ijn 15860 nano structured delivery system for zinc phthalocyanine prep 012411[1]

8/4/2019 IJN 15860 Nano Structured Delivery System for Zinc Phthalocyanine Prep 012411[1]

1/12

2011 da Volta Soas t al, publis and lins Dov Mdial Pss Ltd. Tis is an Opn Assatil wi pmits unstitd nonommial us, povidd t oiinal wok is poply itd.

Intnational Jounal of Nanomdiin 2011:6 227238

International Journal of Nanomedicine Dovepress

submit your manuscript | www.dovpss.om

Dovepress

227

O r I g I N A L r e S e A r c h

open access to scientifc and medical research

Opn Ass Full Txt Atil

DOI: 10.2147/IJN.S15860

Nanostutud dlivy systm fo zinptaloyanin: ppaation, aatization,and pototoxiity study aainst uman lunadnoainoma A549 lls

Maiana da Volta Soas1

Mainaa ranl Olivia1

elisabt Pia dosSantos1

Lyia d Bito gitiana2

gly Mono Babosa3

cala holandino Quasma3

eduado rii-Jnio1

1Dpatmnt of Mdiins,Laboatio d Dsnvolvimntogalnio (LADeg), Faulty ofPamay, 2Laboatoy of Animal andcompaativ histoloy, glyobioloyrsa Poam, Institut ofBiomdial Sin, 3Dpatmnt ofMdiins, Laboatio Multidisiplinad cinias Famautias, Faulty ofPamay, Fdal Univsity of rio dJanio (UFrJ), rio d Janio, Bazil

cospondn: eduado rii-JnioDpatmnt of Mdiins, Laboatiod Dsnvolvimnto galnio(LADeg), Faulty of Pamay,Fdal Univsity of rio d Janio(UFrJ), calos caas Fi lo Avnu,rio d Janio, rJ 21941-590, BazilTl +55 21 2262 6625Fax +55 21 2260 7381email [email protected]

Abstract: In this study, zinc phthalocyanine (ZnPc) was loaded onto poly--caprolactone(PCL) nanoparticles (NPs) using a solvent emulsicationevaporation method. The process

yield and encapsulation eciency were 74.2% 1.2% and 67.1% 0.9%, respectively. The NPshad a mean diameter o 187.4 2.1 nm, narrow distribution size with a polydispersity indexo 0.096 0.004, zeta potential o4.85 0.21 mV, and spherical shape. ZnPc has sustainedrelease, ollowing Higuchis kinetics. The photobiological activity o the ZnPc-loaded NPs

was evaluated on human lung adenocarcinoma A549 cells. Cells were incubated with ree ZnPc

or ZnPc-loaded NPs or 4 h and then washed with phosphate-buered saline. Culture medium

was added to the wells containing the cells. Finally, the cells were exposed to red light (660 nm)

with a light dose o 100 J/cm2. The cellular viability was determined ater 24 h o incubation.

ZnPc-loaded NPs and ree photosensitizer eliminated about 95.9% 1.8% and 28.7% 2.2%o A549 cells, respectively. The phototoxicity was time dependent up to 4 h and concentration

dependent at 05 g ZnPc. The cells viability decreased with the increase o the light dose inthe range o 10100 J/cm2. Intense lysis was observed in the cells incubated with the ZnPc-

loaded NPs and irradiated with red light. ZnPc-loaded PCL NPs are the release systems that

promise photodynamic therapy use.

Keywords: photosensitizer, nanoparticles, photodynamic therapy, lung cancer, phototoxicity,

biodistribution

IntroductionPhotodynamic therapy (PDT) is being actively exploited in many clinical applications

such as cancer treatment, age-related macular degeneration, and inection.1,2 PDT is

based on the administration o drugs known as photosensitizers that are taken up and/

or retained by neoplastic tissues.3,4 The photosensitizers alone are harmless and ideally

have no eect on either healthy or abnormal tissues. Ater a predened time interval

that allows photosensitizer accumulation in the tumor tissue, the illumination o the

tumor site with nonthermal light (600800 nm) leads to the ormation o an excited

photosensitizer. The combined action o the excited triplet photosensitizer and molecu-

lar oxygen results in the ormation o singlet oxygen (1O2), which is the main mediator

o cellular death induced by PDT.2 PDT has been shown to induce both apoptosis

(slow process)5 and necrosis (drastic process).3 Among the most promising second-

generation photosensitizers or PDT are the phthalocyanines. In this study, zinc

phthalocyanine (ZnPc), a metallophthalocyanine, was used because o its very

Number of times this article has been viewed

This article was published in the following Dove Press journal:

