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Chitosan-coated cinnamon/oregano -loaded solid lipid nanoparticles to augment 5-Fluorouracil cytotoxicity for colorectal cancer: extracts standardization, nanoparticles optimization and cytotoxicity evaluation Kamel M Kamel, Islam A Khalil, Mostafa E Rateb, Hosieny Elgendy, and Seham Elhawary J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03093 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Journal of Agricultural and Food Chemistry

Chitosan-coated cinnamon/oregano-loaded solid lipid nanoparticles to augment 5-Fluorouracil cytotoxicity for colorectal cancer: extracts standardization, nanoparticles optimization and cytotoxicity evaluation Kamel M. Kamel 1, Islam A. Khalil

2, 3, §

, Mostafa E. Rateb

4, 5

, Hosieny Elgendy

1

,

Seham

Elhawary 6

1

Department of Pharmacognosy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6th of October, Giza, (12566) Egypt, 2 Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6th of October, Giza, (12566) Egypt, 3Nanomaterials Lab., Center of Material Science (CMS), Zewail City of Science and Technology, 6th of October, Giza, (12588) Egypt, 4School of Science & Sport, University of the West of Scotland, Paisley (PA1 2BE), Scotland, U.K, 5Pharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, Beni-Suef (62511), Egypt, 6 Pharmacognosy Department, Faculty of pharmacy, Cairo University, Cairo, Egypt. §

Author was conducted and analyzed data of nanoparticles part, write and reviewed the

manuscript.

*Correspondence to: Islam A. Khalil, PhD Tel.:+20 1090140748 Fax: +20 238247417 Email: [email protected] ; [email protected]

1 Environment ACS Paragon Plus


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ABSTRACT

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This study aimed to coat lipid-based-nanocarriers with chitosan to encapsulate nutraceuticals,

3

minimize opsonization and facilitate passive-targeting. Phase-one was concerned with

4

standardization according to WHO. Qualitative-analysis using LC-HRMS/MS investigated

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the active-constituents especially reported cytotoxic-agents. Cinnamaldehyde and rosmarinic

6

acid was selected to be quantified using HPLC. Phase-two was aimed to encapsulate both

7

extracts in solid lipid nanoparticles (core) and chitosan (shell) to gain the advantages of both

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materials properties. The developed experimental-model suggested optimum formulation

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with 2% lipid, 2.3% surfactant and 0.4% chitosan to achieve particle-size of 254.77nm,

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polydispersity-index of 0.28, zeta-potential of +15.26, and entrapment-efficiency % of 77.3%

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and 69.1% for cinnamon and oregano, respectively. Phase-three was focused on the

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evaluation of cytotoxic-activity unencapsulated/encapsulated cinnamon and oregano extracts

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with/without 5-Flourouracil on HCT-116 cells. This study confirmed the success of the

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suggested combination with 5-Flourouracil for treating human colon carcinoma with low

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dose leading to decreasing side-effects and allowing uninterrupted therapy.

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Keywords: Cinnamon, Oregano, 5-Fluorouracil, Chitosan, Solid lipid nanoparticles,

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colorectal cancer


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Introduction

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Colorectal cancer (CRC) is considered one of the most fatal diseases throughout the world. It

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always comes in an advanced order in the list of causes of mortality throughout the world

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which represents a huge public health problem. Worldwide, it comes third in the most

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common cancers after lung and breast, and fourth in cancer related mortality cases. Its

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incidence usually starts in people between 40-50 years old and increase with aging 1. The

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current widespread cancer therapy consists of surgery combined with chemo- and/or

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radiotherapy while gene therapies did not show the expected satisfied application results yet

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2

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Capecitabine (Xeloda®), Irinotecan (Camptosar®), Oxaliplatin (Eloxatin®) and Trifluridine

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and tipiracil (Lonsurf®). 5-Fluorouracil (5-FU) alone or in combination with oxaliplatin is

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often used 3. Unfortunately, the current therapies are associated with serious side effect, high

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cost and 50% recurrence rates due to the chemo-resistance 4.

