Engineering a Piperine Eluting Nanofibrous Patch for Cancer

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Engineering a Piperine Eluting Nanofibrous Patch for Cancer Treatment Shubham Jain, Sai Rama Krishna Meka, and Kaushik Chatterjee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00297 • Publication Date (Web): 13 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

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ACS Biomaterials Science & Engineering

Engineering a Piperine Eluting Nanofibrous Patch for Cancer Treatment

Shubham Jain, Sai Rama Krishna Meka, Kaushik Chatterjee*

Department of Materials Engineering Indian Institute of Science, Bangalore 560012 India

*

author to whom all correspondence should be addressed

Email: [email protected] Tel: +91-80-22933408

1 ACS Paragon Plus Environment


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Abstract The objective of this study was to engineer a biodegradable polymeric system for sustained release of piperine for cancer treatment. We prepared nanofibrous patches of poly(ε-caprolactone) (PCL) and gelatin (GEL) blends of different ratios by electrospinning. The PCL/GEL nanofibers were loaded with up to 30 wt% piperine, a phytochemical derived from black pepper, which is believed to exhibit anti-cancer, anti-arthritis, antibacterial, antioxidant and anti-inflammatory properties. Scanning electron microscopy revealed that the fiber diameter was in the range of 300-400 nm. Fourier transform infrared spectroscopy confirmed that the drug was successfully loaded into the nanofiber mats. In vitro release kinetics revealed the sustained release of the drug with 50% release in 3 days from the PCL/GEL (50:50 by weight) blend fibers. The reduced viability and growth of HeLa and MCF-7 cancer cells on the piperine eluting nanofibers demonstrated the anti-cancer activity in vitro. The proliferation of non-cancerous cells such as NIH3T3 cells and human mesenchymal stem cells was affected to a markedly lesser extent. Flow cytometry revealed that the released piperine induced generation of reactive oxygen species (ROS) and cell cycle arrest in the G2/M phase leading to cell death of cancer cells. The findings of this study suggest that piperine loaded nanofiber mats could be developed into implantable biodegradable patches for use in post-surgical cancer treatment.

Keywords: Polymeric nanofibers; Piperine; Cancer; Drug delivery; Phytochemicals


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1. Introduction It is widely recognized that spices and herbs traditionally used in different cuisines offer many health benefits in addition to adding flavor to our meals and this knowledge is the basis of many home remedies. These spices are rich in essential oils, anti-oxidants, minerals, vitamins and phytochemicals. Black pepper, a spice native to southern India is used as a medicine in India 1. Piperine (PIP) is an alkaloid derived from black pepper (Piper nigrum Linn.) and long pepper (Piper longum Linn.)

2

It has been shown that piperine has anti-

depressant, anti-arthritic, anti-inflammatory, antibacterial and anti-oxidant effects

3-5

and

possesses diverse functions such as enhancing the bioavailability of drugs, uptake of drug, lipid metabolism, and gastrointestinal function 1, 6. Globally there is growing interest in the use of such plant derived products for various treatments as they are emerging as potent drugs with fewer side effects even at high dosages 7-8

. In the U.S. alone, approximately 40-50 % of cancer patients are using plant parts and their

derivatives 9 and also imbibing these into their diet to minimize the occurrence of cancer 10-11. Every year more than 12 million people suffer from cancer worldwide

12-13

. Local cancer

recurrence remains a foremost medical problem after post-surgical therapy in numerous cancer types such as those of the brain13, neck 14, lung 15, prostate

16

and breast 17, which is

commonly caused by inadequate resection during surgery 18. Current treatments for cancer include combination of therapies such as radiation therapy, chemotherapy, immunotherapy and surgery

19-20

. Although effective against cancer,

they adversely affect the patient’s quality of life through severe side-effects

14, 21

. A number

of plant products including curcumin from turmeric 22-23, apigenin from parsley 24, resveratrol from grapes 25, gingiberin from ginger 26, and crocetin from saffron 27 are reported to exhibit some anti-tumor properties. Their anti-cancer action is just starting to be understood. Phytochemicals such as curcumin act as anti-cancer agents through multitude of actions



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including changes in cell cycle, apoptosis, metastasis and expression of oncogenes, etc.

