Curcumin-loaded layer-by-layer folic acid and casein coated carboxymethyl cellulose/casein nanogels for treatment of skin cancer

P. Priya; R. Mohan Raj; V. Vasanthakumar; V. Raj

doi:10.1016/j.arabjc.2017.07.010

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Original article

13 (

); 694-708

doi:

10.1016/j.arabjc.2017.07.010

Curcumin-loaded layer-by-layer folic acid and casein coated carboxymethyl cellulose/casein nanogels for treatment of skin cancer

P. Priya^{^a}, R. Mohan Raj^{^a}, V. Vasanthakumar^{^a}, V. Raj^{^a,^b,⁎}

a Advanced Materials Research Laboratory, Department of Chemistry, Periyar University, Salem 11, Tamil Nadu, India

b Centre For Nanoscience and Nanotechnology, Periyar University, Salem 11, Tamil Nadu, India

⁎Corresponding author at: Advanced Materials Research Laboratory, Department of Chemistry, Periyar University, Salem 11, Tamil Nadu, India. alaguraj2@rediffmail.com (V. Raj)

Received: 2017-5-8, Accepted: 2017-7-17, Published: 2017-01-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Targeted drug delivery systems using natural polysaccharide/protein biopolymer for tumor cells are an attractive platform for enriching the therapeutic effects and reducing the side effects of the drug. Carboxymethyl cellulose (CMC) and casein (CA) nanogels (NGs) loaded with curcumin (CUR) were prepared by self-assembly method and fabricated with folic acid (FA) and casein using layer-by-layer (LbL) technique for skin cancer drug delivery. The prepared samples were characterized by techniques like zeta potential, FTIR, XRD, TGA and Cryo-SEM. Both the swelling and in vitro drug release was performed in acidic pH (4.5 and 6.8) and physiological pH 7.4. Hemolysis assays demonstrated that the drug carriers are hemocompatible. Confocal microscope studies indicate facilitated uptake of 2-FA/CA/CUR@CMC-CA NGs in MEL-39 melanoma cancer cell line, which in turn result in a higher potential for apoptosis. Compared to pure CUR and CUR@CMC-CA NGs, the 2-FA/CA/CUR@CMC-CA NGs has lower IC50 value and superior cytotoxicity in MEL-39 cells because of folate-receptor mediated endocytosis evaluated by the cellular viability quantification using MTT assay and optical microscope images. Finally from in vitro skin permeation experiments, 2-FA/CA/CUR@CMC-CA NGs showed 3.47 ± 0.03 to 4.15 ± 0.25 μg/ml CUR concentrations at the stratum corneum, epidermal and dermal layers. Overall, our results put forth 2-FA/CA/CUR@CMC-CA NGs as an aspiring candidate to achieve enhanced anticancer effects against melanoma skin cancer.

Keywords

Carboxymethyl cellulose

Casein

Folic acid

Curcumin

Nanogels

Skin cancer

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1 Introduction

Skin cancer, a cutaneous malignancy, is one of the most prevalent forms of cancer and can lead to disfigurement and even in death if not diagnosed and treated early. Over the past decades, skin cancer incidence has grown dramatically; the reason is likely to multifactorial including increased UV exposure, inflammation agents, environmental carcinogens, tumor promoters, family history and multiple moles (Saladi and Persaud, 2005). Skin cancers are named according to the cell from which they originate and become malignant. The three commonest types are basal cell carcinomas, squamous cell carcinomas and melanomas (Hayano et al., 2012). The first two types are less common known as non-melanoma skin cancer. Melanoma is one of the most aggressive skin cancers originated from the malignant transformation of melanocytes (pigment cells) (Einspahr et al., 2002).

Development and progression of metastatic melanoma to other organs are very fast, leading to a hindered response to traditional chemotherapeutic drugs and a decrease in the long term survival rate of patients. Treatment of melanoma involves some combination of chemotherapy, surgery, radiation therapy, immune therapy, biotherapy and targeted therapy (O’Day et al., 2002). For treating cutaneous pathological conditions topical application of therapeutics is an attractive strategy because skin is always considered as a prominent gateway of entry for chemicals into the body. This is because of its ease of self-administration, selective targeting to pathological location, non-invasiveness, lower dosage and lower systemic toxicity for attaining the therapeutic effect (Alkilani et al., 2015). However, significant penetration barrier provided by the stratum corneum (SC) composition has limited the scope in development of successful topical/transdermal drug delivery systems.

To overcome this barrier, various colloidal carriers including lipid nanoparticles, nanovesicles and nanocapsules have been studied notoriously for improving low drug absorption from the skin surface (Pegoraro et al., 2012). Among them, nanogels (NGs) feature unique structural and mechanical properties, which are similar to those of tissues and to the extracellular matrix of the skin. NGs already reported as a carrier for skin cancer have the limitations of spilling their contents over time and aggregation (Kaminski et al., 2016). In order to prevent this process, the NGs can be coated with polymers to achieve the desired release kinetics.

Carboxymethyl cellulose (CMC) is a water-soluble ubiquitous polysaccharide derivative with carboxymethyl (—CH2—COONa) groups and hydroxyl groups on the cellulose backbone (Ninan et al., 2013). CMC has numerous advantages over several biopolymers such as its viscosity, nontoxicity, biodegradability, cost effectiveness and hypoallergenic nature (Sivakumar et al., 2013; Wang and Wang, 2016). Casein (CA) is a commercially available water-soluble phosphoprotein, produced by acid precipitation of raw skim milk (Bajpai et al., 2016). It is low-cost and bio-safe natural product that has good dispersibility, high amphiphilicity and ability to reform quickly in physiological media (Elzoghby et al., 2011). Therefore, it seems that CMC and CA are ideal candidates for preparing NGs by self-assembly.