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All these limitations keep the treatment of CRC a clinical challenge and push towards finding

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and developing new safer treatment strategies that can help overcome chemo-resistance and

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sensitize cancer cells towards chemotherapy drugs 5. Nutraceuticals, natural products, both

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total extracts and single constituents, showed the ability to enhance or synergize the

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anticancer activity of standard chemotherapy drugs. Their mechanisms of actions were

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proved at the molecular level; this makes the scientists to look upon them as alternative or

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complementary support for the traditional chemotherapy drugs 2.

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Cinnamon is one of the most promising anticancer herbs. It is widely used as spice, flavoring

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and preservative in food industry. It has been used in medieval medicine for treatment of a

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variety of diseases including arthritis, coughing, sore throats, etc.

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derived from different sources and it is the most common form of cinnamon. 7 Cassia is the

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predominant variety found in retail trade and pastry shops.

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have cytotoxic activity against various types of cancer cell lines like human cervix carcinoma

. Some common drugs used for colorectal cancer include 5-Fluorouracil (5-FU),

8

6

Cinnamomum cassia is

Its aqueous extract proved to



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Cinnamomum cassia, showed potent cytotoxic activity against human promyelocytic

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leukemia cells. Its mechanism was revealed via transduction apoptosis via ROS generation

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followed by mitochondrial permeability transition and cytochrome c release to the cytosol 10.

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2'-Hydroxycinnamaldehyde,

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benzoyloxycinnamaldehyde, prepared by the reaction of 2'-Hydroxycinnamaldehyde and

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benzoyl chloride; both proved to be strong inhibitors for in vitro growth of 29 kinds of human

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cancer cells and in vivo growth of SW-620 human tumor xenograft without the loss of body

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weight in nude mice 11.

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Oregano (Origanum vulgare) is also a widely used spice known by its volatile oil. It has been

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used traditionally for treatment of respiratory disorders, indigestion, and rheumatoid arthritis

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12

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in both time- and dose- dependent manner via activation of both intrinsic and extrinsic

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apoptotic pathways. The more important is that these effects were selective for cancer cells,

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and achieved by whole extract not by a specific component 13. In vivo, aqueous extract was

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tested for its effect on anti-oxidant status in l,2-dimethylhydrazine-induced rat colon

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carcinogenesis. It succeeded in reversing the levels of anti-oxidants superoxide dismutase,

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catalase, reduced glutathione, glutathione reductase, glutathione peroxidase and glutathione-

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S-transferase 14.

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Various nutraceuticals have limited bioavailability due to low solubility which affects human

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body absorption. Furthermore, stability issue of nutraceuticals like hydrolysis, oxidation, and

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photolysis urge the need for stabilization. The limited bioavailability and stability of

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nutraceuticals can be controlled by nanoencapsulation like phytososmes, micelles, and

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polymer nanoparticles using different carrier categories 15.

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Encapsulation of nutraceuticals into micro- and nanocarriers have recently emerged as

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suitable techniques for the protection of its bioactive compounds and improve biological

. Cinnamaldehyde which is the main active compound isolated from the stem bark of

isolated

from

Cinnamomum

cassia

and

2'-

. Its ethanolic extract arrested growth and killed cells of colon adenocarcinoma Caco2 cells


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activity which can be achieved by several methods. One of the famous approaches for

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encapsulation is solid lipid nanoparticles technique (SLNs) which is able to control drug

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release, improve drug permeation into mucosa and enhance therapeutic activity

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Nevertheless, SLNs physicochemical properties are still needed to be optimized like size as

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key factor in the biodistribution of long-circulating nanoparticles, tissue extravasation, tissue

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diffusion, and avoid hepatic filtration and kidney excretion. Furthermore, SLNs are easily

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recognized by immune system hydrophobicity and surface negativity which determines the

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level of opsonization and subsequent clearance. Therefore, chitosan as a cationic polymer

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with low molecular weight and hydrophilicity makes it suitable to minimize the opsonization

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through coating SLNs with chitosan. Furthermore, the positive charge with nanoscale size

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makes the core/shell system suitable for passive targeting of loaded cargo.