International Journal of Nanomedicine

24 January 2011
http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/mailto:[email protected]:[email protected]://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/


2/12

Intnational Jounal of Nanomdiin 2011:6submit your manuscript | www.dovpss.om

Dovepress

Dovepress

228

da Volta Soas t al

strong phototoxicity eect,6 easy availability, and low cost.

ZnPc is stable and produces 1O2

with high yield.7 However,

ZnPc is lipophilic and slightly soluble in water or in a physi-

ologically acceptable vehicle. This hydrophobic characteristic

hinders its systemic administration and restricts its clinical

studies.

ZnPc is a dye and photosensitizer with molecular weight

o 577.91 g/mol. It is a crystalline powder, lipophilic, and

soluble in pyridine, dimethylsuloxide, dimethylormamide,

and dichloromethane (DCM).8 The photosensitizer can be

dissolved in dimethylsuloxide and diluted with ethanol.

However, it suers aggregation in water with reduction o

its photobiological activity.9 It exhibits a high thermal stability,

and,5% o its mass is lost at 500C.9 ZnPc has strongabsorbance in the red region o the spectrum o visible light

(max

= 666 nm in ethanol). It has strong fuorescence emis-sion in the range o 650800 nm.9 Methylene blue (MB) is

a hydrophilic dye and photosensitizer.10 In this study, it was

used as a positive control o the photobiological activity.

It exhibits strong absorbance in the red region (max

= 656 nmin water) and fuorescence emission (

max= 656 nm)10 similar

to ZnPc.

Nanoparticles (NPs) are promising delivery systems or

use in PDT. These systems provide sustained release, avoid

plasmatic fuctuations, decrease side eects, reduce the

requency o administration, and enhance the uptake o drugs

by targeted tissue, increasing the treatment eectiveness.2,1114

Advances in nanobiotechnology have resulted in the develop-

ment o several colloidal carrier systems such as NPs to

achieve these multiple objectives.2,4 The encapsulation o

hydrophobic drugs in NPs is a viable alternative in solving

the solubility problem,12 besides promoting targeted delivery

and sustained release. The liposomes and the nanoemulsions

are examples o drugs carrier. However, they have problems

o physical stability. The liposomes can suer disruption and

have limited capacity to encapsulate lipophilic drugs.15

Nanoemulsions can suer creaming, focculation, and coales-

cence. NPs have excellent physical stability, provide protec-

tion o the actives, and sustain drugs release.15

NPs have been used as a vehicle or the delivery o PDT

agents.2,4,16,17 Several photosensitizers, many o which are

hydrophobic, have been encapsulated in polyesters, such as

poly(lactic-co-glycolic acid) and polylactic acid NPs, to

overcome the problems associated with solubilization.12

For example, meso-tetra(4-hydroxyphenyl) porphyrin,18,19

bacteriochlorophyll-a, 20 verteporn,21 MB,22 hypericin,23 and

various phthalocyanines9,24 have all been encapsulated in such

polyesters. Several studies have proved that the NPs are prom-

ising delivery systems or photosensitizers. Generally, pho-

tosensitizers loaded in NPs induce better phototoxicity than

ree photosensitizers.18,23 Moreover, encapsulation in NPs

decreases the aggregation o the photosensitizer in solution20

or in physiologically acceptable vehicles.

Biocompatible and biodegradable polymers can be used

to produce NPs. Much attention has been ocused on poly-

-caprolactone (PCL), a semicrystalline polyester.9,2527 Thispolymer has been used in the development o release systems

such as microparticles and NPs.2527 PCL NPs have been used

as delivery systems o isradipine,28 primidone,29 paclitaxel,30

griseoulvin,31 tamoxien,32 espironolactone,33 vinblastine,34

and docetaxel.35

The aim o this study was to load ZnPc in PCL NPs to

improve the photobiological activity o the photosensitizer.