Curcumin (CUR) is a natural antioxidant, yellow-colored compound extracted from the rhizomes of turmeric (Curcuma longa) (Datz et al., 2016). It has been widely used by practitioners of Indian traditional medicine, which inhibits many diseases including cough, rheumatism, biliary disorders, hepatic disorders, anorexia, cancer and Alzheimer’s (Hatcher et al., 2008). There are various ongoing clinical trials using CUR for the treatment of different types of cancers such as skin cancer, multiple myeloma, pancreatic cancer and colorectal cancer (Sampath et al., 2014). Although, CUR shows good therapeutic efficacy toward a variety of diseases including cancer, its use is fettered due to its poor aqueous solubility, low bioavailability and rapid degradation (O’Toole et al., 2016).

Few NGs made up of polysaccharide such as Hsp70 chaperone-based gels, chitin nanogels, PEG-chitosan nanogels have been reported for skin cancer (Abkin et al., 2013; Mangalathillam et al., 2012; Sabitha et al., 2013 ). To further improve the transfection and retention effect of therapeutic agent towards the deepest layers of skin, we have coated the NGs with folic acid (FA). Compared with protein-targeting molecules such as antibodies and peptides, FA is stable, cost-effective and non-immunogenic (Fu et al., 2016). Folate receptors (FR) are upregulated in many malignant tumors such as nasopharyngeal, breast, liver, ovarian, colon, cervical and chorion carcinomas including skin cancer, while its expression is limited in healthy tissues and organs (Ohyama et al., 2016; Ogbodu et al., 2015; Li et al., 2015). Previous literature has proved that FA was able to coat the PEI/pDNA and PEI-OA/LOR-2501 complexes electrostatically and taken up by B16-F10, Hela, Kb cells via the FR-mediated pathway (Kurosaki et al., 2011; Yang et al., 2015).

Layer-by-Layer (LbL) engineered NGs are a promising group of therapeutic nanocarriers used in an increasing number of biomedical applications because of its low cost, simplicity and versatility. At each assembly step, the LbL assembling process creates a monolayer of adsorbed polymers (Barbosa-Barros et al., 2014). Using the LbL assembly process peptides, proteins and DNA plasmids, have been deposited onto the surface of medical implants, tissue engineering scaffolds, microcapsules for encapsulation and sustained release of drugs (Rydzek et al., 2015).

In this paper, we report on preparation of uniformly self-assembled CMC-CA NGs (core) coated with bilayer films (shell) composed of CA and FA by LbL assembly method. Till date, there have been only a handful of studies reviewing the use of LbL coating especially a nanogel architecture (Xiao et al., 2016). To assess the suitability of the delivery system, we investigated physicochemical parameters and performed an in vitro release study. Biological studies including hemolysis, cellular uptake, cytotoxicity and apoptosis on melanoma cells (MEL-39) as well the in vitro skin permeation studies were done to measure the retention and penetration effects of CUR.

2 Materials and methods

2.1

2.1 Materials

Sodium carboxymethyl cellulose (molecular weight: 6.62 × 10⁵ g/mol, degree of substitution: 0.68, degree of polymerization: 3062, CAS no: 9004-32-4), folic acid (molecular weight: 441.40, CAS no: 59-30-3) were purchased from Loba chemie, Mumbai, India. Casein (molecular weight: 783.928 g/mol, CAS no: 9000-71-9), curcumin (molecular weight: 368.38, CAS no: 458-37-7) and phosphate buffer were purchased from Sigma Aldrich, India. All other chemicals were of analytical grade and used without further purification.

2.2

2.2 Preparation of CUR loaded CMC-CA NGs

CUR@CMC-CA NGs were prepared by self-assembly method. Briefly, 20 ml of aqueous solution of CA (5 mg/ml) was allowed to stir at room temperature for 1 h. 30 mg of CUR was dissolved in 10 ml of ethanol and then added to the above CA solution and kept in stirring for 30 min. Then 20 ml of aqueous solution of CMC (10 mg/ml) was allowed to stir for 1 h at room temperature and added to the above mixture and stirred for 3 h to obtain CUR@CMC-CA NGs.

2.3

2.3 Preparation of LbL (FA/CA) coated CUR@CMC-CA NGs

LbL-functionalized CUR@CMC-CA NGs were obtained by alternately coating CA and FA on the surface of CUR@CMC-CA NGs by electrostatic interaction of CA and FA. 15 mg of CA (1 mg/ml) dissolved in deionised water was added to the nanogels suspension and stirred gently for 1 h. The NGs were centrifuged at 2000 rpm for 5 min and then washed. Then 15 mg of FA (1 mg/ml) dissolved in 1 M NaOH was added to the suspension and stirred gently for 1 h. 1-FA/CA/CUR@CMC-CA NGs obtained were centrifuged, washed and re-dispersed in deionised water. The whole process was repeated again. Therefore, bilayers of CA and FA coated 2-FA/CA/CUR@CMC-CA NGs was obtained. Finally, the CUR@CMC-CA and FA/CA/CUR@CMC-CA NGs were freeze dried at −50 °C and lyophilized for 48 h for further investigations performed using only powdered samples. The same procedure was adopted for preparation of blank FA/CA/CMC-CA NGs.

2.4

2.4 Physicochemical characterization

2.4.1

2.4.1 Zeta potential measurement

The surface charge of the NGs was determined by zeta potential analyzer (Malvern instruments zetasizer ver. 7.11 MAL 500999) at room temperature in water. The dispersion of NGs was diluted by ultrapure water and sonicated before measurement.

2.4.2

2.4.2 Fourier transform infrared (FTIR)

FTIR spectra of CMC, CA, FA, CUR, 2-FA/CA/CMC-CA, CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs were measured using a Perkin Elmer Spectrophotometer (Perkin Elmer Inc., USA). FTIR spectra were recorded after grinding the sample with KBr into a powder, compressed into a transparent disk and scanned from 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹.

2.4.3

2.4.3 X-ray diffraction (XRD)

X-ray powder diffraction spectra of pure CUR, 2-FA/CA/CMC-CA, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs were obtained using X-ray diffractometer (Bruker AXS D8 Advance) with Cu as a target at Wavelength 1.5406 Å.

