jou rn al h om epage: www.elsev ier .com/ locate /aca
new method for pH triggered curcumin release by applyingoly(l-lysine) mediated nanoparticle-congregation
igambara Patra ∗, Fatima Sleemepartment of Chemistry, Faculty of Arts and Sciences, American University of Beirut, P.O. Box 11-0236, Riad El Solh, Beirut 1107-2020, Lebanon
i g h l i g h t s
New method for encapsulating cur-cumin and pH triggered release aredescribed.Poly(l-lysine)-curcumin mediatednanoparticle-congregation is fast.Present method avoids long hours ofpreparation steps and flow rate con-trolled.Curcumin release is efficient and fol-lows Higuchi model.
g r a p h i c a l a b s t r a c t
r t i c l e i n f o
rticle history:eceived 31 May 2013eceived in revised form 19 July 2013ccepted 29 July 2013vailable online 2 August 2013
We introduce a novel method for encapsulation of curcumin by synthesizing microcapsule containingself-assembled nanoparticles using poly (l-lysine), trisodium citrate and silica sol. Such microcapsulescan only be prepared in neutral and alkaline environment and unencapsulated curcumin can be effec-tively removed by simple centrifugation with encapsulation efficiency 57.34%. The particle sizes are inthe range 0.7–3 �m with an effective diameter 1.05 �m. The structure of the microcapsules is dependentupon the solubility of curcumin in the solvent environment, the microcapsule are spherical when pre-pared in 10% acetone and bowl-shaped/conical when prepared in water suspension, however, the sizeof these curcumin encapsulated microcapsules remain similar. Fluorescence microscope images confirmthat curcumin is predominantly concentrated within the shell wall of the capsules. Photophysical behav-ior of Micro-curcumin with respect to curcumin alone is evaluated. Release of curcumin is found to betriggered by pH where acidic environment trigger maximum release compared to basic and neutral con-ditions. Micro-curcumin is as stable as curcumin. Drug release efficiency is found to be 61.44% and the
drug release profile of Micro-curcumin follow Higuchi model. It is also revealed that �-diketone group ofcurcumin responsible for scavenging activity is retained in the Micro-curcumin, thus suggesting appli-cability of such system as a poorly water soluble drug delivery vehicle. In contrast to other curcumindelivery systems, the presented method is easy, fast and does not need flow rate monitoring device. Inaddition poly (l-lysine) as a non-toxic and highly stable material that prevents metabolic degradation isused in the present preparation method.
Curcumin, a natural diphenolic compound derived from turmericCurcuma longa, is widely used as spice, food coloring agent andtraditional medicine [1,2]. During these two decades, numerousstudies have signified the importance of curcumin as antioxi-
dant [3,4], anti-inflammatory [5,6], antiarthritic [7], antiamyloid[8], hepatoprotective [9], thrombosuppressive [10], anti-HIV [11],antimicrobial [12,13] and antitumor agent [14]. Curcumin hasreceived considerable interest among the natural polyphenols in
ancer prevention and cancer therapeutics [15,16] and is an exten-ively studied natural compound due to its anticancer activityia influencing multiple signaling pathways involved in cancer17–19]. In cancer cells, curcumin has revealed chemo/radio-ensitizing properties [20–23]. No studies in either animals [24,25]r humans [26] have discovered any toxicity associated with these of curcumin. Curcumin is now in the early phase of clinicalrials for cancer treatment [27]. It has a safe profile with no toxic-ty to healthy organs at doses as high as 8 g day−1 in clinical trials28]. However, very little amount of curcumin has been found inlood of a patient when administrated 10–12 g curcumin per day.hen rats are administered curcumin at a dose of 1 g kg−1, it was
ound that about 3/4th of curcumin is excreted in the feces, whileegligible amounts of curcumin appears in the urine [29]. Cur-umin is poorly absorbed from the gut and the quantity of curcuminhat reaches tissues outside the gut is pharmacologically insignif-cant as per the measurements of blood plasma levels and biliaryxcretion. The insolubility of curcumin in water at physiologicalH limits absorption, poor bioavailability, rapid metabolism, andxcretion [30]. The poor water solubility (i.e. 0.0004 mg mL−1 at pH.3) and low bioavailability of curcumin can be overcome by apply-
ng delivery approach based on nanotechnology [31]. Curcumin haseen encapsulated in liposome [32], silk fibroin and chitosan [33],hitosan [34], phospholipids [35], cyclodextrin [36], silica parti-les [37] and polymeric nanoparticles [38–40]. Recently, curcumins also popping up in growing number of dietary supplementss special extract form, such as BCM-95, beside in curcuminoidsorm.