NPs were produced and characterized in terms o their size,

charge, morphology, and encapsulation eciency (EE).

In vitro release studies were carried out to evaluate the

photosensitizer release prole and kinetic study. The pho-

tobiological activity o the ZnPc-loaded NPs and ree pho-

tosensitizer were assessed on human lung adenocarcinoma

A549 cancer cells.

Material and methodsMatialsZnPc (M

W= 577.91), MB (M

W= 373.90) ($82%), hydro-

lyzed polyvinyl alcohol (PVA) (87%89%) (MW

= 13,00023,000), 9,10-anthracenediyl-bis(methylene)dimalonic acid

(ABMDMA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyl

tetrazolium bromide (MTT), and PCL (MW

= 42,50065,000)were purchased rom Sigma-Aldrich (St. Louis, MO). DCM

and acetone were purchased rom Merck (Darmstadt,

Germany). Sodium dodecyl sulate (SDS), NaCl, KCl,

NaH2PO

4, and Na

2HPO

4were purchased rom Vetec (Duque

de Caxias, Brazil). Uranyl acetate was purchased rom

Reagem (Colombo, Brazil). The ollowing cell culture items

were purchased rom Gibco Invitrogen (New York, NY):

Dulbeccos modied Eagles medium (DMEM), glutamine,

etal bovine serum (FBS), sodium bicarbonate, sodiumhydroxide, and Pen Strep (penicillin 10,000 units/mL +streptomycin 10,000 g/mL in aqueous solution).

NPs ppaation and poss yild (%)NPs were prepared by the emulsication and solvent evapo-

ration method. Briefy, ZnPc (3 mg) and PCL (97 mg) were

dissolved in the DCM (8 mL). The organic solution was
http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/


3/12

Intnational Jounal of Nanomdiin 2011:6 submit your manuscript | www.dovpss.om

Dovepress

Dovepress

229

Nanostutud dlivy systm fo zin ptaloyanin

emulsied in 50 mL o aqueous solution o PVA (1.5%, w/w),

or 5 min using an ultrasonicator UP100H (100 W, 30 kHz)

(Hielscher, Teltow, Germany) in an ice bath. PVA is a bio-

compatible polymer and has the unction o a suractant.

DCM was evaporated under reduced pressure using a rota

vapor (Heidolph, Schwabach, Germany) at room temperature

(28C). The NPs were puried thrice by centriugation at20,000 g (Avanti J-25; Beckman Coulter, San Francisco,

CA) or 20 min ollowed by resuspension in water to remove

the suractant. NPs were then transerred into glass vials,

rozen in a liquid nitrogen bath, and lyophilized (Lyophilizer,

FreeZone 4.5-L Benchtop Freeze Dry System; Labconco,

Kansas City, MO). The lyophilized powder was stored at

room temperature (28C) beore analysis.The process yield (%, w/w) was calculated by Eq. 1:

YM

M% ,( ) = 1

2

100 (1)

where Y(%) is the process yield, M1is the mass o recovered

lyophilized powder, and M2

is the mass o the ormulation

(PCL and ZnPc).

caatizationNPs were characterized in terms o their size, polydispersity

index (PI), morphology, surace charge, EE, and drug content

(DC). The size and PI were determined by photon correlation

spectroscopy using a Zetasizer 5000 (Malvern Instruments,

Malvern, UK). Beore measurements, the lyophilized pow-

ders were diluted using puried water. The surace charge

(zeta potential) was measured using the electrophoretic mode

with the Zetasizer 5000. The lyophilized powder was dis-

persed in water. The pH o the suspension was measured at

room temperature (28C). Morphology was evaluated bytransmission electron microscopy (TEM) (Morgagni 268;

FEI, Hillsboro, OR). Lyophilized powder was dispersed in

water and 5 L was transerred to a grid. Uranyl acetatesolution (3%) was added or xing and contrast. Ater 30 sec,

excess liquid was removed using lter paper (Whatman;

Maidstone, Kent, UK). Grids were placed in vacuum desic-

cators (Pyrex; Corning, Lowell, MA) beore analysis.

