Curcumin nanoformulations

Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations Curcumin nanoformulations

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Curcumin nanoformulations: A review of pharmaceutical properties

and preclinical studies and clinical data related to cancer treatment

Ornchuma Naksuriyaa,b, Siriporn Okonogia, Raymond M Schiffelersc, Wim E Henninkb,*

a Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Suthep Rd, Mueang, Chiang Mai 50200, Thailand

b Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, Utrecht 3805 TB, The Netherlands

c Department of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands

a r t i c l e i n f o

Article history:

Received 28 November 2013

Accepted 22 December 2013

Available online 15 January 2014

Keywords:

Curcumin

Cancer

Nanoformulation

Drug delivery

Nanomedicine

Clinical studies

a b s t r a c t Curcumin, a natural yellow phenolic compound, is present in many kinds of herbs, particularly in Cur-cuma longa Linn (turmeric) It is a natural antioxidant and has shown many pharmacological activities such as anti-inflammatory, anti-microbial, anti-cancer, and anti-Alzheimer in both preclinical and clinical studies Moreover, curcumin has hepatoprotective, nephroprotective, cardioprotective, neuroprotective, hypoglycemic, antirheumatic, and antidiabetic activities and it also suppresses thrombosis and protects against myocardial infarction Particularly, curcumin has demonstrated efficacy as an anticancer agent, but a limiting factor is its extremely low aqueous solubility which hampers its use as therapeutic agent Therefore, many technologies have been developed and applied to overcome this limitation In this re-view, we summarize the recent works on the design and development of nano-sized delivery systems for curcumin, including liposomes, polymeric nanoparticles and micelles, conjugates, peptide carriers, cy-clodextrins, solid dispersions, lipid nanoparticles and emulsions Efficacy studies of curcumin nano-formulations using cancer cell lines and in vivo models as well as up-to-date human clinical trials are also discussed

1 Introduction

Curcumin is a natural yellow colored phenolic antioxidant and

was ﬁrst extracted in an impure form by Vogel et al [1] The

structure of curcumin was elucidated and it was synthesized by

Milobedeska et al and Lampe et al., respectively [2,3] Many

different plant species synthesize curcumin and the commercial

product (such as from SigmaeAldrich) is isolated from the rhizome

of Curcuma longa Linn in which it is present in relatively high

concentrations The chemical structure of curcumin is shown in

Fig 1 It should be mentioned that the commercially available

curcumin products also contain structurally related compounds

(w17% demethoxycurcumin, and 3% bisdemethoxycurcumin)

Sandur et al reported that the potency for the suppression of tumor

necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kB)

acti-vation ranked curcumin > desmethoxycurcumin >

bisdesme-thoxycurcumin suggesting a critical role of the methoxy groups on

the phenyl rings [4] Moreover, curcumin has the highest

car-dioprotective, neuroprotective and antidiabetic activities of the

three curcuminoids shown in Fig 1 [5e7] Interestingly, the mixture of curcuminoids has increased nematocidal activity as compared to the individual compounds, suggesting a synergistic effect[8]

For many centuries, curcumin in its crude form has been used as spice and dietary supplement as well as component of many traditional Asian medicines[9] In recent studies, it has been shown that curcumin exhibits a wide range of pharmacological activities against many chronic diseases including type II diabetes, rheuma-toid arthritis, multiple sclerosis, Alzheimer’s disease and athero-sclerosis It also inhibits platelet aggregation, suppresses thrombosis and inhibits human immunodeﬁciency virus (HIV) replication Further, curcumin enhances wound healing and pro-tects against liver injury, cataract formation, pulmonary toxicity andﬁbrosis[10e20] Finally, the anti-cancer activity of curcumin has been extensively investigated and it has been suggested as a potential agent for both prevention and treatment of a great variety

of different cancers, including gastrointestinal, melanoma, genito-urinary, breast, lung, hematological, head and neck, neurological and sarcoma[20e23] At a molecular level, curcumin not only in-hibits cell proliferation and metastasis, but also induces apoptosis

by modulating several pro-inﬂammatory factors (e.g interleukin (IL)-1, IL-1b, IL-12, tumor necrosis factor (TNF)-a and interferon

* Corresponding author Tel.: þ31 30 253 6964; fax: þ31 30 251 7839.

E-mail address: W.E.Hennink@uu.nl (W.E Hennink).

Contents lists available atScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / b i o m a t e r i a l s

Biomaterials 35 (2014) 3365e3383

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(INF)-g), growth factors (e.g epidermal growth factor (EGF),

he-patic growth factor (HGF) and platelet-derived growth factor

(PDGF)), receptors (e.g epidermal growth factor receptor (EGFR),

human epidermal growth factor receptor (HER)-2, IL-8R and Fas-R),

transcription factors (e.g signal transducer and activator of

tran-scription (STAT) 3, nuclear factor (NF)-kB, Wilms’ tumor (WT-1)

and peroxisome proliferator-activated receptor (PPAR) g) and

protein kinases, e.g extracellular signal-regulated kinases (ERK),

mitogen-activated protein kinases (MAPK), protein kinase A (PKA)

B (PKB) and C (PKC)[20e25]

An overview of the different indications for which curcumin has

been investigated is shown inFig 2 It has been suggested that,

because of its many pleiotropic properties, curcumin can be more

effective than single pathway targeted anticancer drugs [26,27]