As a prevailing synthetic approach, self-assembly process ismerging to generate advanced materials out of nanoparticlesuilding blocks where molecular subunits spatially organize intoell-defined supramolecular structures [41–44]. The challenge
mong material scientists is the design of such well-defined archi-ectures with dynamic and stimulus-responsive properties. On flaturfaces highly structured nanoparticles assemblies, such as wire,ings, supper lattice, may be synthesized [45,46]. Self-assemblynto micrometer sized spherical aggregates of functionalized goldanoparticles have been achieved [47]. Cysteine and lysine polymerlocks binding to gold and silica nanoparticle surfaces, respec-ively, to mediate nanoparticle self-assembly while forming hollow
icrospheres [48]. Poly-l-lysine (PLL) chains have been seen tondergo counter ion condensation in certain salt solution to formolymer aggregates which then direct nanoparticle-assembly gen-rating ordered microcapsule structure. We have found in this casehat the microcapsule structure is spherical, in other words, a core,urrounded by a uniform wall, a shell or even a membrane [49–52].esigning curcumin delivery system based on self-assembly ofolymer blocks on nanoparticle surfaces provides a great oppor-unity to explore.
This paper describes a new method for encapsulating cur-umin based on poly(l-lysine)-curcumin mediated nanoparticle-ssemble microcapsules termed as Micro-curcumin and applyts application as a vehicle for curcumin delivery. Advantages ofhe present method are that (i) it is easy to prepare and purify
icro-curcumin; (ii) the template is formed in situ thus not requir-ng a preformed template and polymerization/hydrolysis undercidic conditions unlike earlier delivery systems with silica par-icles [37]; (iii) it is also fast and does not need long hours ofreparation steps and flow rate controlled as used for deliveryystem using other polyelectrolyte [39]; and (iv) in addition, PLLs a non-immunogenic and non-toxic peptide allowing it to be aood carrier of small molecules in contrast to earlier case. Drug
olecules linked to PLL are highly stable, afford a long half life
nd prevent metabolic degradation [53]. The biological activity oficro-curcumin is found to be also promising in terms of scaveng-
ng activity.
ica Acta 795 (2013) 60– 68 61
2. Experimental
2.1. Materials
Poly(l-lysine) hydrobromide (150 kDa), trisodium citrate, andSilica LUDOX® HS-40 Colloidal Silica, 40 wt% suspension in waterprocured from Sigma–Aldrich, Curcumin obtained from AcrosOrganics and used as received. The particle size of the silica parti-cles were 20–24 nm (Sigma–Aldrich). All the solutions for synthesiswere prepared using de-ionized water and pH was monitored whendesired.
2.2. Synthesis and encapsulating curcumin inside themicrocapsules
Encapsulation of curcumin inside the silica microcapsules wasmade using PLL as the structure-directing agent. In this process1.31 mL of PLL (2 mg mL−1) was gently vortex mixed for 10 s with7.81 mL of trisodium citrate solution (0.01 M). The ratio R of totalnegative charge of added salt to total positive charge of the polymerwas 10. To the above suspension 0.5 mL of curcumin (0.01 M, a sus-pension in water or a solution in 10% acetone) was added and agedfor 30 min. Then it was vortex mixed with 7.81 mL of Silica sol for20 s to form the microcapsules encapsulating curcumin. The cloudysolution was allowed to age for 2 h, then centrifuged (4500 rpm,15 min). The fluorescent precipitate of microcapsules was washedthree times with deionized water and dispersed in 10 mL of deion-ized water for further characterization and investigation.