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28

.

The poor absorption and bioavailability of these phytochemicals is a major limitation that limits their use in various treatments underscoring the need to engineer delivery systems to enhance their efficacy. A variety of delivery vehicles have been proposed for the delivery of drugs such as nanoparticles, hydrogels 29, polymeric conjugates 30, composite nanofibers 14, 16 and microspheres

31

. In recent years, electrospun polymeric nanofibers have gained

significant attention in drug delivery due to advantages such as a simple and cost-effective fabrication route, high surface to volume ratio and high drug loading capacity to effectively deliver the drug 14. Reports have shown that electrospun nanofibrous patch incorporated with Doxorubicin along with silica nanoparticles and ZnO nanosphere loaded with multiple cancer drugs were used for post-surgical cancer treatment 14, 32. Studies have proposed that nanofibers loaded with anti-cancer drugs such as Doxorubicin

33

,

Paclitaxel

33

and Cisplatin

14

could be useful for post-surgical tumor

treatment but these drugs show severe side effects. These include cardiotoxicity by Doxorubicin 34, neurotoxicity by Paclitaxel 35 and nephrotoxicity and hepatotoxicity induced by Cisplatin

36

. Many of these drugs are immunosuppressive that can lead to infections.

Moreover, many chemotherapeutics agents harm dividing hematopoietic cells also which led to the neutropenia and cytopenia further compromising immunity 37-38. Thus, there is need for novel approaches and agents which can fight the recurrence of cancer post-surgery with minimal side effects. It is believed that phytochemicals or plant-derived dietary agents like curcumin and piperine will likely cause fewer side effects as they are used as an ingredient in food

1

and such that the human body is adaptable for them. Many phytochemicals are also

antioxidants

39

that can boost the immune system

40

to help in fighting cancer. Dietary

phytochemicals agents can also be combined with chemotherapy drugs to minimize the side effects with increased efficiency

37, 41

. For example, Quercetin is used with the Cisplatin to


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protect the normal tubular renal cells

42

. The controlled release of phytochemicals

incorporated into electrospun nanofibers can thus be an efficient alternative for localized sustained delivery at the tumor site for use as post-surgical implants. It is envisaged that the anticancer phytochemical can kill the cancer cells and the nanofiber mat can provide a constructive microenvironment for the tissue defects and offer long term healing 14, 43. In the present work, we prepared and characterized PIP loaded nanofibers prepared from poly(ε-caprolactone) (PCL) and its blend with gelatin (GEL) (PCL/GEL). PCL is a biodegradable polyester

44

used clinically in medical products

45

. It is amenable for

processing and suited for encapsulating piperine in the form of nanofibers 43. To augment the release of the hydrophobic drug from the slow degrading hydrophobic PCL nanofibers blends with GEL were prepared. From the few studies that have been reported on the anticancer effect of piperine it is observed that concentration of at least 25 µM was required to affect proliferation of cancer cells

46-47

. Thus, we aimed to fabricate fibers to release comparable

concentration of the drug in order to prevent proliferation of cancer cells. We characterized the drug loaded fibers and the in vitro release of piperine from PCL and PCL/GEL nanofibers at different drug loading. The effect of the piperine loaded nanofibers on the viability and proliferation of cancer and non-cancerous cells were assessed in vitro. The mechanisms underlying the observed cell response was investigated to elucidate the mode of action of the released piperine.

2. Experimental 2.1 Preparation of nanofibers PCL (Mn~80,000), piperine and gelatin type B from porcine skin were purchased from Sigma Aldrich. 2,2,2-triflouroethanol (TFE) and glacial acetic acid were purchased from SDFCL, India. 12% (w/v) solutions of neat PCL, neat GEL and mixtures of PCL and GEL (designated as PCL/ GEL) at ratios of 50:50 and 75:25 were prepared in TFE by stirring 5 ACS Paragon Plus Environment


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for 8 hours at 23 oC. For piperine loaded fibers 10, 20 and 30 % of piperine (w/w of total polymer) were added to the solution. Hereafter, PIP loaded fibers are designated by the suffix PIPX where X represents the fraction (wt/wt %) of PIP. Table 1 lists the composition of the various fibers mats prepared in this study. Additionally, 10 µl of acetic acid was added to the PCL/GEL polymeric solutions. Piperine loaded PCL and PCL/GEL blend fibers, neat PCL and GEL nanofibers were prepared by electrospinning (ESPIN NANO, India)