Many preclinical studies have demonstrated that curcumin has

anti-inﬂammatory and anticancer activity [27e30] In a recent

clinical study it appeared that oral administration of curcumin was

well tolerated at doses of 12 g/day which indicates that curcumin is

safe [31] Curcumin can freely pass through cellular membranes

due to its lipophilicity (log P ¼ 2.5)[32] It should however be

mentioned that curcumin has a very low aqueous solubility of only

0.6 mg/ml and is susceptible to degradation particularly under

alkaline conditions[33e35] These characteristics are the cause for

its very low bioavailability resulting in suboptimal blood

concen-trations to achieve therapeutic effects[21,34e36] For instance, in a

study in rats reported by Yang et al a maximum serum

concen-tration of 0.36 0.05mg/ml after an intravenous injection of 10 mg/

kg was reached, whereas 500 mg/kg orally administered curcumin

gave a maximum plasma concentration of 0.06 0.01 mg/ml,

indicating that oral bioavailability was only 1% [37] Similarly,

Shoba et al showed a maximum serum concentration of

1.35 0.23mg/ml at 1 h after administration of an oral dose of 2 g/

kg to rats, whereas healthy man volunteers (weighing 50e75 kg)

receiving a single dose of 2 g curcumin (4 capsules of 500 mg each)

showed an extremely low serum concentration of

0.006 0.005mg/ml at 1 h[38] An obvious approach to improve

the poor biopharmaceutical properties of curcumin is to improve

its aqueous solubility using nanocarriers Nanocarriers have a small size (typically 10e100 nm) and can, besides for solubilization, also

be used for the targeted delivery of drugs[39e44] Nanocarriers can improve the circulation time of the loaded therapeutic agent and may improve its accumulation at the pathological site exploiting the so-called‘enhance permeation and retention (EPR) effect’ [45e48] During the last decades, various types of nano-carriers, such as polymeric micelles and nanoparticles, liposomes, conjugates, peptide carriers etc., for drug delivery/targeting have been investigated and some systems have reached clinical evalua-tions and applicaevalua-tions[49e52] Many studies, as summarized in the next sections, have shown that nanocarriers are suitable for increasing curcumin’s bioavailability and its targeted delivery to tumors and other sites of disease This review focuses on the design and development, the evaluation in preclinical and clinical trials of curcumin nanoformulations, particularly focused on cancer ther-apy In the next section, different curcumin nanoformulations are discussed with emphasis on their pharmaceutical properties In the ﬁnal section of this review the results of curcumin nano-formulations in preclinical studies and clinical evaluations are summarized and discussed

2 Curcumin nanoformulations The nanoformulations discussed in this section primarily aim to achieve increased solubilization of curcumin, but at the same time protect curcumin against inactivation by hydrolysis The formula-tion should be efﬁciently prepared and loaded and should retain curcumin for the required time period Some formulations are aimed for a prolonged release of curcumin, while others have additional mechanisms for cellular delivery or intracellular release 2.1 Liposomes

Liposomes consist of one or more phospholipid bilayers sur-rounding an aqueous core Both lipophilic compounds/drugs (sol-ubilized in the liposomal bilayer) and hydrophilic compounds (soluble in the aqueous core) can be loaded into liposomes Different types of liposomes for targeted drug delivery have been developed and some systems have reached clinical practice[53e56]

Many liposomal curcumin formulations have been developed in recent years (Table 1) and a few studies are highlighted Karewicz

et al prepared curcumin loaded liposomes composed of egg yolk phosphatidyl choline (EYPC), dihexyl phosphate (DHP), and cholesterol prepared by the ﬁlm evaporation technique [57] Because of its lipophilicity, curcumin is solubilized in the lipophilic bilayer By usingﬂuorescent probes, the authors showed that it was indeed located at the hydrophobic acyl side chain and positioned closely to the glycerol groups It was shown that curcumin loaded into the EYPC/DPH/cholesterol liposomal bilayer stabilizes the system proportionally to its content In a follow up study, the li-posomes were coated with the cationic lipid/polymer conjugate N-dodecyl chitosan-N-[(2-hydroxy-3-trimethylamine) propyl] (HPTMA) chloride The obtained liposomes with a size of 73 nm were able to bind to and penetrate cells due to their cationic nature These coated liposomes released their content in a sustained manner in about 10 h Further, the formulations showed a slightly better cell killing activity than free curcumin, likely due to the improved cellular internalization of the cationic liposomes[58]

Re et al developed curcumin loaded liposomes composed of bovine brain sphingomyelin, cholesterol, and 1,2-stearoyl-sn-glyc-ero-3-phosphoethanolamine-N-[maleimide(poly(ethylene glycol)-2000)] and surface functionalized with the apolipoprotein E (ApoE) peptide as targeting ligand The liposomes were prepared by theﬁlm evaporation technique and non-incorporated curcumin

Fig 1 Chemical structures of curcumin (A), demethoxycurcumin (B) and

bisdeme-thoxycurcumin (C).

O Naksuriya et al / Biomaterials 35 (2014) 3365e3383

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was removed by size-exclusion chromatography using a PD-10

column The recovery of lipids was about 90% and the liposomes

had a mean size ofw130 nm It was shown these ApoE-liposomes

enhanced the transport of their curcumin payload through RBE4

brain capillary endothelial cells making these nanocarriers

inter-esting for brain targeting[59] In another study, a cationic

lipo-someepolyethylene glycol (PEG)epolyethylenimine (PEI) complex

(lipoePEGePEI complex, LPPC) was used for the encapsulation of

curcumin Morphological analysis by transmission electron

micro-scopy (TEM) showed a spherical shape of the liposomal

nano-particle with hair like projections on the surface likely originating

from PEG and PEI The size of curcumin loaded LPPC wasw260 nm

and the encapsulation efﬁciency of curcumin was 45% In vitro,

these LPPC released curcumin within 120 h[60]

2.2 Polymeric nanoparticles

Different polymers, particularly biodegradable ones, have been

used for the preparation of curcumin loaded nanoparticles[65]

PLGA (poly(D,L-lactic-co-glycolic) is widely used for drug delivery

purposes due to its biocompatibility and biodegradability[66e72]