2.3. Morphological characterization
The capsules obtained by using curcumin in 10% acetonesolution were used for characterization unless otherwise it wasmentioned. Scanning electron microscopy (SEM) analysis were car-ried out using Tescan, Vega 3 LMU with Oxford Edx detector (IncaXmaW20) SEM. The sample was mounted on an aluminum stubcoated with carbon adhesive for the SEM analysis. Optical micro-scopic analyses were done with Zeiss LSM 410 where Confocalmicroscopy set-up consisted of a computer controlled laser scan-ning confocal microscope with 2 lasers (Ar/Kr and HeNe) and acompliment of high NA DIC and fluorescent lenses. The samplewas scanned using a 63 × 1.4 NA oil objective at 416 nm excita-tion wavelength. The wide-field fluorescence images were takenusing Axiovert 200, Zeiss Fluorescence and optical microscope withZeiss Axiocam hrc and KS 300 V3image analysis software. Particlesize distribution was analyzed using DLS (Brookhaven InstrumentsCorps) technique with a 90Plus Particle Sizing Software Ver. 5.23.
2.4. Spectroscopic measurement
FT-IR spectra were recorded using a Thermo Scientific NicoletiS5 FT-IR Spectrophotometer where the sample was placed on thecrystal plate and sealed with the tip. Absorption spectra were mea-sured using a JASCO V-570 UV-VIS-NIR Spectrophotometer, andthe fluorescence measurements were done using the Jobin-Yvon-Horiba Fluorolog III spectrofluorimeter. The excitation source wasa 100 W Xenon lamp, and the detector used was a R-928 oper-ating at a voltage of 950 V. Excitation and emission slits widthwere 5 nm. The fluorescence lifetime measurements were doneusing the same instrument except a pulsed diode laser of exci-tation wavelength 405 nm was used for excitation. Instrumental
response (prompt) for lifetime measurement was carried out usingcolloidal non-fluorescent particles. The decay data were analyzedusing Data Analysis Software. For a good decay fit, the values of�2 were in between 0.99 and 1.5 within the acceptable range.
62 D. Patra, F. Sleem / Analytica Chim
SP
Tc
E
wCi
3
mctngomPtc
ccftwctw
Fci
cheme 1. Schematic illustration of microcapsules formation by self-assembly ofLL in the presence of trisodium citrate and nanoparticles encapsulating curcumin.
he encapsulation efficiency was estimated spectrophotometri-ally using following equation.
ncapsulation efficiency ={
CURt
CURi
}× 100 =
{CURi − CURw
CURi
}× 100
here CURt is the total amount of curcumin in the microcapsules,URw is the total amount of curcumin lost during washing and, CURi
s the initial amount of curcumin added during preparation.
. Results and discussion
Micro-curcumin was prepared by PLL mediated self-assemblyethod where the first step involved was ionic-linking of the PLL
hains with the multivalent counter anions, in the present caserisodium citrate, to form spherical aggregates [50]. Due to theiret positive charge, these aggregates later on assisted the con-regation of negatively charges silica nanoparticles, to shape therdered microcapsule structure illustrated in Scheme 1. In theicrocaspules formed, the shell wall consists of positively charged
LL chains interspersed with the negatively charged silica nanopar-icles. We added curcumin to the PLL solution to encapsulateurcumin inside the microcapsules.
PLL has a strong binding affinity with curcumin which wasonfirmed by recording the absorption spectra of PLL at constantoncentration ∼1–2 mg mL−1 with various curcumin concentrationrom 0 to 12 �M in water. PLL showed a sharp narrow absorp-ion spectrum with maximum at ∼304 nm as depicted in Fig. 1hich was slightly red shift to ∼306 nm in the presence of 12 �M
urcumin. The absorbance of PLL increased with curcumin concen-ration. The ground state association constant of PLL with curcumin
as estimated by assuming 1:1 complex formation as [54]
1�A
= 1K�ε304[PLL]
(1
Curcumin
)+ 1
�ε304[PLL]
ig. 1. UV–visible absorption spectrum of PLL with increase in concentration ofurcumin. The spectra around 400–500 nm represents the absorption of curcuminn this region. Inset depicts linear relationship to estimate association constant (K).
ica Acta 795 (2013) 60– 68
where �A is the change in the absorbance at 304 nm, K theassociation constant of PLL with curcumin, �ε304 the differentialextinction coefficient at 304 nm and [PLL] and [Cur] represent theconcentrations of PLL and curcumin, respectively. Plot for curcuminbound PLL demonstrated a linear relationship as given in inset ofFig. 1 with an estimated association constant, K = 4.85 × 105 M−1
which is in the same range of binding of curcumin with other pro-tein molecules [55,56].