48

. The solutions were filled in 2 ml

syringes (gauge size 22) and placed in the spinneret at 25°C and 50% humidity. The voltage was set at 10 kV and the distance between syringe and the collector was fixed at 10 cm. The solution was pumped at a rate of 0.5 ml/hour flow rate and the nanofibers were collected onto an aluminum sheet in the form of a plate collector. 2.2 Characterization of electrospun nanofibers The morphology of the electrospun nanofibers was characterized using a scanning electron microscope (SEM, ZEISS ultra 55) after sputtering with gold (≈10 nm thick) at 7.0 kV accelerating voltage. Fiber diameter was calculated using the ImageJ software from the SEM images for at least ten randomly chosen fibers from five different images for each mat. The chemical composition of the fiber was characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, PerkinElmer Frontier IR/NIR systems, USA) in the range of 4000-650 cm-1 and 16 scans were accumulated for each sample. To characterize the water wettability of the nanofibers, 1 µl drop of ultrapure water (Sartorius) was used to measure the contact angle (OCA 15EC goniometer, Dataphysics, USA). 2.3 In vitro piperine release kinetics Piperine loaded nanofiber mats (20 mm x 20 mm x 0.14 mm) were placed in vials containing 20 ml of phosphate buffered saline (PBS, pH 7.4) with 0.5% of Tween-20 in an incubator shaker at 37o C and 100 rpm. The release of piperine from the nanofiber mats was


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measured for up to 5 days at 24 hour intervals. At each time point the solution was replaced with fresh buffer solution. To determine the total amount of drug loaded in the PIP10, PIP20 and PIP30 fibers in PCL/GEL (50:50) blend, the nanofibrous mats of 10 mg weight were placed in 20 ml mixture of PBS and methanol. The pH was initially fixed at 7.4 to dissolve gelatin and changed to 9.0 to dissolve PCL. The concentration of the released PIP was measured using a spectrophotometer (Shimadzu, UV-1700 PharmaSpec) at 342 nm wavelength. The concentration was determined from a standard curve prepared by serial dilutions of a solution of known concentration. Three independent replicates were used for each composition and the data are presented as mean ± S.D. for n=3. 2.4 Cell studies Human cervical cancer cells (HeLa), human breast cancer cells (MCF-7), mouse fibroblasts (NIH3T3) (all ATCC, USA) and primary bone marrow derived human mesenchymal stem cells (hMSCs, Stempeutics, India, 25 year old male donor) were used for this study. Dulbecco's modified Eagle medium (DMEM, Gibco) was used for culturing HeLa, MCF-7 and NIH3T3 cells supplemented with 10% fetal bovine serum (FBS, Gibco). Knockout DMEM (Invitrogen) supplemented with 15% MSC-qualified FBS (Invitrogen) and 1% Glutamax (Invitrogen) was used for the hMSCs. 1% antibiotic mixture of penicillin– streptomycin (Sigma) was added in all media. Cells were cultured in a standard incubator with humidified atmosphere and 5% CO2 at 37° C. The cell culture medium was refreshed every 3 days until cells reached to 70-80% confluency. 0.25% Trypsin-0.5mM EDTA (Gibco) was used to remove adhered cells from the tissue culture flask. Nanofiber mats were cut to fit into individual wells of a 48- well plate in the form of circular discs (≈ 10 mm diameter) and sterilized for 30 min in ultra violet light. Prior to cell seeding, 0.2 ml of complete culture medium was added in each well and centrifuged at 2500 rpm for 2 min to