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The main objective of the current study is to encapsulate nutraceuticals (Cinnamon and

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Oregano) in SLNs coated with chitosan to improve its chemotherapeutic activity and used as

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a complementary therapeutic to conventional chemotherapeutic agents like 5-Fluorouracil.

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Cinnamon and oregano are selected and examined due to their unique mechanism in treating

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CRC due to exclusive diversity of active constituents with cytotoxic activity. Standardization

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was conducted according to WHO guidelines 17. The advantages of SLNs for encapsulation

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of nutraceuticals and limitations of its physicochemical properties directed the study to

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optimize its characteristics first. SLNs coated with chitosan (SLN-Cs) was developed aiming

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at delivery of cinnamon and oregano extracts to improve stability and bioavailability. Several

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factors were studied to optimize SLN-Cs with statistical experimental design (Fig. 1).

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Furthermore, cytotoxic activity evolution on human colon cancer cell line HCT-116 was

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carried out to explore the synergistic activity with 5-FU (standard chemotherapeutic for CRC)

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to be used as complementary or alternative to chemotherapy.

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Materials and Methods

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Materials 5 Environment ACS Paragon Plus

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.


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Cinnamon bark (Cinnamomum cassia) and Oregano leaves (Origanum vulgare) were bought

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from the local market and authenticated by taxonomy department then were grinded to fine

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powder. Ethanol (HPLC grade), Methanol (HPLC grade), Acetonitrile (HPLC grade),

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Dimethylsulfoxide,

5-fluorouracil,

propylene

glycol,

chitosan

(Mw

260,000

Da),

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cinnamaldehyde, rosmarinic acid, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium

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bromide (MTT), acridine orange and ethidium bromide were purchased from Sigma-Aldrich

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(St. Loius, MO, USA). Compritol 888 ATO (glyceryl behenate, a mixture of ∼15% mono-,

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50% di- and 35% triglycerides of behenic acid) and Gelucire 40/14 (PEG glyceride) were

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kind gift of Gatteffose, France. Poloxamer 407 was obtained from BASF (Florham Park, NJ).

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Human colon carcinoma (HCT 116; ATCC® CCL-247™) cells were purchased from

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American Type Culture Collection (ATCC, NY). 3,3',5,5'-tetramethyl benzidine was

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purchased from Kirkegaard and Perry Lab. Gaithersburg, MD, USA. Rabbit polyclonal to

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caspase-3 was purchased from Abcam Inc., Cambridge, MA, USA. Polyclonal Goat anti-

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rabbit peroxidase conjugate was purchased from Jackson Immunsearch Lab, USA.

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MitoTracker® Mitochondrion-Selective probe, Chloromethyl-X-rosamine purchased from

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Life Technologies, Carlsbad, USA. All other reagents were of analytical grade and used as

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received.

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Preparation of Cinnamon and Oregano extracts

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100 gm of commercially available Cinnamomum cassia bark and Origanum vulgare leaves

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were crushed and extracted by 70% Ethanol by maceration aided by sonication for 3

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consecutive times. Extract was then evaporated under suction by rotatory evaporator (Model

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Heidolph rotavapor vv 2000/WB 2000, Germany) then lyophilized (Free-zone, Labconco,

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USA) to dry powder.