The drug extraction method rom NPs was patronized or

determination o the EE and DC. The lyophilized powder

was added in hot acetone (45C) or disruption o the NPsand dissolution o the photosensitizer. The organic solution

was sonicated or 10 min using an ultrasonic bath (T14;

Thornton, Vinhedo, So Paulo, Brazil). The organic solution

was diluted in phosphate-buered saline (PBS) containing 2%

SDS to precipitate the polymer (PCL) and to extract the pho-

tosensitizer. The suspension was centriuged, ltered through

a membrane lter (0.45 m, Millex-FH50

lter unit; Millipore,

Billerica, MA) to remove the PCL, and quantied by fuores-

cence emission. The photosensitizer was diluted in PBS con-

taining 2% SDS at concentrations o 20200 ng/mL. The

solutions were excited at 610 nm, and fuorescence emission

was measured at 680 nm using a Jasco FP-750 fuorimeter

(Jasco Corporation, Tokyo, Japan). The determination coe-

cient (R2) exceeded 0.999, with excellent linearity. Intraday

and interday precision and accuracy o the method showed a

variation coecient and a relative error (E) not .2.3% and

3.1%, respectively.

EE was calculated rom Eq. 2:

EE % ,( ) = M

M

3

4

100 (2)

where EE(%) is the encapsulation eciency,M3

is the mass

o ZnPc measured in lyophilized powder, andM4is the mass

o photosensitizer used in ormulation.

DC in the NPs (g ZnPc/mg NP) was calculated romEq. 3:

DCg

mg Np

ZnPc

=

M

M

5

6

, (3)

where DC is the drug content,M5 is the mass (g) o ZnPcmeasured in lyophilized powder, and M

6is the mass (mg) o

the lyophilized NP.

In vito lasin studis and kintiA known amount o NPs (50 mg NP containing 1 mg o

ZnPc) was dispersed in 60 mL o PBS containing 0.5% SDS,

with pH 7.4, kept at 37C in a thermostatic bath (M214M2;Quimis, Diadema, So Paulo, Brazil). The acceptor solution

was stirred with a magnetic stirrer at a constant rate o

300 rpm using a stirrer (M15; Marte, Santa Rita do Sapuca,

Minas Gerais, Brazil). At given time intervals, six samples

(n = 6) o 3 mL were withdrawn and centriuged at 20,000gor 20 min. The precipitates were resuspended in 3 mL o

resh medium and placed in the respective dissolution ves-

sels. The released photosensitizer was quantied by fuores-

cence emission. The in vitro release prole was obtained by

correlating time (h) versus drug release (%).


4/12


Dovepress

Dovepress

230

da Volta Soas t al

Ater 240 h o release studies, the acceptor solution

containing the NPs was removed rom the dissolution vessel

and centriuged thrice at 20,000 g or 20 min to recover

the nanostructured system. The NPs were lyophilized, and

the photosensitizer content was determined by fuorescence

emission. The extraction and quantication o the photosen-

sitizer o the lyophilized NPs have been described in the

Characterization section.

The release data were tted using the mathematical

models o zero order, rst order, Higuchi, HixonCrowell,

square root o mass, three seconds root o mass, and Baker

Lonsdale.36,37 Equations o the mathematical modeling are

shown in Table 1. The data were tted, and the linear regres-

sion was assessed. The correlation coecient (r) andR2 were

compared to determine the mathematical model that best ts

the release data.

In vito potobioloial ativityThe human lung adenocarcinoma cell line (A549)38 was

chosen or evaluation o the photobiological activity. The

cells were thawed and placed in DMEM supplemented with

10% FBS. Penicillin (100 units/mL) and streptomycin

(100 g/mL) were added to the culture medium. The mediumwas maintained at 37C in a 5% CO

2atmosphere or 48 h.

Cell concentrations were determined by the exclusion test o

trypan blue using a Neubauer counting chamber. Aliquots o

1 105 cells/mL were placed into 96-well dishes containing250 L o culture medium. The cell culture was incubated or24 h at 37C in a 5% CO

2atmosphere beore examination o

the toxicity and phototoxicity o the ree photosensitizer, and

the photosensitizer was loaded in NPs.