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remove the trapped air pockets and wet the mat. The culture medium was refreshed with 0.4 ml of complete medium containing 5 x 103 cells. 2.5 Cell proliferation and morphological characterization Cell attachment and proliferation were studied using a combination of DNA quantification assay and fluorescence microscopy (Olympus IX-71). Cell attachment was observed at day 1 and proliferation was assessed at days 3, 5 and 7. For quantification of DNA, 0.2 ml of the lysis solution (0.02% sodium dodecyl sulfate and 0.2 mg/ml proteinase K) was added to the cells and incubated for 12 hours. Lysate was collected in a 96 well plate and an equal volume of the Picogreen (Invitrogen) dye solution was mixed following supplier’s protocol. The DNA content was quantified by measuring the fluorescence intensity using 485 nm excitation and 525 nm emission in a wellplate reader (Synergy HT, BioTek, USA). A solution of known concentration of dsDNA was serially diluted and a standard plot was generated for calculating the dsDNA content. To fix cells for morphological study using fluorescence microscopy, 3.7% formaldehyde solution used for 20 min. 0.2% Triton X-100 (Sigma) solution was added for permeabilization of the cells. To stain F-actin and nucleus, 6.6 µM Alexa Fluro 546 (Invitrogen) for 30 min and 14.3 mM DAPI for 10 min, respectively, were used. 2.6 Estimation of reactive oxygen species (ROS) and cell death ROS generation in HeLa cells on the fiber mats containing 30 wt% PIP was measured using 2,7-dichlorofluoresceindiacetate (DCFH-DA; Sigma-Aldrich, USA) staining. DCFHDA is non-fluorescent and can readily diffuse into the cells through the plasma membrane where it is hydrolyzed to DCFH and further converted into the highly fluorescent DCF through oxidation, in the presence of H2O2. For estimation of dead cells propidium iodide (PI, Sigma Aldrich) was used. After staining with PI, fluorescence activated cell sorting (FACS, BD FACSVerse) was performed. 2×104 cells/well were seeded onto the mats cut to fit into a


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12 well plate and cultured for 3 days. Subsequently, cells were suspended by trypsin, washed twice with PBS, centrifuged at 2500 rpm for 3 min and the supernatant was discarded. The cells were suspended in 0.3 ml culture medium containing 30 µg/ml of DCFH-DA and 20 µg/ml of PI, and incubated for 30 min at 37 0C in the dark and analyzed using FACS. 2.7 Cell cycle analysis 2×104 HeLa cells were seeded per well onto the nanofibrous mats in 12 well plates and cultured in complete culture medium for 3 days. Cells removed from the mats using 0.25% Trypsin-0.5mM EDTA and washed twice using PBS. Cell pellet after centrifugation was resuspended in 0.5 ml of 70% ethanol (pre-cooled at -20 oC) to fix the cells. The fixed cells were centrifuged at 4000 rpm for 2 min. The supernatant was discarded and cells were suspended in 0.5 ml PBS containing 0.2% Triton X-100 and incubated for 20 min on ice. Thereafter, the cells were collected by centrifugation and resuspended in 0.5 ml PBS containing 10 µg/ml of RNase A (Sigma) and 20 µg/ml of PI, and incubated for 30 min in the dark. Cells were analyzed by FACS. 2.8 Statistical analysis All statistical analysis was carried out using 1-way ANOVA (Analysis of variance) with Tukey’s test for three independent samples. Differences were considered significant for p < 0.05.

3. Results and discussion 3.1 Morphology of nanofibers Drug loaded nanofibers of PCL and PCL/GEL blends were prepared by electrospinning. TFE was used to prepare fibers of PCL/GEL blend by electrospinning. To facilitate homogenous mixing of PCL and GEL in solution for preparation of the blend fibers and thereby improve the morphology of the resultant fibers, a small volume of acetic acid was added to the solution, as suggested earlier 49. After adding PIP no discernable changes in



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the solution viscosity was observed and hence the same parameters were used for all the concentration of PCL/GEL/PIP. The drug release profile from the PCL/GEL (50:50) blend fibers was determined to be better than from neat PCL and the 75:25 blend as discussed below. All the physico-chemical characterization for the nanofibers is thus reported for the 50:50 blend unless otherwise stated. Representative SEM micrographs of nanofibrous mats of PCL/GEL and PCL/GEL_PIP30 are shown in Figure 1(a). The mean diameters of all the fiber mats for PCL and the different PCL/GEL blends are listed in Table 1. There was a marginal decrease in the mean diameter with the addition of piperine although the differences were not statistically significant. 3.2 Chemical analysis and wettability of nanofibers A number of different techniques were used for characterization of the electrospun nanofibers. Figure 1(b) shows the different FTIR spectra. FTIR spectrum of PCL fiber shows characteristic peaks at 2940 cm-1, 2860 cm-1 (C-H bond stretching) and a strong carbonyl bond stretching at 1720 cm-1 (C-O-C). GEL fibers show peaks at 1650 cm-1 (amide I) and 1540 cm-1 (amide II) due to the bending of N-H bond and stretching of the C-N bonds, respectively

50-51

. PCL/GEL blend showed the characteristic peaks of both PCL and GEL.