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Qualitative analysis of Cinnamon and Oregano extracts using LC-HRMS/MS


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Qualitative analysis for cinnamon and oregano extracts were conducted by gradient

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separation using a Poroshell EC-C18 RP analytical HPLC column (2.7µm, 2.1 × 100 mm,

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Agilent, USA) with a mobile phase of 0–100% acetonitrile over 25 min followed by 100%

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acetonitrile over 5 min at a flow rate of 0.5 mL/min. High resolution mass spectral data was

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obtained from a Thermo Instruments MS system (Finnigan LTQ / LTQ Orbitrap) coupled to a

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Thermo Instruments HPLC system (Accela PDA detector, Accela PDA autosampler and

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Accela Pump) with the following conditions: capillary voltage 45 V, capillary temperature

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260 °C, auxiliary gas flow rate 10–20 arbitrary units, sheath gas flow rate 40-50 arbitrary

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units, spray voltage 4.5 kV, mass range 100–2000 amu (maximum resolution 60000) 18.

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Quantitative analysis of Cinnamon and Oregano extracts using HPLC

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Cinnamaldehyde standard, rosmarinic acid standard, cinnamon extract, oregano extract and

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formula (55%:45%w/w for Cinnamon to Oregano, respectively) were analyzed using an

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agilant 1260 with quaternary pump, autosampler, ZORBAX SP-C18 column (4.6 x 150mm x

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5µm, Agilent, Palo Alto, CA, USA), photodiode array detector (PDA) and chemstation

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software.

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Cinnamaldehyde quantitation for Cinnamon extract

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Cinnamaldehyde was quantified in cinnamon extract according to the method developed by

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Gursale and coworkers 19. Mixture of methanol:acetonitrile:1% acetic acid glacial (35:20:45)

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was used at flow rate of 1.0 cm3/min, and detection was done at 221 nm and injection volume

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was 10 µL. Serial dilution of standard stock solution was done and calibration curve of was

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established.

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Rosmarinic acid quantitation for Oregano extract

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Rosmarinic acid was quantified in oregano extract according to the modified method

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developed by Couto and coworkers

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acetonitrile:0.1% acetic acid (50:50) at flow rate of 1.0 cm3/min. Detection was done at 230

20

. Mobile phase was consisted of a mixture of



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nm and injection volume was 5 µL. Serial dilution of standard stock solution was done and

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calibration curve of was established.

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Development of extract-loaded core/shell (SLN-Cs) nanoparticles

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Preparation of SLN-Cs nanoparticles

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Core/Shell nanoparticles composed of SLNs core coated with chitosan as a shell. SLNs

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prepared by hot melt emulsification homogenization/ultrasonication method

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phase, poloxamer 407 was dissolved in propylene glycol to distilled water (1:1) at 75 °C.

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Simultaneously, oil phase was prepared by melting solid lipids (Compritol® 888ATO and

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Gelucire® 44/14 at 1:3 ratio) at 75 °C. The aqueous phase was added gradually to oily phase

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while homogenization at 10,000 rpm (GLH 850, Omni inc.,USA) followed by sonication (3

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min, 3s on/off and 50% voltage efficiency; Model LC 60/H, Elma, Germany). During

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sonication, the obtained emulsion was cooled ice bath to decrease emulsion temperature

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gradually. The suspension was then cooled to room temperature. Appropriate amounts

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chitosan was dissolved in 1% acetic acid solution followed by adding to SLNs suspension

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gradually during mixing for 30 min at room temperature. The obtained suspension was stored

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at 4 °C. For extract-loaded core/shell nanoparticles, the required amount of extract was mixed

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with molten oily phase before emulsification with aqueous phase.

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Experimental design

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This study was designed to optimize SLN-Cs formulations followed be loading cinnamon and

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oregano extracts in optimum formulation. Box-behnken design was applied to optimize the

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formulation variables using Design Expert® 10.0.3.1 software (Stat-Ease, Inc., Minneapolis,

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MN, USA.). The selected independent variables lipid phase (mixture of Compritol® 888ATO

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and Gelucire® 44/14) concentration, poloxamer concentration and chitosan concentration as

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presented in Table 1. A total of 17 runs were required (12 factorial and 5 center points). The

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dependent variables were analyzed using analysis of variance (ANOVA) to identify of the 8 Environment ACS Paragon Plus

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. For aqueous

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significance of each variable and interaction between variables (P

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