Shaikh et al reported on curcumin loaded PLGA nanospheres

pre-pared by emulsion-evaporation method using PVA as surfactant The

obtained particles had a size of 264 nm and 77% entrapment ef

ﬁ-ciency resulting in 15% loading capacity of curcumin The particles

showed a biphasic release pattern characterized by a relatively rapid

initial release of about 24% of the loading in 24 h followed by

sus-tained release of about 20% of the loading during the next 20 days An

in vivo study in rats revealed that the curcumin loaded PLGA

nano-spheres improved the oral bioavailability of curcumin at least 9 fold

when compared to curcumin administered with piperine The latter compound was co-administered to improve curcumin availability as

it inhibits curcumin inactivation by hepatic and intestinal glucur-onidation[73] Yallapu et al encapsulated curcumin in PLGA nano-particles by a nanoprecipitation method using poly(vinyl)alcohol (PVA) and poly(L-lysine) as stabilizers (nano-CUR 1e6)[74] It was found that the size of the nanoparticles decreased from 560 to 76 nm with increasing PVA concentration Further, the particles had a neutral zeta-potential, although for poly(L-lysine) coated nano-particle a positive zeta-potential is expected The absence of charge

on the particle surfaces might be ascribed by the improper way the measurements were done (in distilled water with no pH control), whereas a low ionic strength buffer is preferred[75]or even the absence of the polylysine coating The nanoparticles showed after a small burst of around 20% of the loading a sustained release of cur-cumin for 25 days (Fig 3) The particles prepared with the highest concentration of PVA showed the slowest release and the authors hypothesized that the surface adsorbed PVA acts as a barrier and consequently controls the release rate The Nano-CUR6 formulation that released 64% of the loaded curcumin in 25 days was selected for further in vitro studies (outcome discussed in section3)[74] Ghosh

et al developed curcumin-loaded PLGA nanoparticles (Nano Cur) for the treatment of diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) in rats Nano Cur was prepared by emulsion-diffusion-evaporation method and atomic force microscopy (AFM) showed that the particles had an average diameter of 14 nm The optical density of Nano Cur was measured atlmaxof 422 nm to calculate the encapsulation efﬁciency which was 78% Fourier transform infrared (FTIR) analysis revealed that there were no strong interactions between curcumin and the polymer matrix, but no

Fig 2 Indications for which curcumin has been investigated.

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release data were reported It was also not commented why the

particles had a small size, but probably the strong surfactant

(didodecyldimethylammonium bromide) in combination with the

high-speed homogenizer that was used to produce the emulsions

might be an explanation [76] Anand et al prepared

curcumin-loaded PLGA nanospheres using a nanoprecipitation method and

polyethylene glycol (PEG)-5000 as stabilizer Curcumin was almost quantitatively entrapped in particles of 81 nm However, no in vitro release data were reported[77] Polylactic-co-glycolic acid (PLGA) and PLGAepolyethylene glycol (PLGAePEG) nanoparticles contain-ing curcumin were obtained by a scontain-ingle-emulsion solvent evapora-tion technique[78] The encapsulation efﬁciency was over 70% and particles with a sizew150 nm were formed The PLGAePEG particles released 21% of curcumin in 24 h, followed by a sustained release to 57% of the loading over 9 days On the other hand, the PLGA particles showed a continuous release of 40% of the loading in 9 days The authors hypothesized that the faster release of curcumin from the PLGAePEG nanoparticles is attributed the higher water-absorbing capacity of this matrix compared to PLGA only[78] NIPAAm, N-vi-nyl-2-pyrrolidone, poly(ethyleneglycol) monoacrylate and N,N0 ,-methylene bis acrylamide were copolymerized in water resulting in crosslinked nanoparticles with a size of 50 nm An aqueous disper-sion of these nanoparticles was vortexed with a solution of curcumin

in CH3Cl and a very high 90% entrapment efﬁciency was obtained The loaded nanoparticles released 40% of their content in 24 h[79] Natural polymers have also been used to prepare curcumin nanomedicines Liu et al developed curcumin loaded chitosan/ poly(Ɛ-caprolactone) (chitosan/PCL) nanoparticles by a precipita-tion method The mean diameter of the obtained nanoparticles was between 220 and 360 nm whereas the encapsulation efﬁciency and loading of curcumin were 71 and 4%, respectively The curcumin chitosan/PCL nanoparticles released 68% of their content over 5 days

in a sustained manner[80] Rejinold et al described chitosan-g-poly(N-isopropylacrylamide) for the development curcumin-loaded nanoparticles[81] This polymer is temperature sensitive because of the presence of the pNIPAAm grafts[82,83] Below the lower critical solution temperature (LCST) (38 C), chitosan-g-poly(N-isopropylacrylamide) was fully soluble in water whereas the polymer solution became turbid above the LCST Particles of

Table 1

Some recent curcumin liposomal formulations.

efﬁciency (%)

Size (nm)

Release kinetics Status of

investigation

Curcumin loaded liposomes

coated with N-dodecyl

chitosan-HPTMA chloride

Not reported

73 >80% in 10 h In vitro Non-toxic for murine ﬁbroblasts

(NIH3T3) whereas toxic for murine melanoma (B16F10) cells.

[58]

Curcumin loaded liposomes

coupled with the ApoE

peptide

Not reported

132 Not reported In vitro Increased accumulation of

curcumin in RBE4 cell brain capillary endothelial cells.

[59]

Curcumin loaded lipoe

PEGePEI complex

45 269 90% in 120 h In vitro/vivo The cytotoxic activity of the

nanoformulation was higher than free curcumin on both curcumin-sensitive cells and curcumin-resistant cells.

60e90% inhibition of tumor growth in mice inocolated with CT-26 or B16F10 cells.

[60]

Curcumin loaded

silica-coated ﬂexible liposomes

91 157 Not reported In vivo Increased 3.3-fold bioavailability

compared with curcumin loaded liposomes in mice through gavage administration.

[61]

Curcumin-conjugated

nanoliposomes

Not reported

207 Not reported In vivo Down regulated the secretion

of amyloid peptide (Ab) and partially prevented Abinduced toxicity in mouse model of Alzheimer disease.