An NP suspension was prepared in culture medium

(DMEM) at 1 mg/mL. The cells were washed with PBS

and incubated with 250 L o the NP suspension (dose = 5 go ZnPc) at 37C in a 5% CO

2atmosphere or 4 h. Ater

incubation, the cells were washed twice with PBS and resh

culture medium (250 L) was added in each well. Finally,ater cellular uptake o the ree ZnPc or ZnPc-loaded NPs, the

cells were exposed to red light (660 nm) or 90 sec, with a

light dose o 100 J/cm2 (Photon Lase I; DMC, So Carlos, So

Paulo, Brazil). Ater light exposure, the cell culture was incu-

bated or 24 h at 37C in a 5% CO2

atmosphere, and the

cellular viability was determined by MTT assay. The NP

suspension without photosensitizer (empty NPs), control

(PBS) solution, and photosensitizer solution were also

assessed in the phototoxicity studies. The dark toxicity

was also assessed with the same samples. The photobiologi-

cal activity o the light was evaluated. The cell culture was

exposed to red light (660 nm) with light dose o 0100 J/cm2,

and cell viability was determined by the MTT assay. Briefy,

ater removing the cell medium, 50 L o MTT solution(1 mg/mL) was added to each well and incubated or 3 h.39

Then, the ormazan crystals were dissolved with 100 L o10% SDS in 0.01 M HCl in each well.39 The absorbance was

determined at 595 nm by a microplate reader. For each sample,

the cellular viability was calculated rom the data o 10 wells

(n = 10) and expressed as a percentage, compared with theuntreated cells (100%). Comparison o the mean optical den-

sity between the untreated (100%) and treated cells 24 h ater

illumination allowed the evaluation o the phototoxicity.

MB was used as a positive control o the photobiological

activity. The infuence o dierent parameters on the in vitro

phototoxicity was investigated by varying the time (16 h),

concentration o photosensitizer (010 g), and light dose(0100 J/cm2).

Mopoloial valuationAliquots o 1 105 cells/mL were placed in 24-well dishescontaining culture medium. A coverslip was placed on the

bottom o the well or growth o cells in monolayer. The cell

culture was incubated or 24 h at 37C in a 5% CO2

atmo-

sphere beore phototoxicity assay. The cells were washed

with PBS and incubated with NPs suspension (dose = 5 go ZnPc) (at 37C in a 5% CO

2atmosphere) or 4 h. Ater

incubation, the cells were washed twice with PBS. Culture

medium was added in each culture plate. Finally, the cells

were exposed to red light (660 nm) or 90 sec (light dose o

100 J/cm2) and incubated or 24 h (at 37C in a 5% CO2

atmosphere) beore microscopic analysis. The empty NPs

and control solution (PBS) were also assessed.

For microscopic analysis, the cells were xed in Bouins

liquid or 5 min and washed with alcohol (70%, v/v) and water

Table 1 Matmatial modls usd fo linaization of t

photosensitizer release prole data

Mathematical models Equation

Zo od F=k0tFist od ln(1 F) =k

ft

hiui F=kht1/2

BakLonsdal 3/2[1 (1 F)2/3] F=k3/2t

hixoncowll 1 (1 F)1/3=k1/3t

Squa oot of mass 1 (1 F)1/2=k1/2t

T sonds oot of mass 1 (1 F)2/3=k2/3t

Notes:Fdnots fation (%) of du lasd up to tim t. k0, k

f, k

h, k

3/2, k

1/3, k

1/2,

and k2/3

a onstants of t matmatial modls.


5/12


6/12


Dovepress

Dovepress

232

da Volta Soas t al

describing the release o the photosensitizer rom PCL NPs.

These results suggest that the release o ZnPc rom PCL NPs

is controlled by diusion.