2.4.4

2.4.4 Cryo-scanning electron microscope (Cryo-SEM)

For the Cryo-SEM imaging, a droplet of NGs suspension was rapidly immersed in liquid nitrogen at −210 °C for 5 min and the frozen sample was fractured in the cryo chamber at −180 °C. Then the sample was etched for 90 s at 100 °C and coated with platinum (5 nm thickness). Following coating, specimen was transferred to the cooled stage and viewed at −130 °C.

2.4.5

2.4.5 Thermogravimetric analysis (TGA)

TGA was used to measure the degradation temperature of pure CUR, 2-FA/CA/CMC-CA, CUR@CMC-CA, and 2-FA/CA/CUR@CMC-CA NGs using Perkin Elmer, Diamond TG/DTA STA 6000 instrument under nitrogen atmosphere (45 ml/min) at a heating rate of 10 °C min⁻¹ at a temperature range up to 700 °C.

2.5

2.5 Swelling studies

Swelling behavior of the CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs was examined by placing 100 mg of the NGs into the tea-bag that was made of nylon wire mesh. Then the tea-bag was fully immersed in buffer solution (pH 4.5, 6.8 and 7.4) at 37 °C for a predetermined time interval. Then the tea-bag with swollen NGs was suspended in the air for 15 min to remove the surface water of NGs before weighing on a balance (Lu et al., 2016). The swelling ratio of the NGs was determined by equation: $Swelling (%) = \frac{Final weight of the NGs after swelling - Initial weight of the NGs}{Initial weight of the NGs} \times 100$

2.6

2.6 In vitro drug release

The drug loaded NGs equivalent to 100 mg of CUR were immersed in a defined volume of pH 4.5, 6.8 and 7.4 buffer solutions in a shaker incubator at a shaking speed of 100 rpm at 37 °C. At predetermined time intervals the solution was centrifuged at 4000 rpm for 10 min to separate the released CUR from NGs. 5 ml of each solution was collected for determination of CUR content using a UV visible spectrophotometer by reading the absorbance of the solution at 420 nm (UV–vis spectrophotometer, Varian 4000, USA) and an equal volume of the same medium was added back to maintain a constant volume. The concentration of released CUR was calculated from a calibration curve of CUR. The release was calculated as follows using equation: $Release (%) = \frac{Released amount of curcumin}{Total amount of curcumin} \times 100$

2.7

2.7 In vitro hemolysis assay

For hemolysis analysis, 200 μl of packed RBCs was diluted in 4 ml PBS with 5% hematocrit. 200 μl of diluted RBC suspension mixed with samples (concentration of 1 mg/ml) were gently vortexed and incubated at 37 °C for 4 h. The mixture was centrifuged at 1000g for 5 min. The supernatant free hemoglobin was transferred to a cuvette and measured at absorbance of 540 nm using UV–vis Spectrophotometer. Triton X-100 and saline were used as the positive and negative control. The percentage of hemolysis was calculated using the following formulae: $Hemolysis (%) = \frac{{OD}_{sample} - {OD}_{negative control}}{{OD}_{positive control} - {OD}_{negative control}} \times 100$

2.8

2.8 In vitro cellular uptake studies

Cellular uptake of CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs and CUR was investigated by confocal laser scanning microscope (CSLM). MEL-39 cells were seeded in a 96 well plates at a density of 1 × 10⁴ cells per well in DMEM medium and cultured for 24 h. After 24 h, the culture medium was removed and NGs with a concentration 5 μg/ml CUR were incubated at 37 °C for predetermined time intervals. Then the cells were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min at room temperature. Finally, the cellular uptake was visualized by staining with 1 μl of lysotracker green dye for 10–15 min. PBS wash was given to remove the excess of dye and the slides were mounted and visualized with confocal microscope (Olympus FV-1000).

2.9

2.9 In vitro cytotoxicity

The cells were cultured in a petri dish comprising DMEM medium containing 10% fetal bovine serum and 1% antibiotic kept in a 5% CO₂ incubator. The percentage of viable cells after exposure to samples was estimated by MTT assay. For this, MEL-39 cells were seeded in a 96 well plates at a density of 1 × 10⁴ cells per well in 200 μl medium. After 24 h, the culture medium was replaced with a serial concentration of samples. The cells were grown for another 48 h. Then, 20 μl of MTT assay stock solution in PBS was added to each well and kept at 37 °C for 4 h in dark for formation of formazon crystals. After 4 h, the medium containing MTT was removed and the formed purple formazon crystals were dissolved in 300 μl of DMSO. Absorbance of purple formazon product was measured at 570 nm using a microplate reader and IC₅₀ values were obtained by plotting optical density versus concentration. The percentage of cell viability was expressed by the equation as follows: $Cell viability (%) = \frac{Absorbance of control cells}{Absorbance of treated cells} \times 100$

2.10

2.10 Apoptosis assay

For the analysis of apoptosis, MEL-39 cells were seeded in a 96 well plates at a density of 1 × 10⁴ cells per well in DMEM medium and cultured for 24 h. Then CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs and free CUR were added and the cells were incubated for 48 h. After treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde with 0.1% triton X-100. The fixed cells were then washed with PBS twice and stained with DAPI to observe nuclear morphology of cells for 10 min. The cells were finally washed with PBS thrice and analyzed using confocal microscope (Olympus FV-1000). Percentage of apoptosis was quantified using the Annexin V-FITC Apoptosis Detection Kit. The apoptotic index was calculated as a ratio of the apoptotic cell number to the total tumor cell number in high-power field.