[62]

Curcumin loaded soybean

phosphati-dylcholine

liposomes

Not reported

176 37% in 48 h In vivo Decreased parasitemia and

increase survival of Plasmodium berghei infected mice (anti-malarial therapy).

[63]

Curcumin loaded egg

phosphatidyl-choline

liposomes

Not reported

Not reported In vivo Exhibited cytoprotection for

renal ischemiaereperfusion injury.

[64]

Fig 3 Curcumin release proﬁles of nano-CUR 1e6 The red circle indicates the burst

release Nano-Cur 1 to 6 were prepared with different concentrations of PVA in the

aqueous phase (0e1% w/v) Reprinted from Journal of Colloid and Interface Science, Vol.

351/1, M.M Yallapu, B.K Gupta, M Jaggi, S.C Chauhan, Fabrication of curcumin

encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer

cells, pp 19e29, Copyright (2010), with permission from Elsevier (For interpretation of

the references to colour in this ﬁgure legend, the reader is referred to the web version

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chitosan-g-poly(N-isopropylacrylamide) with a size of 180e220 nm

were formed by ionic crosslinking using pentasodium tripoly

phosphate (TPP) in the presence of curcumin added to the mixture as

a solution in ethanol Only 5% of the loaded amount of curcumin was

released below the LCST in 35 h, whereas above this temperature

100% drug release was observed within 35 h The authors

hypoth-esized that below the LCST hydrogen bonds exist between the

phenolic hydroxyl groups of curcumin and the amide groups of the

pNIPAAm blocks that retain curcumin in the polymer matrix Above

this temperature the interpolymer interactions dominate and as a

consequence curcumin-polymer interactions are weakened, which

in turn results in release of the active[81] In a recent study of Anitha

et al., curcumin-loaded nanoparticles of dextran sulfate and chitosan

were prepared by coacervation method resulting in spherically

shaped and stable nanoparticles of 200e220 nm which are hold

together by electrostatic interaction between the two oppositely

charged polymers and the curcumin encapsulation efﬁciency and

loading capacity were 74 and 5%, respectively[84] The drug release

pattern was characterized by a burst release in theﬁrst 3 h followed

by a sustained release of curcumin that reached 70% of the loaded

amount within 120 h The release was faster at pH 5 than at pH 7 due

to the protonation of the amine groups of chitosan at low pH

resulting in swelling of the polymer matrix[84]

2.3 Polymeric micelles

Polymeric micelles are composed of amphiphilic block

co-polymers that spontaneously form micelles with a size ranging

between 20 and 100 nm in aqueous solution above the critical

micellar concentration (CMC) The hydrophobic core can

accom-modate hydrophobic drugs and therefore polymeric micelles have

been extensively used for solubilization and targeted delivery of

drugs [85e92] Song et al loaded curcumin into micelles of

amphiphilic methoxy poly(ethylene glycol)-b-poly(

Ɛ-capro-lactone-co-p-dioxanone) by a solid dispersion method[93] These

micelles had a small size (30 nm) with a narrow size distribution,

whereas the entrapment efﬁciency was more than 95% and the

loading capacity was 12% The micelles slowly releasedw80% of

their content without a burst in 300 h[93] A poly(D,L

-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide)

(PLGAePEGePLGA) triblock copolymer was synthesized by

ring-opening polymerization ofD,L-lactide using PEG as macroinitiator

[94] Curcumin loaded triblock copolymer micelles were prepared

by a dialysis method and it was shown that the CMC at room

temperature was 2.8 102mg/ml The drug loading capacity and

entrapment efﬁciency were 4 and 70%, respectively TEM analysis

showed that the micelles were spherically shaped and had a size of

26 nm which was conﬁrmed by dynamic light scattering

mea-surements [94] No release data were reported, but these

nano-particles were evaluated in vivo (discussed in Section4) Zhao et al

used a central composite design to optimize a formulation of mixed

micelles composed of Pluronics P123 and F68[95] The average size

of the mixed micelles was 68 nm, and the encapsulation efﬁciency

and loading capacity for curcumin were 87% and 7%, respectively It

was shown that 50% of the loaded curcumin was released from the

micelles in 72 h demonstrating that this formulation had sustained

release properties[95] Samanta et al conducted a molecular

dy-namics study of curcumin with pluronic block copolymers and they

concluded that the hydrophobic PPO chains cover the curcumin

molecule leaving the hydrophilic PEO chains exposed, resulting in

solvation of curcumin in water[96] Gong et al reported on the

encapsulation of curcumin in monomethyl poly(ethylene

glycol)-poly(Ɛ-caprolactone) (MPEGePCL) micelles by a one-step solid

dispersion method[97] Micelles with a mean diameter of 27 nm

were obtained that were well dispersible in water after

freeze-drying The encapsulation efﬁciency and drug loading capacity were 99 and 15%, respectively The release study was done by dialysis method using phosphate buffered saline (PBS) and 0.5% of tween 80 as external medium, and these micelles released about 58% of the loading in 14 days[97] Ma et al loaded curcumin in micelles of different PEO-PCL block copolymers by a cosolvent evaporation technique [98] It was reported that the PEO5000 -PCL24500 showed the highest solubilization capacity whereas PEO5000-PCL13000had the best drug retention capacity resulting in the slowest release kinetics The authors also found that the release was faster in the presence of HSA which is probably due to the high

afﬁnity of curcumin for HSA[98] 2.4 Conjugates

Conjugation of curcumin to small molecules (particularly amino acids) and as well as to both natural and synthetic hydrophilic polymers has been exploited to increase its aqueous solubility Several amino acids among which proline, glycine, leucine, isoleucine, alanine, phenylalanine, phenyl glycine, valine, serine and cysteine were coupled to curcumin[99] These conjugates were synthesized in dry dioxane using e.g N,N0 -dicyclohex-ylcarbodiimide (DCC) as coupling agent, and (4-dimethylamino-pyridine (DMAP) and triethylamine (TEA)) as catalysts, and puriﬁed