The chemical structure of piperine is shown in the inset and its characteristics peaks in the FTIR spectra are seen at 1633cm-1, 1580 cm-1 and 1449 cm-1 due to the aliphatic and aromatic C=C and N-C=O stretching vibration and C-H bond stretching

52

. These piperine

peaks are also seen for PCL/GEL nanofibers containing piperine at 10 wt%, 20 wt % 30 wt% although the peaks in the 10 wt % and 20 wt % PIP are weak. These data confirm that the drug was successfully loaded into the blend polymeric nanofibers. Figure 1(c) shows the water wettability of different fibers including PCL, the PCL/GEL blend and PIP loaded blend. Results showed that PCL nanofibrous mat is hydrophobic showing a contact angle of 119° + 3° consistent with the known hydrophobic


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character of the polymer

53

. After the addition of GEL, water wettability increased as

expected and is reflected by the decrease in the contact angle. This is due to the presence of hydrophilic groups such as amine and carboxyl in gelatin 54. GEL tends to readily dissolve in water and in the blend its dissolution is expected to facilitate the hydrolytic degradation of PCL augmenting the drug release as well as cell attachment. The addition of piperine into the blend did not significantly change the contact angle. 3.3 Piperine release kinetics The drug release profile from different weight ratios of PCL and GEL and the effect of the drug loading are compiled in figures 2(a) and 2(b), respectively. The motivation of blending GEL with PCL was to facilitate rapid drug release. It was envisaged that the hygroscopic GEL will increase water wettability of the fibers (figure 1(c)) thereby augmenting hydrolytic degradation of PCL and subsequent drug release. Different blends ratio of PCL and GEL was prepared to maximize the release of piperine. As seen in figure 2(a), the fraction of piperine released scales with the GEL content in the fibers with the slowest release from neat PCL fibers. Within 5 days, PCL/GEL (50:50) released 84% of the piperine whereas in PCL/GEL (75:25) and neat PCL released only 47% and 10% of the drug, respectively. Figure 2(b) compiles the release of piperne from PCL/GEL (50:50) fiber mats containing 10, 20 and 30 wt% of the drug. All the fiber mats released ≈20% of the incorporated drug within 1 day and ≈ 50% by day 3. At day 5, the fraction of the encapsulated piperine released was 65 %, 72 % and 84 % for the fibers with 10%, 20% and 30% drug, respectively. The fractional release is essentially independent of drug loading (Figure 2(b)). The concentration of the eluted drug in solution is plotted in Figure S1. Note that the piperine concentration in solution was highest for PCL/GEL (50:50) fibers and thus were used for the cell studies for maximal anti-cancer activity. The total drug loaded (mass of