2.11

2.11 In vitro skin permeation studies

In vitro skin permeation experiment was performed using full thickness porcine skin in a vertical Franz diffusion (FD) cell to evaluate transdermal absorption of CUR by NGs. Fresh pig ears were obtained from a local slaughter house. After washing thoroughly, the skin from the inner face of the ear was excised into appropriate sizes using a surgical blade, wrapped in a aluminum foil and stored at −80 °C until use. The receptor chamber was filled with 5.5 ml of phosphate buffer pH 4.5. The diffusion cell was maintained in occlusive conditions at 32 ± 1 °C using a circulating water jacket and the receptor solution was stirred continuously at 100 rpm. 1 ml of the sample under test (the NGs dispersion as well as control CUR solution, both containing 500 μg of CUR) was placed in the donor chamber on the stratum corneum side of the skin. The skin layers were separated by stripping after the exposure period of samples (area 1 cm²) and placed on a cork disc. CUR was extracted using methanol by centrifugation at 5000 rpm for 15 min. 20 μl of filtered supernatant was injected into the HPLC system to determine the CUR concentration penetrated. All experiments were performed in triplicate.

3 Results and discussion

In this study, we prepared LbL-NGs by means of a very simple and easily reproducible preparative method without the addition of crosslinkers. LbL assembling process is performed in an aqueous medium, does not require harsh conditions and is generally amenable to biomaterials. FA is used as a targeting ligand coated on the surface of CUR@CMC-CA NGs expressing folate receptors on the surface of tumor. The schematic illustration of preparation of CUR@CMC-CA NGs and 2-FA/CA/CUR@CMC-CA NGs is shown in Fig. 1.

Schematic illustration of preparation of curcumin-loaded folic acid/casein coated carboxymethyl cellulose-casein NGs.

3.1

3.1 Physicochemical characterization

3.1.1

3.1.1 Surface charge

To study the change in the surface charge of NGs by FA and CA coating, we analyzed the zeta potential of FA and CA coated and uncoated NGs. Measurement of zeta potential is an important tool to predict the stability of a colloidal nanocarriers in medium. Coating of alternate layers was confirmed by reversal of charge from +ve to −ve and vice versa as shown in Fig. 2(a). Before the coating process, CUR@CMC-CA NGs exhibited extremely low potential −31.6 mV and the zeta potential was increased to about −15.3 mV when the CA layer was deposited on the CUR@CMC-CA NGs core due to presence of amino group and again zeta potential value reduced to −25.6 mV for the consequent adsorption of FA during first layer deposition due to the presence of carboxylic groups. After second layer coating of CA and FA, again the charge has been reversed from 5.4 to −21.6 mV. The alternative changes of zeta potential values to each other demonstrate the successful alternate double layer coating of CA-NH₂ and FA-COOH onto the surface of CUR@CMC-CA NGs. High negative surface charge tends to have better stability by preventing the aggregation of particles to each other (Campardelli et al., 2016).

(a) Zeta potential of CUR@CMC-CA, 1-CA/CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA, CA/FA/CA/CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA and 2-FA/CA/CMC-CA NGs. (b and c) FTIR spectra of FA, CMC, CA, CUR, 2-FA/CA/CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA, 2-FA/CA/CMC-CA and CUR@CMC-CA NGs. (d) XRD pattern of 2-FA/CA/CMC-CA, CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs and CUR.

3.1.2

3.1.2 FTIR spectra

Fig. 2(b) and (c) shows FTIR spectra of FA, CMC, CA, CUR, CUR@CMC-CA, 2-FA/CA/CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs. The spectrum of FA shows significant peaks at 3600 cm⁻¹ and 3000 cm⁻¹ due to the hydroxyl (OH) stretching and —NH stretching vibration bands, 1695 cm⁻¹ is for CO bond stretching vibration of —CONH₂ group, 1401 cm⁻¹ corresponds to characteristic OH deformation band of phenyl skeleton and 1473 cm⁻¹ is attributed to absorption band of the phenyl ring. FTIR spectrum of CMC exhibits bands at 3510 cm⁻¹ due to OH stretching vibrations, 1609 cm⁻¹ and 1418 cm⁻¹ are due to asymmetric and symmetric modes of stretching vibration of carboxylic groups and asymmetric stretching vibration of ether bonds are found in the range of 1020–1080 cm⁻¹. CA exhibits bands at 3488, 1609, 1539 and 1220 cm⁻¹ that arise from N—H stretching and amide bending vibrations. CUR shows peaks at 3512 cm⁻¹ due to phenolic OH, 1641 cm⁻¹ is due to C⚌O stretching, 1510 cm⁻¹ is due to aromatic C⚌C, 1422 cm⁻¹ is due to alkanes C—H and 1039 cm⁻¹ is due to C—O stretching vibration (Shelma and Sharma, 2013).

Nearly all peaks are observed in NGs formulation that appeared in FA, CMC, CA and CUR spectra with some shifts. The peak of secondary —NH bending of CA was shifted from 1539 cm⁻¹ to 1507 cm⁻¹ and the band associated with carboxylic groups of CMC is shifted from 1659 to 1607 cm⁻¹. The appearance of shifted peaks in 2-FA/CA/CUR@CMC-CA NGs from individual polymers is ascribed to strong electrostatic interaction between amino groups of CA and carboxyl groups of CMC. Briefly, all major peaks of both CUR and 2-FA/CA/CMC-CA NGs have been shifted in CUR loaded 2-FA/CA/CMC-CA NGs, thus confirming encapsulation of CUR in 2-FA/CA/CMC-CA NGs. No notable changes in peaks has been observed after first LbL (CA/FA) coating on CUR@CMC-CA NGs and a slight shift with increase in intensity of peaks has been observed in bilayer coated 2-FA/CA/CUR@CMC-CA compared to monolayer coated 1-FA/CA/CUR@CMC-CA NGs due to overlay of carboxylate group of FA and CA amine moieties.

3.1.3

3.1.3 XRD studies

X-ray diffraction studies were carried out for CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA, 2-FA/CA/CMC-CA NGs and CUR to understand the nature of CUR in NGs and their XRD pattern are shown in Fig. 2(d). Since CUR is a hydrophobic drug, it has a tendency to form crystals in polymer matrix which will hinder the drug elution and release pattern will be irregular (Anitha et al., 2011). XRD pattern of CUR contains numerous peaks in the 2θ range of 20–30° stating its crystalline nature. However, the peaks of CUR totally disappeared in CUR@CMC-CA, and 2-FA/CA/CUR@CMC-CA NGs diffractograms. It can be attributed to the successful incorporation and molecular level dispersion of CUR into the system. Similar observation has been made in XRD spectra of CUR loaded folic acid tagged aminated starch/zinc oxide coated iron oxide nanoparticles (Saikia et al., 2017). In addition, CUR loaded CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA and blank 2-FA/CA@CMC-CA NGs are amorphous in nature.