by column chromatography These amino acid conjugations increased curcumin’s aqueous solubility to 1e10 mg/ml[99] Manju

et al reported on the conjugation of hyaluronic acid and curcumin both dissolved in a water/DMSO mixture using DCC and DMAP as coupling agent and catalyst, respectively [100] Although hyal-uronic acid is very well soluble in water, the conjugates were amphiphilic due to the hydrophobic curcumin groups and as a result they self-assembled into particles with a size between 300 and 600 nm and a negative zeta-potential (25 to 75 mV) It was found that curcumin conjugated to hyaluronic acid remained intact for 90% once incubated in aqueous solution at pH 7.4 for 8 h whereas free curcumin showed 60% degradation within 25 min

[100] Tang et al conjugated curcumin to two short oligo(ethylene glycol) chains viab-thioester bonds that are labile in the presence

of intracellular glutathione and esterases (Curc-OEG;Fig 4B (top))

[101] These Curc-OEG conjugates contained 25% by weight cur-cumin and formed micelles with a size of 37 nm that released less than 12% of the conjugated amount of curcumin by hydrolysis in

24 h at pH 7.4 and 5.0 indicating a good stability of this system in PBS On the other hand,Fig 4B (bottom) shows that more than 25%

of the conjugated curcumin at pH 7.4 and 35% at pH 5.0 was released within 10 h in a medium containing reduced glutathione (GSH) and more than 80% of Curc-OEG hydrolyzed within 2 h at pH 7.4 in medium containing 30 U esterase The authors argued that Curc-OEG will be stable in the blood circulation and release cur-cumin once in the cell catalyzed by a combination of GSH and esterase[101] In a recent study, three curcumin molecules were covalently linked to the distal end of a block copolymer of methoxy poly(ethylene glycol) (mPEG) and PLA via a tris (hydroxyl methyl) aminomethane (Tris) spacer (mPEGePLAeTriseCur) (Fig 5)[102] Also, a block copolymer of (mPEG) and PLA to which one molecule

of curcumin was coupled was synthesized Micelles with a size from

60 to 100 nm were prepared by a dialysis method and they con-tained both conjugated and solubilized curcumin with a high loading (up to 20%; only 2% loading for mPEGePLA micelles) The release was studied using a Franz cell and PBS (pH 7.4) containing 5% sodium dodecyl sulfate as the acceptor medium It was found that the release of curcumin was due to a combination of diffusion

of physically loaded curcumin and hydrolysis of the ester bond that connects curcumin and the polymer (Fig 5) The authors reported that mPEGePLAeCur and mPEGePLAeTriseCur showed a rapid

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release of curcumin during theﬁrst 12 h which then leveled off The

authors argued that the release was controlled by hydrolysis of the

ester bond connecting the active and the polymer but that

simul-taneously degradation of released curcumin occurred resulting in a

steady state concentration of the compound However, no

convincing data were presented to substantiate this explanation

[102] Wichitnithad et al coupled curcumin via different carboxylic

ester spacers to mPEG 2000 The authors reported a log-linear

release of curcumin in time for all conjugates tested Further, it

was shown that as compared to the half-life of free curcumin

(0.56 h at pH 7.4 and 37C), PEG bound curcumin had a substantial

better stability (t1/2isw3 to 13 h)[103]

2.5 Peptide/protein carriers

Beta casein, an amphiphilic polypeptide with molecular mass

of 24,650 Da, spontaneously forms micelles (CMC at 37 C is

8 mM) When curcumin was loaded in the hydrophobic core of

these casein micelles, its solubility increased 2500 fold [104]

However, no data regarding size and release properties were

re-ported Nanoparticles of cross-linked human serum albumin (HSA)

have shown good biocompatibility and have been used for drug

delivery purposes[105,106] Kim et al presented curcumin-HSA nanoparticles that were prepared by homogenization of a mixture of HSA in water and curcumin in chloroform[107] The mean size of curcumin-loaded HSA particles was 135 nm and the loading capacity was 7.2% The authors speculated that the parti-cles were formed by crosslinking of albumin molecules via disul-ﬁde exchange due to heating associated with cavitation produced

by the high-pressure homogenizer Curcumin was likely solubi-lized in hydrophobic cavities of albumin[108,109]resulting in a

300 fold increase in solubility However, the release characteristics

of the nanoparticles were not investigated[107] 2.6 Cyclodextrins

Cyclodextrins are cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic cavity that can solubilize hydrophobic drugs and other small hydrophobic compounds such as curcumin

[110,111] Yadav et al used 2-hydroxypropyl-g-cyclodextrin (HPgCD) to complex curcumin by a pH shift method Curcumin was dissolved in an alkaline solution containing HPgCD and subse-quently the pH was adjusted to 6.0[112] Due to this pH change curcumin becomes hydrophobic and consequently partitioned in

Fig 4 Synthesis of curcumin amino acid conjugates (A) Reprinted from Food Chemistry, Vol 120/2, K Parvathy, P Negi, P Srinivas, Curcumineamino acid conjugates: Synthesis, antioxidant and antimutagenic attributes, pp 523e530, Copyright (2010), with permission from Elsevier (B) top; chemical structure of Curc-OEG, middle; synthesis of Curc-OEG, bottom; degree of hydrolysis of Curc-OEG at different conditions (B) Reproduced from Nanomedicine, Volume 5, Issue 6, pp 855e865 with permission of Future Medicine Ltd.