The photobiological activity at dierent incubation times

o ree ZnPc, ree MB, and ZnPc-loaded NPs is shown inFigure 3. No toxicity was observed in the dark. The photo-

toxicity was time dependent up to 4 h. No signicant dier-

ence (P. 0.05) in the phototoxicity o the encapsulated ZnPc

was observed or the incubation time o 4 and 6 h. Thus, the

incubation time o 4 h was chosen or other phototoxicity

studies (infuence o the light dose and concentration). The

cell viability o the ree ZnPc slowly decreased in the range

16 h. The photobiological activity o the MB was observed

ater 2 h o incubation with activity maximum in 6 h. ZnPc-

loaded NPs exhibited phototoxicity signicantly higher than

ree photosensitizers (P, 0.05) (Figure 3).

The photobiological activity o dierent concentrations

o ZnPc-loaded NPs was evaluated in cell cultures and

compared with the ree photosensitizers. MB was used as

control. No toxicity was observed in the dark. In the assays

without photosensitizer, ZnPc-loaded NPs and ree photo-sensitizer were replaced by empty NPs and PBS, respectively.

The cell viability decreased with the increase in drug con-

centration (Figure 4). No signicant dierence (P. 0.05) in

the photobiological activity o the encapsulated ZnPc was

observed or the concentrations o 5 and 10 g/mL. The pho-totoxicity o the ree ZnPc was not concentration dependent

(Figure 4). The phototoxicity o the MB was observed only

0

0

0

5

10

15

20

25

0 10 15

y = 1.2748x

R2 = 0.9865

r = 0.9932

20

0

3

6

9

12

15

18

21

24

20 40 60 200

Time (min)

A

B

Time (min)1/2

ZnPcreleased(%)

ZnPcreleased(%)

100 120 140 260240220200180160

Figure 2 A) In vitro releasing prole of the ZnPc from nanostructured system. B) gapi psntation of t hiui modl obtaind aft ssion. Man and SD of

n = 6 dtminations.Abbreviations: ZnP,zin ptaloyanin; SD, standad dviation.

Table 2 rlasd du in t apto solution and mainin

ontnt of potosnsitiz in t nanopatils

Released

drug (%)1Remaining drug in the

nanoparticles (%)1Total (%)2 Loss (%)2

22.1 0.24 70.8 0.48 92.9 7.1

Notes:1Mdia standad dviation of n = 6 dtminations; 2Mdia of n = 6 dtminations.

Table 3 Determination coefcient (R2) and correlation coefcient

(r) obtaind aft lina ssion of t las data

Mathematical models R2 r

Zo od 0.9556 0.9775

Fist od 0.9663 0.9830

hiui 0.9861 0.9932

BakLonsdal 0.9782 0.9890

hixoncowll 0.9629 0.9812

Squa oot of mass 0.9612 0.9824

T sonds oot of mass 0.9594 0.9794


7/12


Dovepress

Dovepress

233


in 5 and 10 g/mL. ZnPc-loaded NPs exhibited phototoxicitysignicantly higher than ree photosensitizers (P, 0.05)

(Figure 4).

The infuence o the light dose (J/cm2) on the photobio-

logical activity was evaluated using 5 g o ZnPc (volumeo 250 L) and incubation time o 4 h. No toxicity wasobserved in the dark (without irradiation). Photobiological

activity o the ZnPc-loaded NPs was observed at 10 J/cm2.

The light dose increase was responsible or the improvement

o the biological eect. The ree ZnPc exhibited photobio-

logical activity in the light dose o 50100 J/cm2. Phototoxic-

ity o the MB was observed at 20100 J/cm2. Besides,

ZnPc-loaded NPs exhibited a phototoxic eect signicantly

higher than ree photosensitizers (P, 0.05) (Figure 5).

No morphological change was observed in cells treated

with saline (control group) (Figure 6A, B) and empty NPs

(no photosensitizer) (Figure 6C, D) ater illumination with

visible light (red light, 660 nm, or 90 sec, with light dose o

100 J/cm2). The cells incubated with NPs containing ZnPc

and irradiated with visible light showed morphological

changes. Morphological alterations o the cell induced by

photobiological action o ZnPc included the ollowing

changes: cytoplasmic condensation with signicant reduction

o cell volume, raried cytoplasmic matrix, extensive cell lysis,

and loss o plasma membrane extensions (Figure 6E, F).