3.1.4

3.1.4 Cryo-SEM analysis

Morphology of the prepared CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs have been analyzed using cryo-SEM in Fig. 3(a)–(c). Cryo SEM technique provides benefit of analyzing the structural morphology of samples by conserving the spatial structure of NGs that can be used for further investigations. Crystallization and nucleation of water can be prevented (Dykstra and Reuss, 2011). The network structure formed by self-assembly of CMC and CA in CUR@CMC-CA NGs exhibits a random porous structure with a diameter less than 1 μm forming a 3-D networks. After coating of NGs with first layer of FA/CA on CUR@CMC-CA NGs, a difference can be seen as a smooth interconnected surface morphology has been obtained and almost the inner NGs structure has become invisible. After second layer coating, there is formation of flat thick layer with a reduction in number of pores in 2-FA/CA/CUR@CMC-CA NGs compared to 1-FA/CA/CUR@CMC-CA NGs.

Cryo-SEM images of (a) CUR@CMC-CA, (b) 1-FA/CA/CUR@CMC-CA and (c) 2-FA/CA/CUR@CMC-CA.

3.1.5

3.1.5 TG analysis

TGA of FA, CMC, CA, CUR as well as prepared 2-FA/CA/CMC-CA, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs were studied and their TGA curves are presented in Fig. 4a and DTG curves in Fig. 4b. According to TGA results, raw materials such as FA, CMC, CA, CUR as well as prepared CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs show three degradation stages. CMC exhibits a weight loss of 5% at 106 °C, 12% at 257 °C and 42% at 340 °C. CA shows weight loss of 6% at 106 °C and 13.6% at 252 °C and a loss of 49% at 321 °C. First stage of weight loss is due to loss of moisture content, second and third stage is due to splitting and scission of backbone polymer chains. The observed first stage of weight losses until 100–160 °C of CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs is attributed to loss of surface moisture content and water imbibed with the NGs. Secondly, between 210 and 300 °C weight loss occurred is due to fragmentation and rupturing of CMC and CA network to its monomers. After 300 °C, a major weight loss (approximately 62.0%) is mostly because of the intensive pyrolysis and breakage of backbone chains (Anirudhan et al., 2011). A residual weight of approximately 30% remains even after 700 °C for CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs indicating not all the composite is completely degraded within 700 °C.

(a and b) TGA and DTG curves of FA, CMC, CA, CUR, 2-FA/CA/CMC-CA, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs.

DTG values of CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs are 310 and 330 °C whereas DTG values of FA, CMC, CA, and CUR are below 300 °C. An increase in thermal stability of 2-FA/CA/CUR@CMC-CA NGs is observed compared to CUR@CMC-CA, probably because of multilayer coating of CA and FA on the surface of CUR@CMC-CA NGs. Thermal stability of 2-FA/CA/CUR@CMC-CA NGs is significantly high compared to raw CMC and CA. This can be credited to the electrostatic interaction between —COOH groups of CMC and —NH₂ groups of CA and FA/CA coatings.

3.2

3.2 Swelling analysis and in vitro drug release

The swelling study was carried out at pH 4.5, 6.8 and 7.4 shown in Fig. 5(a)–(c). The —NH₂ groups on CA chains in the nanogels are positively charged due to the protonation of amine groups in acidic media. Due to the repulsion between —NH₃⁺ groups on CA chains, the nanogels swell and increases in size at acidic pH than neutral pH, which is required condition for release of drug in acidic environment of melanoma treatment (Verma et al., 2016). At three pH regions, swelling rate of 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA are smaller compared to CUR@CMC-CA because of the additional layers present on the surface of NGs.

(a–c) swelling ratio and (d–f) in vitro drug release of CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA (pH = 4.5. 6.8 and 7.4).

Since curcumin is a highly hydrophobic drug that tends to form crystals when curcumin solution is added to an aqueous solution. If the nano sized curcumin crystals are formed inside the nanocarrier matrix, then the drug elution from the carrier will be obstructed and the release profile will be irregular (Anitha et al., 2011). But in the nanogel formulations of CUR, there were no crystalline peak as confirmed by XRD. Therefore, the LBL-NGs could enhances markedly the solubility of Cur. It is worth mentioning that other synthesized nanogels like dextrin nanogel, gel-core hyaluosome, chitin nanogels (Goncalves, 2012; El-Refaie et al., 2015; Mangalathillam et al., 2012 ) were reported to enhance solubility of CUR after loaded into nanogels. CUR has another disadvantage of degradation at an alkaline pH. But the nanogel formulation increased the stability of curcumin at physiological pH 7.4 and retarded rapid invitro release of CUR (Zhou et al., 2016).