Addition of the citrate anions to PLL solution caused PLL to formglobular aggregates and these aggregates did not have any hol-low structure. Encapsulation inside the microcapsules was done byadding curcumin to the suspension of PLL-citrate aggregate beforeaddition of silica nanoparticles. These aggregates were allowed tostabilize for 30 min during which the curcumin molecules by virtueof their negative charge are expected to interact with PLL therebypenetrating into the PLL-citrate aggregates. Because in the elec-tronic ground state curcumin exists in two tautomeric forms: ketoand enol in which enol form is the more stable form in the solu-tion [57,58]. In the next step, the colloidal silica sol was added toassemble around these aggregates to form the microcapsule struc-ture encapsulating curcumin (Micro-curcumin). Upon addition ofsilica nanoparticles these globular aggregates yielded microcapsulestructure because even if the ratio R of total negative charge ofadded salt to total positive charge of the polymer is negative, thesurface is a heterogeneous mix of PLL and citrate salt, therefore,negatively charged shell material can still be associated with thePLL [41]. It should be noted that the pH of the solution increasedfrom 7.0 to 9.8 while adding silica sol but it did not influence onmicrocapsule formation. A control experiment by combining silicananoparticles and PLL resulted in randomly structured aggregates.The size of the capsule was also influenced by amount of citratepresent in the solution and the size increased with the amount ofcitrate. Rana et al. have carried out a thorough investigation by com-paring different citrate salts [49]. It is observed that citrate ion in theform of Hcit2− and cit3− yield microcapsule structure but H3cit andH2cit− species do not form aggregates. Thus, PLL aggregates pro-ceeds within pH window defined by pKa values of the citrate andPLL. Similarly smaller silica particles diffuse deeper in to the PLLaggregate than large particles. The silica nanoparticles penetratethe surface exterior of the PLL aggregate and the penetration depthdetermines the shell thickness [49]. Patwardhan et al. have shownthat PLL produces amorphous silica [59]. Dilution of the sampledecreases the particle size whereas aging time increases the cap-sule size. We have also compared effect of temperature on the sizeof microcapsule formation by comparing two different samples at25 and 6 ◦C keeping all other parameters constant. The size of themicrocapsules as measured by SEM decreased from ∼3–4 �m to∼1.5–2 �m while lowering the temperature from 25 to 6 ◦C, whichis also in accordance with similar system reported by Yu et al. [60].Initially 1.84 mg of curcumin was loaded to 2.62 mg of PLL, but theamount of curcumin loaded in the microcapsules was found to be210 mg g−1 with an encapsulation efficiency of 11.44%. However,the encapsulation efficiency increased to 57.34% when the initialconcentration of curcumin was reduced to half. The low encapsu-lation efficiency in the presence of 0.01 M compared to 0.005 Mof curcumin could be due to saturation of curcumin concentra-tion at much lower concentration while interacting with PLL. Theencapsulation efficiency did not change appreciably while chang-ing solvent for curcumin from 10% acetone to water suspension.The unencapsulated drug was easily and efficiently separated bysimple centrifugation and washing for three times. It is clear fromthe optical images that the Micro-curcumin are well dispersed as a
colloidal sol in the solution. As in Fig. 2 (left), SEM image of Micro-curcumin are spherical but the capsules are hollow in nature asobserved in confocal fluorescence images (right). It is establishedearlier [49,50] that during assembly of silica nanoparticles the
D. Patra, F. Sleem / Analytica Chimica Acta 795 (2013) 60– 68 63
F M resm revio
nfstaStsnWrtc
tgiwocctMnwma6swtHW
Fw
ig. 2. SEM image (left) of curcumin encapsulated microcapsules with 10 �M and 2 �icrocapsules where the extreme right side shows 3D orientation of the image of p
egative charge on silica drags the PLL chains toward the surface toorm the hollow structure. To prove this, the formation of microcap-ules structure was imaged within 2 min to 120 min by examininghe microstructure of hybrid materials consisting of PLL, curcumin,nd citrate salt upon addition of silica solution. The correspondingEM images after 2 min, 20 min, 60 min and 120 min upon addi-ion of silica solution is shown in Fig. 3. It was found that thetructure of hybrid materials immediately after addition of silicaanoparticles was irregular and smaller but having conical nature.ith time the size of Micro-curcumin grew into a larger size with
egular conical/spherical shape, which indicate that silica nanopar-icles drag PLL-curcumin, complex to the surface to give regularonical/spherical shape.