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drug/ mass of polymer) was determined to be 6.0 + 2%, 14.0 + 3% and 23.0 + 4% for PIP10, PIP20 and PIP30 fibers, respectively, where the data are shown as mean ± S.D. for n=3. 3.4 Biological studies 3.4.1 Cytotoxicity of cancer cells Cytotoxicity of the eluted PIP was studied on HeLa and MCF-7 cells, which are well established models for cervical and breast cancer, respectively 55. The DNA content taken as a measure of the number of cells was assessed at days 1, 3, 5 and 7 after seeding the cells on these nanofiber mats and is compiled in figures 3(a) and 3(b), respectively. Figure S2 presents the fluorescence micrographs of the stained nuclei of HeLa cells. For HeLa cells, on day 1, there was no statistically significant difference in the DNA content between control (without drug) and 10 wt % PIP (figure 3(a)). But fewer cells (less DNA content) were seen on the fibers with 20 wt% and 30 wt% PIP. At days 3, 5 and 7, fiber mats with piperine showed significantly fewer cells compared to the control fibers where the cell numbers increased rapidly with time. Cell number increased on the 10% and the 20% PIP fibers albeit markedly slower than on the control fibers. On the 30% PIP fibers the cell number at day 3 was marginally higher than at day 1 and dropped by day 5. Fluorescent micrographs in figure S2 qualitatively corroborate these trends. Whereas the cells had proliferated to a confluent layer on the control, fewer cells (stained nuclei) were seen with increasing drug loading. These differences were especially discernable at days 5 and 7. MCF-7 cells exhibited trends similar to that of HeLa cells as seen in figure 3(b). On day 1, the DNA content on the control fibers (without drug) was higher than the drug loaded fibers. On days 3, 5, and 7 the cells numbers on the 10, 20 and 30 wt% PIP fibers were significantly lower compared to the control and the cell number scaled inversely with the increase in drug content. Taken together, the DNA content assay and the fluorescent


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microscopy suggest that piperine loaded nanofibers can profoundly retard the proliferation of cancer cells. 3.4.2 Morphology of cancer cells Cell morphology reveals important characteristics of cell viability and function

56

.

Fluorescence microscopy revealed morphological changes for HeLa cells on the nanofibers (Figure 4). On the control fibers (PIP0), cells appeared well spread and enlarged whereas on piperine loaded mats cells were round and less spread. Upon increasing the concentration of PIP the number of rounded cells also increased. Cells are more rounded on the PIP30 fibers compared to PIP10 and PIP20. These morphological changes also suggest that the cancer cells tend to be less viable and unhealthy on the PIP eluting fibers unlike the control fibers. 3.4.3 Cytocompatibility against non-cancerous cells To investigate the compatibility of the piperine eluted from the nanofibers against non-cancerous normal cells we assayed the response of NIH3T3 fibroblasts and hMSCs. Since PIP30 was most effective in killing cancer cells hence we measured the proliferation of the normal cells on PIP30 and compared with PIP0 by measuring the DNA content at day 1, 3, 5 and 7 (figures 5(a) and 5(b)). There was no significant difference in cell attachment of NIH3T3 and hMSCs (day 1). With prolonged culture (day 3, 5 and 7) we observed fewer cells on PIP30 than on PIP0 although the cells proliferated and the DNA content increased on both PIP30 and PIP0. At day 7, we observed that the DNA content on PIP30 was approximately one third lower than on the control for both fibroblasts and hMSCs. Fluorescent micrographs revealed that most of these normal cells were well spread and healthy on PIP30 similar to PIP0 as seen in the representative images in figure S3 unlike the rounded cancer cells on PIP30 (figure 4). These results indicate that piperine eluted from the PIP30 fibers is highly potent in inhibiting proliferation cancer cells with more than two-third reduction in cell numbers



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compared to the control (PIP0) at day 7. The drug modulated the proliferation of the normal cells but was less effective leading to one-third reduction in cell numbers than the control. Similar observations have been reported by others. Whereas piperine inhibited the proliferation of prostate cancer cells at 80 µM and was more effective at 160 µM it was toxic to normal prostate epithelial cells at higher concentration (160 µM)

57

. Thus, the piperine

loaded fibers prepared here may be considered for use in cancer treatment. Interestingly, we observed significant anticancer activity at much lower concentration than has been reported earlier. We attribute this to the difference in the mode of exposure. In previous studies the drug was added to the medium in soluble form and the bulk concentration was representative of the drug exposure to the cells. In this work, the cells are cultured on the drug eluting fiber mats. Thus, the local drug concentration in vicinity of the cells is likely to be markedly higher than the bulk concentration shown in figure S1. 3.4.4 Role of ROS and estimation of cell death in cancer cells ROS are the by-products of cellular metabolic activity such as mitochondrial respiratory chain and ß- oxidation in peroxisomes

58-59

. Whereas ROS has many important

physiological roles, a large increase in cellular ROS can induce cell death and often constitutes the basis of action of many anti-cancer drugs60-62. A marginal increase in ROS level may lead to fleeting cellular variations whereas high amount of ROS results in oxidative stress, which causes oxidative damage of the biological macromolecules such as nucleic acid, proteins, lipids, and cellular organelles