Drug release from LbL assembled nanogel matrices is a combination of many factors including erosion, diffusion, nanogel degradation, and swelling. The drug release study of CUR from CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs were carried out at acidic pH 4.5, 6.8 the pH at the tumor site and physiological pH 7.4 depicted in Fig. 5(d)–(f) at predetermined time intervals. CUR@CMC-CA NGs exhibit a biphasic release pattern, with 36% of drug release at pH 4.5, 41% at pH 6.8 and 18% at pH 7.4 within the first 5 h. Drug release reached greater than 60% in 12 h at pH 4.5, 72% at pH 6.8 and 36% at pH 7.4 followed by sustained release over the next 48 h. In contrast, LbL-NGs showed a similar release profile but with reduced initial burst than CUR@CMC-CA NGs, followed by a more sustained release. 1-FA/CA/CUR@CMC-CA showed a 20% drug release within the first 5 h at pH 4.5, 28% at pH 6.8, 13% at pH 7.4 and 44% drug release in 12 h at pH 4.5, 54% at pH 6.8 and 30% at pH 7.4. 2-FA/CA/CUR@CMC-CA NGs showed 14% drug release in 5 h at pH 4.5, 18% at pH 6.8, 9% at pH 7.4 and in 12 h 35% drug was released at pH 4.5, 40% at pH 6.8, 23% at pH 7.4 followed by sustained drug release. At the end of 48 h, the drug released from CUR@CMC-CA, 1-FA/CA/CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA at pH 4.5 were 82%, 65% and 54%, at pH 6.8 drug release were 89%, 78%, 64% and drug release at pH 7.4 were 64%, 54%, 40% respectively. The faster release from CUR@CMC-CA NGs might be due to shorter path length, provoking partitioning to the release media because of the absence of protective LbL layers around the core nanogels. During the LbL assembly process, it is possible that some of the incorporated drug partitioned between the core and the shell assembly, which is a reason for initial burst release from LbL-NGs. In LbL-NGs in addition to core matrix, the multiple LbL layers increased the diffusional distance thus hindering contact between the drug and the release media considerably for a longer time (Ramasamy et al., 2014). Consequently, the multilayer LbL assembly effectively controlled the drug release rate, reducing the initial burst release that can greatly reduce systemic toxicity and a constant release for a prolonged period to attain a constant therapeutic value. Moreover, an enhanced CUR release profile was observed at acidic pH 4.5 and 6.8 representing the conditions within endosomes/lysosomes compared with physiological pH 7.4, a larger amount of CUR would be taken up into the cell through receptor-mediated endocytosis. It is seen that the amount of CUR released at the physiological pH condition was relatively low.

3.3

3.3 In vitro hemolysis assay

The hemolytic potential of 2-FA/CA/CUR@CMC-CA at various concentrations of 0.2, 0.6, 0.8 and 1 mg/ml were estimated by testing the amount of the hemoglobin (Hb) released from RBC in the supernatant as given in Fig. 6. Hemolysis assay was carried out as an indication of blood compatibility of prepared sample. The percentage of hemolysis even at higher concentration of 1 mg/ml 2-FA/CA/CUR@CMC-CA is found to be 2.6%. Generally, hemolysis of less than 5% is regarded as nontoxic and safe according to ISO/TR 7406, critical safe hemolytic ratio for bio-materials (Mangalathillam et al., 2012). This has proved that 2-FA/CA/CUR@CMC-CA NGs showed negligible hemolysis and possesses good hemocompatibility which is suitable for drug delivery through transdermal route.

In vitro hemolysis assay 2-FA/CA/CUR@CMC-CA NGs.

3.4

3.4 Cellular uptake

To understand the targeting capability of FA coated NGs, cellular uptake assays of CUR and NGs were carried out using MEL-39 melanoma cancer cell lines. Folate receptor is over-expressed in melanoma MEL-39 cell line (Skinner et al., 2016). Fig. 7(a)–(e) shows typical confocal images of MEL-39 cells after incubation with CUR, CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs in FA-free culture medium and 2-FA/CA/CUR@CMC-CA NGs in a medium with FA. Since CUR has an innate fluorescent property, it was used directly without any additional fluorescent markers for cellular imaging. With the same incubation time and equivalent CUR concentration (10 mg/ml) in each formulation, 2-FA/CA/CUR@CMC-CA showed significantly higher fluorescence intensity than either free CUR or CUR@CMC-CA NGs. The high uptake efficiency of 2-FA/CA/CUR@CMC-CA is probably due to the coating of FA residue on the surface of the NGs compared to FA uncoated formulation (CUR@CMC-CA NGs). No green fluorescence spot is observed in control image. These results are consistent with those reported previously on the FA-receptor dependent cellular uptake of (You et al., 2016). Further, the cells were incubated with 2-FA/CA/CUR@CMC-CA in medium containing free FA in order to assess the role of FA in the cellular uptake. 2-FA/CA/CUR@CMC-CA incubated in medium containing FA shows less fluorescence intensity compared to 2-FA/CA/CUR@CMC-CA in medium without FA. This implies that the targeting efficiency of FA coated NGs was suppressed because free FA prevents 2-FA/CA/CUR@CMC-CA from entering into MEL-39 cells by means of competitive binding between free FA and 2-FA/CA/CUR@CMC-CA to folate receptors on the cell surface (Zhao et al., 2016). These outcomes signify that FA coated 2-FA/CA/CUR@CMC-CA NGs have potential as a targeted drug carrier to cancer cells via FR-mediated endocytosis.

Cellular uptake confocal microscope images of (a) control, (b) CUR (c) CUR@CMC-CA, (d) 2-FA/CA/CUR@CMC-CA (free FA) and (e) 2-FA/CA/CUR@CMC-CA + FA.

3.5

3.5 In vitro cytotoxicity

The in vitro cytotoxicity of CUR, CUR@CMC-CA, 2-FA/CA/CMC-CA and 2-FA/CA/CUR@CMC-CA NGs was analyzed toward MEL-39 cell lines treated with four different concentrations (25, 50, 75 and 100 μg/ml) for 48 h by MTT assay represented in Fig. 8(g). Specifically, throughout all the concentrations, 2-FA/CA/CUR@CMC-CA showed superior anticancer effect compared to that of free CUR and CUR@CMC-CA NGs. IC₅₀ values for free CUR, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs were calculated to be 18.3, 15.8, and 13.1 μg/ml, respectively. The results showed that 2-FA/CA/CUR@CMC-CA NGs had better uptake profile than for free CUR, CUR@CMC-CA because of folate-mediated targeting and thus greater cytotoxicity (Skinner et al., 2016). Treating cells with blank 2-FA/CA/CMC-CA NGs exhibited negligible cytotoxicity (viability was above 91%) up to 100 μg/ml, which signified good biocompatibility. In correlation with cellular uptake study, 2-FA/CA/CUR@CMC-CA NGs (medium without FA) exhibited higher cytotoxicity than 2-FA/CA/CUR@CMC-CA NGs (medium with FA). The anticancer effect of each formulation was further investigated qualitatively by microscopic imaging (Fig. 8(a)–(f)). As seen, control cells were intact with its typical morphology and stained uniformly. As expected, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs treated cells clearly showed marked blebbing with morphological changes of cells. The result of optical microscopic images of cell cytotoxicity is consistent with the cytotoxicity by MTT assay.