Curcumin is bound to PLL and is expected to be pulled towardhe shell along with PLL. The fluorescence image confirmed that thereen fluorescence is coming mainly from the shell wall suggest-ng that curcumin is encapsulated and predominantly is confined
ithin the shell wall. We have observed that during the assemblyf silica nanoparticles, the negative charge on silica pulls the PLLhains toward the surface to form the hollow structure [50]. As cur-umin (enol form) is strongly and ionically bound to PLL, we expecthat curcumin will be pushed toward the shell along with the PLL.
icro-curcumin were found to be structurally stable, displaying nooticeable damage after being subjected to repeated centrifugation,ashing and/or microscopy sample preparation and measure-ents. The particle size distribution of the Micro-curcumin was
nalyzed by DLS with an effective diameter 1.05 �m, half-width3 nm and polydispersity 0.358. The higher polydispersity of theample is due to its multimodal distribution. The size distribution
as found to be 0.7–3 �m. The pzc was 4–7 and the thickness of
he shell of Micro-curcumin was estimated to be 200–300 nm [50].owever, preparation of the Micro-curcumin was influenced by pH.hen the pH of the solution was low Micro-curcumin deformed in
ig. 3. SEM image of microstructure of hybrid materials consisting of PLL, curcumin, andere taken in 5 �M resolutions.
olutions. Confocal fluorescence microscope images (right) of curcumin encapsulatedus image.
shape. Micro-curcumin did not form in acidic environment whereasin alkaline medium at pH 8.00 it could be prepared. The shape ofthe Micro-curcumin was also influenced by the way curcumin wasprepared. For instance, spherical structures were obtained whencurcumin was prepared in 10% acetone, but in pure water, wherecurcumin was poorly soluble and formed dispersion, we observedthe structure to be bowl or conical in shape as shown in Fig. 4.However, curcumin remained confined and concentrated withinthe shell wall in this case as well.
In order to study the interaction of PLL, curcumin and silicananoparticles (SiO2) during the formation of Micro-curcumin, theFTIR spectra of PLL, curcumin and Micro-curcumin were furtherinvestigated and are given in Fig. 5. Although the frequency regionof both the phenolic �(OH) vibrations of the curcumin was com-puted to be at 3595 cm−1 [61], this band shifted to lower frequencyat 3500 cm−1 due to intramolecular and intermolecular H-bonding.Theoretical calculation also shows enolic �(OH) mode at 2979 cm−1
and this band disappeared as we carried out the FTIR measurementin solid state. Diketo form is preferred in solid phase of curcuminwhereas enol form in solution [62]. A prominent band was foundat 1500 cm−1 could be due to mixing of �(C C) and �(C O) ofthe benzene ring as predicted earlier [61]. In the FTIR spectrumof PLL, the absorptions near 1645 cm−1 was assigned to the amide Iband of PLL. A band due to carboxylate groups at the C-terminus ofthe polypeptide chain was detected at 1538 cm−1 [63]. The bandat 3288 cm−1 was also due to amide group whereas the bandsat 3047–2923 cm−1 were due to aliphatic group [64]. However,in Micro-curcumin we could not resolve the OH stretching bandof curcumin at ∼3500 cm−1, the band at 3288 cm−1 of PLL was
also not detectable. Instead a band at ∼2900–2800 cm−1 could bedetected, which is, in the same region for enolic �(OH) mode of cur-cumin as predicted earlier and also in the same region of aliphaticgroup of PLL. This region is also for Si OCH3 band, for example, of
citrate salt upon addition of silica solution in different time interval. The images
64 D. Patra, F. Sleem / Analytica Chimica Acta 795 (2013) 60– 68
F in encp
iniT∼117
ftflnepdc
F
ig. 4. Wide field fluorescence microscope (left) and SEM (right) image of curcumrepared in water-curcumin suspension.
nteraction of methoxy group of curcumin and nanoparticles couldot be ruled out. A new band at ∼2280–2330 cm−1 was obtained
n Micro-curcumin which was not observed for curcumin or PLL.his band could be due to Si N CO, on the other hand, the band1600 cm−1 could be due to C N stretching, and weak bands at350–1550 cm−1 were due to aliphatic group. The intense band at090 cm−1 could be attributed to SiO2 absorption band whereas at80 cm−1 band was due to Si C stretching vibration [65].