As shown in Figure 7, the chemical measurements

showed a signicant decrease in the absorbance at 400 nm

or ZnPc-loaded NPs with ABMDMA ater irradiation, sug-

gesting a high eciency in the generation o1O2

(Figure 7).

For the ree ZnPc, the photobleaching caused by 1O2

was

lower than that o ZnPc-loaded NPs. There was no decrease

in the absorbance or a sample containing ABMDMA and

empty NPs (Figure 7).

DiscussionNPs have been used as a possible method to target and control

drug delivery, and prevent the side eects associated with

undesirable biodistribution o the ree orm o drugs.11,15

Moreover, there are many options or modiying the biodistri-

bution o NPs by appropriate selection o the polymer, surace

modication (hydrophilic coating), and particle size.11Among

the morphounctional eatures o solid tumors are the

0

0

20

40

60

80

100

*

*

*

* *

**

*

**

****

120

Free ZnPc lightFree ZnPc dark ZnPc-loaded Np dark

ZnPc-loaded Np light Free MB lightFree MB dark

140

1 2 4 6

Time (h)

Cellviability(%

)

Figure 3 Inuence of the incubation time (h) in the phototoxicity of free ZnPc or ZnPc-loaded nanoparticles. The cells were incubated with 5 (250 L) of f ZnP andZnP-loadd nanopatils at diffnt tims, wasd, and iadiatd at a lit dos of 100 J/m2. Dak toxiity was studid fo all sampls. T MTT assay was pfomd

24 aft lit xposu. ea data point psnts t man (SD) of n = l0 dtminations.Notes: *Signicantdiffn (P, 0.05), **No signicant difference (P. 0.05).

Abbreviations: ZnP,zin ptaloyanin; MTT, 3-(4,5-dimtyl-tiazol-2-yl)-2,5-bipnyl ttazolium bomid; MB, mtyln blu; SD, standad dviation.


8/12


Dovepress

Dovepress

234

da Volta Soas t al

abnormal unction o the lymphatic vasculature, which causes

fuid retention, and the increased permeability o blood vesselwalls. Both these characteristics lead to a higher retention o

NPs in the tumor area in comparison with normal tissues,

resulting in a passive selectivity o the system.42 Thus, nano-

metric size, which is directly related to the technique o NPs

obtention, is crucial or the desired tissue penetration.40

The size o the NPs obtained depends on many actors

such as the stirring speed, solution viscosity, organic/aqueous

phase ratio, and suractant concentration in the external

aqueous phase. In this study, the use o solvent emulsication-

evaporation method (SEEM) associated with sonication

produced the photosensitizer-loaded NPs o appropriate size.NPs containing photosensitizer were produced with success

using PCL by SEEM. The encapsulation method produced

particles in nanometric scale (,200 nm) with narrow size

distribution (IP , 0.1), spherical shape, and excellent EE

(.60%). The low release depends on the nature o both the

polymer and the encapsulated molecule.38 ZnPc presented a

low and sustained release. The release data were tted using

the mathematical models o zero order, rst order, Higuchi,

HixonCrowell, square root o mass, three seconds root omass, and BakerLonsdale. Zero-order kinetics can be used

to describe the drug release rom several types o delivery

systems such as matrix tablets or drugs with low solubility44

and osmotic systems where drug release would be directly

proportional to time. The rst-order model describes the

release prole rom the delivery systems containing drugs

dispersed in porous matrices where drugs would be released

at rates proportional to the amount o drug remaining in the

interior o the delivery system.36 Hixon and Crowell recog-

nized that the particle regular area is proportional to the cubic

root o its volume.45

This relationship between area andvolume plus the release and remaining amount o the drug

can infuence the release kinetics. The shape actor or cubic

or spherical particles should be kept constant i the particles

are dissolved in an equal manner on all sides. When this

model is used, it is assumed that the release rate is limited

by the drug particle dissolution rate and not by the diusion

that might occur through the polymeric matrix. 45

0

0

20

40

60

80

100

*

*

**

*

**

*

** **

120

Free ZnPc lightFree ZnPc dark ZnPc-loaded Np dark

ZnPc-loaded Np light Free MB lightFree MB dark

140

2.5 5 10

Dose (g photosensitizer)