In vitro cytotoxicity represented by optical microscopic images of MEL-39 cells (a) control, (b) CUR (c) 2-FA/CA/CMC-CA (d) CUR@CMC-CA, (e) 2-FA/CA/CUR@CMC-CA (free FA), (f) 2-FA/CA/CUR@CMC-CA + FA and (g) represents the graphical percentage of cell viability of MEL-39 cells.

3.6

3.6 Apoptosis assay

The apoptotic profile of CUR, CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs on MEL-39 cell line was evaluated qualitatively after 48 h exposure and shown in Fig. 9(a)–(e). To understand the effect of CUR loaded and FA functionalized 2-FA/CA/CUR@CMC-CA NGs on CUR-induced apoptosis cell death morphologically in MEL-39 cells, we performed DAPI staining. The result demonstrated that both the nuclei and cytoplasm of CUR untreated cells (control) showed homogeneous fluorescence with no fragmentation and condensation after DAPI staining. After 48 h, 2-FA/CA/CUR@CMC-CA NGs showed a remarkable shrinkage and apoptotic nuclei more obviously than that of CUR@CMC-CA and free CUR. Blebbing and formation of apoptotic bodies was clearly perceptible after treatment with 2-FA/CA/CUR@CMC-CA NGs. Morphological changes of cells undergoing typical alterations are indicated by arrow marks through bright field images and flashily display the dead process of cells. Free CUR diffuses through the cell membrane and cannot accumulate into the cells due to the ATP-dependent efflux pumps on the cell membrane. However, 2-FA/CA/CUR@CMC-CA NGs are taken by folate receptor mediated endocytosis and when CUR is loaded into nanogels, it can escape from the efflux pumps and can accumulate in the cells and cause apoptosis rapidly (Banu et al., 2015; Gunduz et al., 2014). Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells are as follows: control, CUR, CUR@CMC-CA, and 2-FA/CA/CUR@CMC-CA NGs was 7.1 ± 2.70%, 22.5 ± 0.65%, 33.4 ± 1.20% and 48.3 ± 3.20% (Fig. 9(f)). Additionally, the suppressed cellular uptake of 2-FA/CA/CUR@CMC-CA NGs after addition of free FA into the culture media resulted in a significant decrease in the percentage of apoptotic cells to 43.7 ± 2.20% compared to apoptotic percentage obtained for 2-FA/CA/CUR@CMC-CA NGs in FA free culture media. The superior apoptotic activity of 2-FA/CA/CUR@CMC-CA NGs could be due to the combination of sustained release behavior, greater accumulation and the targeting effect by FA receptor mediated uptake inside the tumor cells and is also consistent with the in vitro cytotoxicity assay.

Apoptotic images of MEL-39 cells (a) control, (b) CUR, (c) CUR@CMC-CA, (d) 2-FA/CA/CUR@CMC-CA + FA, (e) 2-FA/CA/CUR@CMC-CA and (f) represents the graphical percentage of apoptotic of MEL-39 cells.

3.7

3.7 Skin permeation studies

Curcumin due to its high antioxidant activity can be used as a topical agent for skin cancer but its delivery is highly limited because of poor skin permeability due to its aqueous insolubility (Rachmawati et al., 2014). In the specific case of melanoma, as the nanogels enhance skin permeation, CUR loaded NGs (2-FA/CA/CUR@CMC-CA) has been examined as a topical therapy for melanoma by in vitro skin permeation study using vertical Franz diffusion cell. Nanogels may hydrate the skin by creating hydrophilic pathways with loosening skin structure to penetrate skin without losing the integrity of skin. Moreover bilayer coated core gel, provide the system better stability to withstand surrounding environment preventing nanogel damage and early drug leakage. Consequently, CUR might also be protected inside the nanogels that may be localized inside the skin. It was evident that the drug release from nanogels across the skin barrier occurrs by a combination of mechanism like drug partition, drug diffusion, matrix erosion and polymer swelling that control the persistent drug release. As shown in Table 1, the in vitro skin permeation has been carried out in the three layers of the skin: stratum corneum, epidermis and dermis. The CUR concentration in the three layers is found in the range 2.20 to 4.15 μg/ml. Strateum corneum is the first check for any nanoformulations delivered topically, transdermally and percutaneously. High amount of drug released by 2-FA/CA/CUR@CMC-CA (4.15 ± 0.25 μg/ml) in SC layer caused by interruption of SC confirms the penetration potential of 2-FA/CA/CUR@CMC-CA is high compared to free CUR (2.30 ± 0.28 μg/ml) and CUR@CMC-CA (3.40 ± 0.28 μg/ml) NGs. Free CUR due to low solubility, its permeation ability inside the skin layers is low compared to CUR@CMC-CA and 2-FA/CA/CUR@CMC-CA NGs which shows the inability of CUR to penetrate skin efficiently or to loacalize in the skin for prolonged period. Whereas, fitness of nanogels permeation inside the skin model may be explained by increased solubility of CUR in the nanogel matrix. The disruption of crystalline nature of CUR may have increased the rate of diffusion which translates to higher topical delivery. The CUR concentration in deeper layer of epidermis and dermis is greater in the case of 2-FA/CA/CUR@CMC-CA (3.67 ± 0.04 and 3.47 ± 0.03 μg/ml) compared to CUR@CMC-CA and CUR solution as depicted in previous literature. 5-Fluorouracil loaded double walled nanogels shows superior permeability in epidermis and dermis layer compared to plain 5-Fluorouracil drug (Sahu et al., 2017). Usually, an efficient percutaneous drug delivery system should be able to transport the drug across the entire layers of skin rather than retention at any depth of skin and here this enhanced CUR delivery in all the three skin layers may be advantageous to treat diseases like skin cancers such as melanoma which affect all the layers of skin and spreads to other region in a short span (Bodenham, 1968). This phenomenon may be probable due to the permeable nature of nanogels and affinity toward folate receptors due to coating of FA on the surface of NGs.