As shown in Fig. 6, the absorption maxima of curcumin shiftedrom 427 nm in water to 430 nm in the presence of PLL. In absorp-ion maximum of Micro-curcumin was noted as 422 nm. Theuorescence excitation spectra of curcumin bound to PLL showedo major difference compared to curcumin in water, however, themission maxima shifted from 543 nm in water to 545 nm in the
resence of PLL (see Fig. 7A). As shown in Fig. 7B, Micro-curcuminemonstrated a remarkable blue shift compared to that of cur-umin in water or curcumin bound PLL with emission maxima at
ig. 5. FT-IR spectra of PLL, curcumin and curcumin encapsulated microcapsule.
apsulated microcapsules with 10 �M (right) and 5 �M (extreme right) resolutions
465 nm. The Stokes’ shift, the difference in absorption and emis-sion maxima in wave number scale, was estimated to be muchsmaller in Micro-curcumin, 2191 cm−1, compared to curcumin inPLL, 4907 cm−1. The shorter emission/absorption wavelength ofcurcumin encapsulated in microcapsule illustrated a completelydifferent microenvironment inside the capsule by increasing theenergy gap between the ground state and excited state. The fluores-cence lifetime decay of Micro-curcumin (not shown) as determinedat excitation wavelength 405 nm and emission wavelength 465 nm(for pure curcumin in water the emission wavelength used was543 nm) exhibited bi-exponential decays in accordance with othermedia [66], however the major constituent (79%) was a short com-ponent with a fluorescence lifetime of �1 = 581 ps and the minorconstituent (21%) was a long component with �1 = 4.04 ns. Thefluorescence lifetime and Stokes’ shift data suggest a microenviron-ment, which is slightly more polar than THF [66]. The fluorescenceemission maximum of curcumin could be related to polarizability(P) of the solvent environment where p = n2 − 1/n2 + 2 and n isrefractive index of the solvent environment. The plot of fluores-cence emission maximum vs. P for curcumin is shown in Fig. 7C.The P estimated for Micro-curcumin from this plot was found to be∼0.25 which gives a refractive index value ∼1.414. In case of chargetransfer, ET (30) of the solvent can be applied to relate with spectro-scopic parameter [66], the plot of emission maximum of curcuminvs. ET (30) is presented in Fig. 7D. ET (30) estimated from this plot
for Micro-curcumin was ∼37.5.
Curcumin is less stable in alkaline solution. Stability of Micro-curcumin was compared with that of curcumin in water at pH 10.The absorbance of curcumin and Micro-curcumin was measured
350 40 0 450 500 550 60 0
Abs
orba
nce
(nor
mal
ized
)
Wav elength (nm)
Curcumin in Water Curcumin + PLL
Fig. 6. Comparison of UV–visible absorption spectra of curcumin in water and inPLL solution. The concentration of curcumin used was 5 �M.
D. Patra, F. Sleem / Analytica Chimica Acta 795 (2013) 60– 68 65
350 400 450 500 550 600 650 7000.0
0.2
0.4
0.6
0.8
1.0
PLL
Water
PLLWater
Excitati on Spectr a
Fluo
resc
ence
inte
nsity
(a.u
.)
Wav elen gth (n m)
Emissi on Spectra
A
400 450 500 550 600
B
Fluo
resc
ence
inte
nsity
(a.u
.)
Wavelength (nm)
0.20 0.2 2 0.2 4 0. 26420
440
460
480
500
520
540
λ em (m
ax)
λ em (m
ax)
P = (n2 - 1)/( n2 + 2)
C
30 35 40 45 50 55 60420
440
460
480
500
520
540 D
ET (30)
Fig. 7. (A) Normalized excitation (left) and emission (right) fluorescence spectra of curcumin in water and PLL; (B) fluorescence emission spectrum of Micro-curcumin; (C)p lvent(
wFlsioDambaw
Fc
lot of fluorescence emission maximum of curcumin vs. polarizability (P) of the so30) value of various solvent.