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the hydrophobic cavity of the CD Yallapu et al developed ab

-cyclodextrin (b-CD)-curcumin inclusion complex by solvent

evap-oration method.b-Cyclodextrin (CD) was dissolved in deionized

water and varying amounts of curcumin in acetone were added

while stirring overnight to evaporate acetone Then,b-cyclodextrin

(b-CD)-curcumin inclusion complexes were recovered by freeze

drying Analysis showed that 1e2 curcumin molecules were

encapsulated perb-CD cavity[113] The same group synthesized

poly(b-CD) (molecular weight from 2900 to 4100 Da) which was

subsequently loaded with 5e10% of curcumin Poly(b

-CD)/curcu-min self-assembled formulations were prepared by drop-wise

precipitation method [114] TEM analysis showed that a

curcu-min/poly(b-CD) inclusion complex (loading: 10e30%) self

assem-bled into nanoparticles with a size of 250 nm An in vitro stability

study was performed in PBS and it was noted that>70% of the

loaded curcumin was retained in the nanoparticles during 72 h of

incubation at pH 7.4 and 37C, demonstrating a good compatibility

of curcumin and its carrier[114]

2.7 Solid dispersions

Solid dispersions are dispersions of a drug/compound (either

molecularly dissolved in amorphous or (semi) crystalline form) in

an inert matrix[115,116] Solid dispersions are prepared by melt

method or solvent evaporation technique and used to enhance the

solubility and dissolution rate of poorly water-soluble drugs[117e

120] Lyophilized 2-hydroxypropyl-b-cyclodextrin (HP-b

-CD)-cur-cumin co-precipitates were prepared by a solid dispersion method

[121] HP-b-CD and curcumin (molar ratios from 0.5 to 2.8) were dissolved in methanol and converted into an amorphous co-precipitate which was subsequently lyophilized The lyophilisates had a porous structure that showed enhanced hydration and dissolution It was further shown that solutions of the curcumin solid dispersions showed a pronounced decrease in curcumin concentration up to 90% of the loaded amount after storage for

168 h, indicating that supersaturated curcumin solutions were formed upon dissolution of the lyophilisates These HP-b -CD-cur-cumin co-precipitates signiﬁcantly inactivated Escherichia coli after exposure to blue light (400e500 nm), most likely caused by the photosensitizing activity of curcumin[121] Seo et al reported on curcumin-polyethylene glycol-15-hydroxystearate (SolutolÒHS15) solid dispersions which were prepared by a solvent evaporation method and it was shown that the solubility of curcumin increased

to 560mg/ml Upon incubation in buffer, 90% the loaded amount of curcumin released/dissolved within 1 h[122]

2.8 Miscellaneous nanoformulations Mohanty et al prepared curcumin loaded nanoparticles composed of glycerol monooleate and Pluronic F127 [123] The entrapment efﬁciency was around 90% and size of the nano-particulates was 192 nm with a high negative zeta potential (32 mV) that ensured long term stability and avoided aggregation

of the particles When dispersed in buffer, these nanoparticles enhanced the stability of curcumin by protecting it against hydro-lysis[123] Anuchapreeda et al prepared a curcumin nanoemulsion

Fig 5 mPEGePLAeTriseCur: synthetic route and loading and release of curcumin Reprinted from Pharmaceutical Research, Vol 29/12, R Yang, S Zhang, D Kong, X Gao, Y Zhao, Z Wang, Biodegradable polymerecurcumin conjugate micelles enhance the loading and delivery of low-potency curcumin, pp 3512e3525, Copyright (2012), with permission from Springer.

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based on soybean oil, hydrogenated l-a-phosphatidyl choline from

egg yolk and co-surfactants (tween 80 and polyoxyethylene

hy-drogenated castor oil 60, Cremophor-HR30) with a mean particle

diameter of 47e55 nm and with a concentration of curcumin of

0.9 mg/ml This formulation was stable for 60 days at 4C Further,

25% of the loaded amount was released from these nanoemulsions

in 72 h when dispersed in PBS, pH 7.4, containing 25% human

serum[124] In another study, curcumin-loaded lipid-core poly(

Ɛ-caprolactone) nanocapsules coated with polysorbate 80 (C-LNCs)

were prepared by interfacial deposition of preformed polymer The

particles had a mean size of 96 nm, a negative zeta potential of

w10 mV and showed 100% encapsulation efﬁciency[125] These

C-LNCs released 35% of the loaded amount within 2 h[125]

3 In vitro studies of curcumin nanoformulations

The cytotoxicity of curcumin nanoformulations has been

stud-ied in many types of cancer cell lines Interpretation of the

rele-vance of the results is often difﬁcult due to the prolonged exposure

of cells to high static concentrations of curcumin (either in its free for or as nanoformulation) that however are not necessarily related

to the concentrations achieved in vivo

Yallapu et al demonstrated that the intracellular drug retention

of Nano-CUR6 formulation was better than free curcumin (dis-solved in DMSO) due to the sustained release of the active This formulation also increased the cellular uptake 2 and 6 fold in MDA-MB-231 metastatic breast cancer cells and A2780CP cisplatin resistant ovarian cancer cells, respectively, compared to free cur-cumin The 50% inhibitory concentrations (IC50) of Nano-CUR6 were 13.9 and 9.1 mM against A2780CP and MDA-MB-231 cells, respectively, whereas the IC50’s of free curcumin were higher than Nano-CUR6 (15.2mMand 16.4mMagainst A2780CP and

MDA-MB-231 cells, respectively)[74] Apoptosis induction of KBM-5 human chronic myeloid leukemia cells upon incubation with curcumin-loaded PEG-5000-PLGA nanoparticles was investigated by Anand

et al.[77] Curcumin-loaded PEG-5000-PLGA nanoparticles were more potent than free curcumin in inducing apoptosis which could

be related to the higher intracellular curcumin concentration upon

Fig 6 Viability of different cancer cells after incubation with curcumin loaded PEG-5000-PLGA for 24 h Reprinted from Biochemical Pharmacology, Vol 79/3, P Anand, H.B Nair, B Sung, A.B Kunnumakkara, V.R Yadav, R.R Tekmal, B.B Aggarwal, Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo, pp 330e338, Copyright (2010), with permission from Elsevier.

