophilized to obtain dried powder.
Characterization of prepared hydrogel nanoparticles
Morphological analysis of the free and curcumin loaded FA-CLAP nanoparticles were then characterized using transmission electron microscopy (TEM, JEOL 1011, and Japan). The samples of the nanoparticle suspension in water milli-Q at 25°C were dropped on to formvar-coated grids and measurements were taken only after the samples were completely dried.
Folic acid conjugation in the parent polymer was confirmed using FTIR spectroscopy. FTIR spectroscopy was performed on a Spectrum 65 (Perkin Elmer). Spectra were recorded between 4000 and 600 cm−1 wave number range. Dried samples were mixed with KBr and further compressed in to pellets for making measurements.
Differential Scanning Calorimetry (DSC) was done to analyze the thermal behavior of the FA-CLAP and Curcumin loaded FA-CLAP hydrogel. DSC thermograms obtained were then analyzed using an automatic thermal analyzer system (Pyres 6 DSC, Perkin-Elmer, USA). Samples were placed in standard aluminum pans and heated from 20 to 250°C at a rate of 10°C/minute under constant purging of N2 at 10 mL/minute. An empty pan, sealed in the same way as that of the sample, was used as a reference.
(AER = Measured drug wt/ Nanoparticle wt & TER = Initial drug wt/ drug wt & polymer wt). Where (AER) is Actual entrapment ratio and (TER) is Theoretical entrapment ratio. Nanogel wt means the weight of the nanogel with curcumin taken for calculating entrapment efficiency and Initial drug weight means drug initially taken for the entrapment.
In vitro release kinetics
For in vitro release study, a known amount of curcumin loaded FA-CLAP were dispersed in 10 mL of P.B.S (pH 7.4) and was then left in a shaking incubator at 37 ± 0.5°C. A known quantity of sample was then withdrawn and replaced with fresh medium in a predetermined time intervals for maintaining the total volume constant. The amount of curcumin released from the hydrogel nanoparticle was then measured using UV spectrophotometer (Perkin Elmer, USA) at 420 nm.
Cell uptake studies
Cellular uptake of curcumin and folic acid-conjugated PAA (FA-CLAP) nanocurcumin were studied using confocal microscopy. Briefly, 2.0 × 104 HeLa cells were grown on cover slips placed in 24 well plates. After overnight incubation, when the cells attained their morphology, they were treated with curcumin dissolved in dimethyl sulfoxide (DMSO) (25 μM), folic acid-conjugated PAA nanocurcumin (25 μM) was suspended in aqueous medium and blank polymer. After 2 h of incubation the cells were washed with 1X PBS, fixed with PFA and the nuclei were stained with propidium iodide and were mounted using DPX. Cells were examined for intracellular fluorescence of curcumin using confocal laser scanning microscope in the FITC channel (488 nm).
Acridine orange (AO)/ethidium bromide (EB) staining
Acridine orange/ethidium bromide (AO/EB) double staining was used to detect apoptosis . Briefly, 5 × 103 cells/well were seeded in a 96-well plate and treated with curcumin in DMSO (25 μM) or folic acid-conjugated PAA (FA-CLAP) nanocurcumin (25 μM) for 24 h. After washing with 1X PBS, the cells were stained with acridine orange (100 μg/ml) and ethidium bromide (100 μg/ml) solutions for 2 min. The cells were then washed with 1X PBS, viewed under an inverted fluorescent microscope (Nikon Eclipse, TE-300) and were photographed.
Authors are thankful to Department of Biotechnology, Government of India, for financial support and Arun Kumar T Thulasidasan and Ashwanikumar Narayanan for Council of Scientific and Industrial Research, New Delhi, India for providing Senior Research Fellowship.
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