ith various time intervals at pH 10 and the data are presented inig. 8. In the first 25 min, Micro-curcumin was found to be simi-ar or slightly less stable compared to curcumin but after 40 min,tability of Micro-curcumin increased with respect to curcuminndicating that Micro-curcumin is as stable as curcumin. Releasef drug is very crucial for application as drug delivery system.elivery of curcumin in various environments was investigated byltering pH of the solution and the release of curcumin from the
icrocapsules was estimated by absorption spectrophotometry. In
rief, Micro-curcumin was dissolved in a desired pH solution andged for a given time interval before centrifugation. The precipitateas rejected and the solution was used to measure absorbance of
0 20 40 60 80 1000.0
0.2
0.4
0.6
0.8
1.0
C/C
0
Time (min )
Curcu min- MC Curcumin
ig. 8. Comparative study on stability of curcumin and Micro-curcumin in alkalineondition. C0 and C are respectively initial and final (at a given time) concentrations.
environment; and (D) plot of fluorescence emission maximum of curcumin vs. ET
curcumin for quantification. It was found that the curcumin isreleased from microcapsules in acidic (pH = 5.0), basic (pH = 8.0)and neutral (pH = 7.0) conditions as shown in Fig. 9A. However,release of curcumin was more triggered in acidic environment thanbasic (63% relative to acidic environment) and neutral (35% rela-tive to acidic environment) condition. We found that the amountof precipitated left over in acidic environment was less compared tobasic and neutral conditions indicating Micro-curcumin was moststable in neutral condition. Silica particles have negative charge inalkaline medium, but at pH 5 or in acidic environment silica parti-cles start losing its negative charges since the isoelectronic point ofsilica nanoparticle is ∼4. Thus, acidic environment leads to weak-ening of ionic interactions between the silica nanoparticles and PLLthat facilitates collapse of Micro-curcumin structure. Many of theMicro-curcumin also deformed in acidic environment when keptfor couple of days suggesting that the Micro-curcumin is not verystable in acidic environment. At the same time protonated form ofcurcumin in acidic environment may cause weakening of interac-tions between curcumin and PLL, therefore, higher rate of curcuminrelease at pH 5 is expected.
In neutral condition, leaching out of curcumin from the micro-capsules could not be ruled out [53]. The amount of drug release wasestimated to be 61.44% of initial concentration after one day. Thedrug release profile of curcumin was monitored overnight in acidic,basic and neutral environment. It was found that there is a linearrelationship between amounts of curcumin-released vs. time stud-ied for 24 h. Higuchi [67] proposed an equation where drug release
rate is related to physical constants based on simple diffusion lawsas R = KH t1/2 where R is the amount of drug release, t is the time andKH is the Higuchi dissolution constant. We applied the equation forthe curcumin release in acidic environment and the plot is shown
66 D. Patra, F. Sleem / Analytica Chimica Acta 795 (2013) 60– 68
4 5 6 7 8 90
5000
10000
15000
20000
In re
lativ
e flu
ores
cenc
e in
tens
ity s
cale
Rel
ease
of C
urcu
min
pH
A
0 2 4 60
5000
10000
15000
20000
In re
lativ
e flu
ores
cenc
e in
tens
ity s
cale
Da ta pH 5 Da ta pH 7 Da ta pH 8 Linear Fit pH 5 Linear Fit pH 7 Linear Fit pH 8
Rel
ease
of C
urcu
min
Hour1/2
B
Fig. 9. (A) Release of curcumin measured in fluorescence intensity scale from themicrocapsule in different pH condition and (B) temporal evolution of curcuminrf
ir
wudmdwoatawcn(bcDatpMwr
0.0
0.1
0.2
0.3
0.4
2 mg /mL1 mg/mL
1 mg/m L
Micro-curcumin
Micr o-curc umin
DPP H
Curc umin Abs
roba
nce
stable material that prevents metabolic degradation, the present
elease in fluorescence intensity scale. Data were fitted with Higuchi model [67]or drug release.
n Fig. 9B for 24 h. The Higuchi correlation plot showed a linearelationship with a good linear regression coefficient (R = 0.99).