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incubation with the nanoparticles due their excellent cellular

internalization as compared to intracellular concentrations

ob-tained after exposure to free curcumin The uptake of

curcumin-loaded PEG-5000-PLGA nanoparticles and free curcumin by

KBM-5 cells was investigated byﬂuorescence microscopy

PEG-5000-PLGA nanoparticles were taken up already after 5 min exposure

and reached a maximum at 30 min In contrast, the uptake of free

curcumin could only be detected after 30 min incubation The

mechanism of cellular uptake of the nanoparticles was not

inves-tigated, but they most likely entered the cells by endocytosis

[126,127] However, no differences in viability were observed after

explore of the cells to either free curcumin or curcumin-loaded

PEG-5000-PLGA nanoparticles (Fig 6) The authors explained that

apoptosis was examined after 24 h of incubation, whereas

prolif-eration was examined at 72 h [77] Liu et al reported that

curcumin-loaded chitosan/polycarpolactone nanoparticles

exhibi-ted cytotoxicity on HeLa cervical cancer cells and OCM-1 human

choroidal melanoma cells to the same extent as free curcumin after

48 h incubation[80] Furthermore, Wichitnithad et al revealed that

mPEG 2000ecurcumin conjugates had IC50values in the range of

3e6mMagainst Caco-2 colon adenocarcinoma cells and IC50values

in the range of 1e3mMagainst KB oral epidermoid carcinoma, MCF7

breast adenocarcinoma, and NCIeH187 small lung carcinoma cells

mPEG 2000ecurcumin conjugates showed a potency comparable

to free curcumin (IC50values in the range of 1e3mM) on all cancer

cells used in this study [103] These studies demonstrate that

nanoparticle-encapsulation of curcumin is not always beneﬁcial

This is also underlined by cytotoxicity studies of curcumin-loaded

nanoemulsions on B16F10 mouse melanoma and leukemic cell

lines (K562, Molt4, U937 and HL60) by Anuchapreeda et al.[124] It

was shown that the 50% inhibitory concentration values (IC50)

ranged from 3.5 to 53.7mM On the other hand, free curcumin

dis-solved in DMSO showed lower IC50in B16F10 cells and also in

leukemic cell lines as compared to that of curcumin-loaded

nano-emulsions The authors argued that the lower activity of

curcumin-loaded nanoemulsions was due to the incomplete release during

24e72 h (incubation time of the formulations with the cells) In the

same study, it was shown that leukemic cell lines were less

sensi-tive to curcumin both in its free form and as nanoemulsion than

B16F10 cells It was hypothesized by the authors that the

pheno-type of B16F10 melanoma cells is responsible for this difference

However, in the same study it was shown that there were no

dif-ferences in the IC50 values of free and curcumin-loaded lipid

nanoemulsion in four leukemic cell lines (K562, Molt4, U937 and

HL60)[124] It was found by Bisht et al that the cytotoxicity of

curcumin loaded micelles based on cross-linked random

co-polymers of NIPAAm with N-vinyl-2-pyrrolidone and

poly(-ethyleneglycol) monoacrylate (nanocurcumin) against pancreatic

XPA-1 cells was lower than curcumin in its free form[79] Dhule

et al developed curcumin loaded HP-g-cyclodextrin liposomes that

showed 50% encapsulation efﬁciency with a size of 98 nm[128]

The cytotoxic activity of these liposomes against KHOS

osteosar-coma and breast cancer MCF-7 cell lines (IC50¼ 6 and 12mg/ml,

respectively) was higher than that of curcumin in DMSO (IC50¼ 23

and 20mg/ml, respectively) Interestingly, non-cancerous

mesen-chymal stem cells and skin ﬁbroblasts were unaffected by the

nanoformulation but were affected by free curcumin, indicating an

improved safety proﬁle Of note, a RFOS osteosarcoma cell line

derived from an untreated osteosarcoma patient was resistant

against free curcumin as well as its nanomedicine formulation The

authors argued that this resistance was because of the low

curcu-min uptake (either in free form or as nanoformulation) because

RFOS cells have a very slow growth rate and a low uptake capacity

which is caused by the low metabolic activity of the cell[129] In a

recent study, it was shown that curcumin loaded Pluronic/

polycaprolactone (Pluronic/PCL) block copolymer micelles with a size of 196 nm released 60% of the loaded amount in 108 h[130] The loaded Pluronic/PCL micelles were evaluated for their uptake

by Caco 2 cells usingfluorescence microscopy based on curcumin’s intrinsicfluorescence Cells incubated with the curcumin-loaded micelles showed a higherfluorescence intensity than those incu-bated with free curcumin, demonstrating good cellular internali-zation of the micelles, that, as hypothesized by the authors, occurred via endocytosis They argued that the insertion of the hydrophobic part of pluronic block copolymers into the lipid bi-layers of cell membranes resulted in a lower membrane micro-viscosity and internalization of micelles [130] Park et al loaded curcumin into nanoparticles based on the R7L10 peptide which is composed of a 7-arginine stretch and a 10-leucine stretch which were prepared by an oil-in-water (O/W) emulsion/solvent evapo-ration method[131] The cationic arginine groups of these peptide micelles were used to make complexes with plasmid DNA Inter-estingly, the authors found synergistic effects of curcumin on transfection (Fig 7) The authors hypothesized that the hydro-phobic curcumin stabilizes the structure of the complexes by facilitating the formation of R7L10 micelles These stable R7L10e curcumin plasmid complexes may show increased endocytosis and cellular internalization resulting in enhanced transfection The R7L10ecurcumin plasmid complexes also showed

anti-inﬂammatory activity by reducing the TNF-a levels of LPS-activated Raw264.7 macrophage cells Moreover, it was shown that after intratracheal injection of this R7L10-curcumin formula-tion, a stronger decrease in TNF-alevels in lung tissue in an acute lung injury mouse model was observed than after administration of free curcumin whereas no liver toxicity was detected[131] Yallapu investigated the hemocompatibility of various curcumin nano-formulations based on PLGA, b-cyclodextrin, cellulose, poly-N-isopropylacrylamide and polyamidoamine dendrimer [132] It was found that the curcumin dendrimer nanoparticles adsorbed more proteins than the other mentioned formulations and had higher lytic activity towards red blood cells, likely caused by the positive surface charge of the dendrimer particles[132]