62

. This oxidative stress leads to the modulation of

biological functions and may cause cell death. In cancer cells, ROS production is typically a little higher than in normal cells resulting from the higher metabolic activity, increased signaling receptor and oncogenic activity63-64. This increased level of ROS is essential for cancer cell survival since the cancer cell cycle progression is mediated by growth factors and receptors such as tyrosine kinase, which is needed for ROS activation63, 65. However, it has


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been shown that very large amounts of ROS in many cancer cells can lead to cell death via apoptosis, autophagy and DNA fragmentation65-67. In general, the removal of ROS is maintained by the scavenging systems of the cell such as glutathione, pyridine nucleotide and thioredoxin, which participate in cell signaling and inflect the cell function as well as apoptotic cell death

66

. Phytochemicals such as

curcumin and apigenin that have been investigated for anti-cancer activity are reported to induce apoptosis in cancer cells through ROS generation 68-69. Similar role of ROS in piperine induced apoptosis in HRT-18 cells was reported by Yaffe et al. 46 where HRT-18 cells treated with ROS scavenger such as N-acetylcysteine showed the reduction in piperine induced apoptosis. Exposure of cells to piperine is shown to induce large increase in cellular ROS 46. Interestingly, piperine induced ROS generation and DNA fragmentation in lung cancer cells but not in non-cancerous lung fibroblasts 47 suggesting that piperine may show potent activity specifically against cancer cells with minimal side effects on non-cancerous cells. To investigate the production of cellular ROS by the piperine eluted from the nanofibers, the ability of cells to convert DCF into the green fluorescent DCFF-DA was used as a measure of ROS. Fluorescence micrographs of the stained HeLa cells show large increase in ROS level in cells on the piperine loaded fibers compared to the cells on the control fibers without the drug (figure 6 (a)). Thus, the death of cancer cells on the piperine loaded fibers (figures 2(a) and 2(b)) may be ascribed to the ROS generation by the eluted drug. Flow cytometry was used to further quantify ROS generation and cell death and to investigate a possible cause. The flow cytometry histogram in figure 6(b) and the bar plot in figure 6(c) indicate that the ROS accumulation in cells on the PCL/GEL_PIP30 fibers increased as compared to the control PCL/GEL fibers from 49.3+ 2.1 % to 66.0 + 3.4 %. Live cells that possess intact membrane can eliminate a variety of dyes including PI 70. Since PI is



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a membrane impermeant dye, it is not taken up by the viable cells but can enter into cells if with compromised membranes 70-71. It can be seen that the fraction of dead cells (PI stained) on the control fibers is ≈ 11.0 % whereas as it is ≈ 26.6 % on PCL/GEL_PIP30. This increase is in good agreement with the increased cell death on the piperine eluting fiber mats as described above (figures 3(a) and 3(b)). The fraction of dead cells not expressing ROS stayed essentially unchanged at ≈10% on both the fibers. The fraction of cells that stained for both ROS and PI was 3.0 + 2.1% on the control fibers which increased to 19.6 + 3.5 % on the drug loaded fibers suggesting increased cell death due to ROS production. 3.4.5 Cell cycle analysis in cancer cells Cell cycle check points play a crucial role in controlling the intracellular mechanisms and eventually cell functions. We analyzed the effect of piperine on possible changes in the cell cycle of HeLa cells on the PCL/GEL_PIP30 fibers at day 3 by flow cytometry. In the control fibers without piperine, the fraction of HeLa cells in the G0/G1, S and G2/M phases and dead cells were found to be 70.9 + 1.2 %, 11.9 + 1 % , 15.6 + 1.3 % and 2.9 + 0.5 %, respectively (figures 7(a), and 7(c)). The cells on the piperine loaded fibers showed significant changes in the cell cycle distribution and dead cells number also increased with values of 54.3 + 0.9 %, 7.8 + 1.1%, 30.5 + 1.5% and 7.8 + 0.4 % for G0/G1, S and G2/M phases and dead cells, respectively. The accrual of the cells in the G2/M phase increased whereas in the G0/G1 phase decreased (figure 7(b) and (c)) indicating that piperine induced cell cycle arrest in the G2/M phase of the HeLa cells. Lin et al reported that piperine caused G2/M phase arrest in A549 lung cancer cells