Besides drug release profile, biological activity of drug moleculeithin the delivery system, could be of immense importance. Tonderstand biological activity of Micro-curcumin, the DPPH (2,2-iphenyl-1-picrylhydrazyl) scavenging activity of curcumin waseasured with and without microcapsules by adopting a proce-
ure reported earlier [68] where curcumin samples were mixedith DPPH in the double distilled deionized water. The changes
f absorption of DPPH at 520 nm were recorded at room temper-ture. Fig. 10 gives the values of absorbance of DPPH at 520 nm inhe presence of 1 mg mL−1 curcumin, 1 mg mL−1 Micro-curcuminnd 2 mg mL−1 Micro-curcumin compared with DPPH alone. Itas found that the presence of curcumin (1 mg mL−1) with DPPH
ould reduce the absorption of DPPH about 70% from its origi-al value, which suggests a strong scavenging ability of curcuminlower absorbance indicates higher scavenging ability). This cane attributed to the donation of H from the �-diketone group ofurcumin to DPPH [69]. It should be noted that the absorbance ofPPH in the presence of 1 mg mL−1 Micro-curcumin had a lowerbsorption compared to free DPPH, but the absorbance value inhe presence of 1 mg mL−1 Micro-curcumin is higher than in theresence of 1 mg mL−1 of curcumin. However, when 2 mg mL−1
icro-curcumin was used the reduction in absorbance of DPPHas almost similar to that of 1 mg mL−1 curcumin. These results
eveal that �-diketone group of curcumin, which is loaded near the
Fig. 10. DPPH absorption at 520 nm and in the presence of 1 mg mL−1 curcumin,1 mg mL−1 Micro-curcumin and 2 mg mL−1 Micro-curcumin in water at room tem-perature.
outer layer of the Micro-curcumin, has the strongest H-donatingability to reduce radical DPPH and the discrepancies of scaveng-ing activity of the same amount of Micro-curcumin compared tocurcumin alone could be due to the difference in total curcuminconcentration present in the Micro-curcumin, which could be over-come by increasing total Micro-curcumin concentration such asusing 2 mg mL−1 Micro-curcumin. However, the size of the micro-capsules normally used in drug delivery is in the range of 100 nm.Thus, the size of present Micro-curcumin may affect the cell mem-brane penetration efficiency. Nevertheless, the present study opensup a new approach that can be modified to reduce the particle sizeby changing the synthesis or reagent conditions, the work in thisdirection is already under progress. Similarly cell experiments forpotential anti-cancer effect of Micro-curcumin will be crucial fordirect applicability of the present system.
4. Conclusion
A novel method for encapsulation of curcumin inside themicrocapsule containing self-assembled nanoparticles by usingpoly(l-lysine), trisodium citrate and silica sol was successfullyachieved. However, such microcapsules could not be prepared inacidic condition. It was also observed that solubility and solventenvironment of curcumin influenced the structure of Micro-curcumin. Spherical microcapsules were formed when curcuminwas prepared in 10% acetone whereas bowl-shaped/conical micro-capsules were accomplished when curcumin were prepared inwater suspension, however, the size of the curcumin encapsulatedmicrocapsules remained similar in both the cases. Unencapsu-lated curcumin was effectively removed by simple centrifugationwith encapsulation efficiency 57.34%. The location of curcumin inMicro-curcumin was mainly concentrated within the shell wall.The particle sizes were in the range 0.7–3 �m with an effectivediameter 1.05 �m. Micro-curcumin was found to be stable anddegradation of curcumin could be suppressed in longer time period.Release of curcumin from Micro-curcumin was triggered by pHwhere acidic environment gave maximum release of curcumincompared to basic and neutral conditions. Release of curcumin fol-lowed Higuchi model. Scavenging activity of curcumin is retained inthe Micro-curcumin, thus �-diketone group of curcumin responsi-ble for such scavenging activity was not altered in Micro-curcumin,concluding that applicability of Micro-curcumin as drug deliveryvehicle. Besides advantage of poly(l-lysine) as non-toxic and highly
method is easy, fast and does not need flow rate monitoring deviceunlike other curcumin delivery systems. However, one of the limi-tation could be the size of the microcapsules, as in its current form
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D. Patra, F. Sleem / Analytic
icro-curcumin may affect the cell membrane penetration effi-iency compared to hundred nanometer particles commonly usedn drug delivery. Yet, the present study opens up a new approachhat can be modified to reduce the particle size by changing theynthesis conditions, similarly cell experiments for potential anti-ancer effect of Micro-curcumin is future prospect.
cknowledgements
Financial support provided by American University of Beirut,ebanon through URB and Lebanese National Council of Scien-ific Research (LNCSR), Lebanon to carry out this work is greatlycknowledged. We appreciate Prof. Marwan El-Sabban, Faculty ofedicine, American University of Beirut, for Confocal microscope
mages.
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