Table 1 Permeation of CUR and CUR from CUR@CMC-CA, 2-FA/CA/CUR@CMC-CA NGs in the three layers of skin.

Nanogels	Stratum corneum (μg/ml)	Epidermis (μg/ml)	Dermis (μg/ml)
CUR	2.30 ± 0.28	2.20 ± 0.26	2.61 ± 0.08
CUR@CMC-CA	3.40 ± 0.28	3.20 ± 0.17	3.10 ± 0.00
2-FA/CA/CUR@CMC-CA	4.15 ± 0.25	3.67 ± 0.04	3.47 ± 0.03

Reported as means of triplicates (±S.D.).

4 Conclusion

In this work, the carboxymethyl cellulose-casein nanogels (CMC-CA NGs) were prepared as the core and coated layer by layer with casein and folic acid (CA/FA) for the delivery of curcumin for skin cancers. The NGs were characterized by FTIR, XRD, cryo-SEM and TGA. XRD indicated the amorphous nature of drug in its molecular dispersion form in the polymer matrix. The thermal stability of 2-FA/CA/CUR@CMC-CA is relatively higher than raw CMC and CA. The blood compatibility was confirmed by in vitro hemolysis. LbL-NGs showed increased swelling and drug release at acidic pH. Cellular uptake and cytotoxicity studies reveal that 2-FA/CA/CUR@CMC-CA NGs has greater cellular uptake when compared to CUR@CMC-CA NGs, resulting in enhanced cytotoxicity and apoptotic against MEL-39 melanoma cancer cells overexpressing folate receptors. 2-FA/CA/CUR@CMC-CA exhibited good skin penetration and retention properties in all three layers of skin. The developed system will find excellent application for melanoma or other skin cancers where a high selective uptake of CUR through the transdermal route will make the treatment effective.

Acknowledgment

One of the authors (P. Priya) would like to acknowledge the DST-INSPIRE division for providing the INSPIRE fellowship (IF150169).

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Show Sections

[1] Abkin S.V., Pankratova K.M., Komarova E.Y., Guzhova I.V., Margulis B.A., . Hsp70 chaperone-based gel composition as a novel immunotherapeutic anti-tumor tool. Cell Stress Chaperones. 2013;18(3):391-396.
[Google Scholar]

[2] Alkilani A., Mccrudden M., Donnelly R., . Transdermal drug delivery: innovative pharmaceutical developments based on disruption of the barrier properties of the stratum corneum. Pharmaceutics. 2015;7(4):438-470.
[Google Scholar]

[3] Anirudhan T., Tharun A.R., Rejeena S.R., . Investigation on poly(methacrylic acid)-grafted cellulose/bentonite superabsorbent composite: synthesis, characterization, and adsorption characteristics of bovine serum albumin. Ind. Eng. Chem. Res.. 2011;50(4):1866-1874.
[Google Scholar]

[4] Anitha A., Deepagan V., Rani V.D., Menon D., Nair S., Jayakumar R., . Preparation, characterization, in vitro drug release and biological studies of curcumin loaded dextran sulphate–chitosan nanoparticles. Carbohyd. Polym.. 2011;84(3):1158-1164.
[Google Scholar]

[5] Bajpai S.K., Bajpai M., Shah F.F., . Alginate dialdehyde (AD)-crosslinked casein films: synthesis, characterization and water absorption behavior. Des. Monomers Polym.. 2016;19(5):406-419.
[Google Scholar]

[6] Banu H., Sethi D.K., Edgar A., Sheriff A., Rayees N., Renuka N., Faheem S., Premkumar K., Vasanthakumar G., . Doxorubicin loaded polymeric gold nanoparticles targeted to human folate receptor upon laser photothermal therapy potentiates chemotherapy in breast cancer cell lines. J. Photochem. Photobiol., B. 2015;149:116-128.
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[7] Barbosa-Barros L., Garcia-Jimeno S., Estelrich J., . Formation and characterization of biobased magnetic nanoparticles double coated with dextran and chitosan by layer-by-layer deposition. Colloids Surf. A Physicochem. Eng. Asp.. 2014;450:121-129.
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Curcumin-loaded layer-by-layer folic acid and casein coated carboxymethyl cellulose/casein nanogels for treatment of skin cancer

Abstract

Keywords

Carboxymethyl cellulose

Casein

Folic acid

Curcumin

Nanogels

Skin cancer

1 Introduction

2 Materials and methods

2.1 Materials

2.2 Preparation of CUR loaded CMC-CA NGs

2.3 Preparation of LbL (FA/CA) coated CUR@CMC-CA NGs

2.4 Physicochemical characterization

2.4.1 Zeta potential measurement

2.4.2 Fourier transform infrared (FTIR)

2.4.3 X-ray diffraction (XRD)

2.4.4 Cryo-scanning electron microscope (Cryo-SEM)

2.4.5 Thermogravimetric analysis (TGA)

2.5 Swelling studies

2.6 In vitro drug release

2.7 In vitro hemolysis assay

2.8 In vitro cellular uptake studies

2.9 In vitro cytotoxicity

2.10 Apoptosis assay

2.11 In vitro skin permeation studies

3 Results and discussion

3.1 Physicochemical characterization

3.1.1 Surface charge

3.1.2 FTIR spectra

3.1.3 XRD studies

3.1.4 Cryo-SEM analysis

3.1.5 TG analysis

3.2 Swelling analysis and in vitro drug release

3.3 In vitro hemolysis assay

3.4 Cellular uptake

3.5 In vitro cytotoxicity

3.6 Apoptosis assay

3.7 Skin permeation studies

4 Conclusion

Acknowledgment

References

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