47

. Similarly, another tumor inhibitory

phytochemical curcumin shows cell cycle arrest at G1/S phase in human prostate cancer cells PC-3 and LNCaP

72

and G2/M phase arrest in T24 bladder cancer cells

73

. The exact

molecular mechanism underlying the action of phytochemicals on cell cycle progression in


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cancer cells to inhibit their growth is not well established although it is believed to play an important role in inducing apoptosis in many cancer cells 7, 74. As shown in figure 7(b), piperine loaded fibers induced elevated ROS levels in the cancer cells. It has been reported that high ROS level can damage to the DNA and can lead to cell cycle arrest47, 75-76 inducing apoptosis. Similar findings have been reported for curcumin, a phytochemical extracted from turmeric, another spice routinely used in many Asian dishes with demonstrated anti-cancer activity

77

. Taken together, the results of this study suggest

that the sustained release of piperine from the polymeric nanofibers induces generation of ROS and cell cycle arrest in the cancer cells, which appear to induce cell death on the cancer cells cultured on the piperine loaded fibers. In addition to the data presented here for cervical and breast cancer cells, piperine is reported to show anti-cancer activity against a variety of cancer cells including prostate cancer, colon cancer and lung cancer

47, 57, 78

. Thus, such a

nanofiber mat could be developed toward a post-surgical patch for clinical use in cancer treatment for localized sustained delivery of piperine to minimize relapse while serving as a scaffold for tissue repair and regeneration after removal of the tumor.

4. Conclusion Nanofibers of PCL/GEL blends loaded with piperine were prepared by electrospinning. The nanofibers exhibited sustained release behavior of the encapsulated drug and the release kinetics could be tailored by varying the PCL/GEL ratio. The eluted drug is shown to inhibit the growth of HeLa and MCF-7 cancer cells through generation of ROS and cell cycle arrest. The drug loaded fibers is shown to have significantly less inhibitory effect on non-cancerous cells than on cancer cells. Thus, such a piperine loaded fiber mat can be developed further for clinical use in cancer treatment.

Acknowledgements



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This work was funded by the Council for Scientific and Industrial Research (CSIR), India. K.C. was supported by the Ramanujan Fellowship from the Department of Science and Technology (DST), India. The Advanced Facility for Microscopy and Microanalysis (AFMM) and the Central Flow Cytometry Facility of IISc are acknowledged for access to equipments.

Supplementary Information Release profile showing concentration of piperine in solution released from the nanofibers, fluorescence micrographs of stained nuclei (blue) for HeLa cells (Scale bar = 0.1 mm) and representative fluorescence micrographs at day 3 showing actin filament (red) and nuclei (blue) (Scale bar = 100 µm) (a) NIH3T3 cells (b) hMSCs. This information is available free of charge via the internet at http://pubs.acs.org/.


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List of Tables: Table 1: Sample nomenclature and fiber diameter Constituent (g/ g of mat)

Fiber diameter (nm)

PCL

Gelatin

Piperine

0.50 0.45 0.40 0.35 0.70 0.53

0.50 0.45 0.40 0.35 0 0.17

0 0.10 0.20 0.30 0.30 0.30

Mean ± S.D. 320+80 310+90 270+100 260+85 380+110 290+120

Sample Code PCL/GEL_PIP0 PCL/GEL_PIP10 PCL/GEL_PIP20 PCL/GEL_PIP30 PCL/PIP30 PCL/GEL(75:25)_PIP30


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Figure captions: Figure 1: Characterization of nanofibers: (a) SEM micrograph of PCL/GEL fibers containing 0, 10, 20 30 wt % piperine. (b) FTIR spectra of neat piperine, neat PCL fibers, GEL, PCL/Gelatin blend fibers, and PCL/GEL_PIP30 fibers. Chemical structure of piperine is shown at the top. (c) Water contact angle of PCL, PCL/GEL and PCL/GEL_PIP30. Statistically significant difference with respect to neat PCL (p

Engineering a Piperine Eluting Nanofibrous Patch for Cancer

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