Several studies have given evidence that drug-resistant cancer cells are sensitive to curcumin Zhang et al demonstrated that curcumin showed a similar cytotoxic effect against A549/DDP multidrug-resistant human lung adenocarcinoma cells compared

to non-resistant cells[133] The IC50of curcumin at 48 h was 16mM for A549 cells and 18mM for A549/DDP cells indicating that the multidrug resistant cells were still sensitive to curcumin The au-thors also found that curcumin inhibited the expression of miR-186*, a miRNA that targets caspase-10 mRNA Inhibition of this miRNA resulted in increased apoptosis as a result of the increased caspase-10 activity in these cells[133] Duan et al co-encapsulated doxorubicin and curcumin in poly(butyl cyanoacrylate) nano-particles (CUR-DOX-PBCA-NPs, size 133 nm) prepared by emulsion polymerization and interfacial polymerization [134] The results showed that CUReDOXePBCA-NPs inhibited the growth of multi-drug resistant human breast cancer cells (MCF-7/ADR) for 97% which was substantially higher than observed for cells incubated with a cocktail of free curcumin and doxorubicin (cell growth in-hibition was only 20%) It is important to notice that the adminis-tration of a nanoformulation loaded with both doxorubicin and curcumin achieved the strongest down-regulation of P-glycopro-tein activity, which is considered to be a major mechanism in multidrug resistance in MCF-7/ADR cells, compared to the combi-nation of free curcumin and doxorubicin This higher cytotoxicity was ascribed by the authors to the high concentration of curcumin near the cell membrane that bound to P-glycoprotein resulting in inhibition of the dox efﬂux[134] In another study, the cytotoxicity

of cationic PEGePEI liposomes loaded with curcumin against

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different cell lines, curcumin-resistant B16F10 murine melanoma

cells and CT26 colorectal adenocarcinoma cells (obtained by

continuously culturing the parental tumor cells in growth media

containing 5mMof curcumin) was investigated[60] It was found

that this liposome formulation, likely because of its rapid cellular

internalization, was substantially more cytotoxic (IC50ofw1mM)

than free curcumin (IC50ofw25mM) Also, these liposomes showed

antitumor activity in tumor bearing mice after intravenous

administration (Fig 8)[60]

4 In vivo studies of curcumin nanoformulations: kinetics and

efﬁcacy

The pharmacokinetic, biodistribution and therapeutic efﬁcacy of

different curcumin nanoformulations have been investigated in

many animal studies in order to get insight into the potential value

of these systems for the treatment of different diseases It has been

shown in many studies that oral or intravenous administration of

curcumin nanoformulations resulted in a larger area under the

concentrationetime curve (AUC) than after administration of

cur-cumin in its free form A more than 40 fold increase in the maximum

concentration (Cmax) and a 10 fold increase in AUC in mice were

observed after an oral dose of 1 g/kg of a curcumin nano-emulsion

composed of PEG 600 and Cremophor EL when compared with a

suspension of curcumin in 1% methylcellulose[135] Khalil et al

showed that curcumin loaded PLGA and curcumin loaded PLGAe

PEG nanoparticles displayed better pharmacokinetics proﬁles

compared to a curcumin aqueous dispersion after a single oral dose

of 50 mg/kg in rats (Fig 9)[78] The mean half-lives of curcumin

loaded PLGA and curcumin loaded PLGAePEG nanoparticles were 4

and 6 h, respectively, compared to a half-life of free curcumin of 1 h

It was also shown that for the same formulations the Cmaxvalues

were 2.9 and 7.9 fold, respectively, higher than free curcumin and

the AUCs were 15.6 and 55.4 fold, respectively, higher than free

curcumin According to the authors, the better performance of the

PLGAePEG nanoformulation was due to the more rapid release of

curcumin from PLGAePEG nanoparticles than from PLGA

nano-particles making the drug quicker available in blood They also

refered to the lack of interaction of the PEGylated systems with

Fig 7 Comparison of transfection activity of R7L10-curcumin/pDNA formulation with other carriers as measured by luciferase assay Reprinted from Biomaterials, Vol 33/27, J.H Park, H.A Kim, J.H Park, M Lee, Amphiphilic peptide carrier for the combined delivery of curcumin and plasmid DNA into the lungs, pp 6542e6550, Copyright (2012), with permission from Elsevier.

Fig 8 The effect of curcumin/LipoePEGePEI complex (LPPC) on tumor growth in vivo (A) Balb/c mice were inoculated with CT26 cells and subsequently treated with 2.1 mg/

kg of free curcumin, cationic PEGePEI liposomes (LPPC) or curcumin/LPPC; (B) C57BL/ 6J mice bearing B16F10 tumors were treated with 40 mg/kg of curcumin or curcumin/ LPPC Reprinted from Nanomedicine : Nanotechnology, Biology and Medicine, Vol 8/3, Y.L Lin, Y.K Liu, N.M Tsai, J.H Hsieh, C.H Chen, C.M Lin, K.W Liao, A LipoePEGePEI complex for encapsulating curcumin that enhances its antitumor effects on curcumin-sensitive and curcumin-resistance cells, pp 318e327, Copyright (2012), with

permis-O Naksuriya et al / Biomaterials 35 (2014) 3365e3383

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