Cellviability(%)

Figure 4 Inuence of the photosensitizer concentration in the photobiological activity of free ZnPc, free MB, or ZnPc-loaded nanoparticles. The cells were incubated with

250 L of f potosnsitiz (ZnP o MB) and ZnP-loadd nanopatils fo 4 , wasd, and iadiatd at a lit dos of 100 J/m 2. Dak toxiity was studid fo allsampls. T MTT assay was pfomd 24 aft lit xposu. ea data point psnts t man (SD) of n = 10 dtminations.Notes: *Signicantdiffn (P, 0.05), **No signicant difference (P. 0.05).

Abbreviations: ZnP,zin ptaloyanin; MB, mtyln blu; MTT, 3-(4,5-dimtyl-tiazol-2-yl)-2,5-bipnyl ttazolium bomid; SD, standad dviation.


9/12


Dovepress

Dovepress

235


The BakerLonsdale model was developed rom the Higuchi

model and describes drug controlled release rom a spherical

matrix.46

This mathematical model has been used or linear-ization o release data rom several ormulations o micropar-

ticles and NPs. The mathematical models o squared root and

three seconds root o mass were also tested in the release

kinetic study.36,37 The Higuchi model has been based on the

Ficks law where the release occurs by the diusion o drugs

within the delivery system.36,47 In this case, the released

amount o the drug is proportional to the square root o time.

The Higuchi model gave the highest value oR2 andr, indi-

cating that this mathematical model was the most suitable

or describing the release o the photosensitizer rom PCL

NPs (Table 3). These results suggest that the release o ZnPcrom PCL NPs is controlled by diusion. From these results,

the mechanism o the photosensitizer release rom PCL NPs

can be considered as ollows: 1) Water penetrates into poly-

meric matrix o the NPs through pores, slowly dissolving the

photosensitizer. 2) ZnPc is released by diusion into acceptor

solution. 3) The photosensitizer is hydrophobic and accumu-

lates into SDS micelles.

The in vitro phototoxicity studies showed that the

ZnPc-loaded NPs were more phototoxic than the ree ZnPc

(Figures 35). The photobiological activity is highly depen-dent on the photosensitizers cellular uptake mechanism and

its subcellular localization. In aqueous medium, a ree lipo-

philic photosensitizer is generally absorbed by diusion

across the plasmatic membrane (lipophilic) leading to a low

intracellular concentration. Moreover, lipophilic photosen-

sitizers such as ZnPc tend to aggregate in aqueous medium

with reduction o its photodynamic activity.48 Due to its

instability in water, the photobiological activity o the ree

ZnPc was not concentration dependent (Figure 4). The MB

was less phototoxic than the ZnPc-loaded NPs (P, 0.05)

(Figures 35). MB is hydrophilic and has diculty in cross-ing the lipophilic membrane o the cells, leading to a low

intracellular concentration. However, NPs are able to enter

the cells by endocytosis, releasing the photosensitizer in the

cell cytoplasm. The intracellular localization o the photo-

sensitizer, an important actor in PDT, can induce dierent

damage in the cells. Due to its hydrophobic characteristics,

intracellular ZnPc is able to accumulate in the cytoplasmic

0

0

20

40

60

80

100**

**

**

*

*

*

*

*

*

*

*

*

120PBS Empty Np Free ZnPc ZnPc-loaded Np Free MB

140

10 20 50 100

Light dose (J/cm2)

Cellviability(%

)

Figure 5 Inuence of light dose on phototoxicity of free ZnPc and ZnPc-loaded nanoparticles. The cells were incubated for 4 h, at an equivalent drug dose of 5-pg ZnPc

(250 L), wasd, and iadiatd wit d lit (660 nm). MTT assay was pfomd 24 aft lit xposu. ea data point psnts t man (SD) of n = 10dtminations.

Notes: *Signicantdiffn (P, 0.05), **No signicant difference (P. 0.05).

Abbreviations:ZnP,zin ptaloyanin; MTT, 3-(4,5-dimtyl-tiazol-2-yl)-2,5-bipnyl ttazolium bomid; SD, standad dviation.


10/12


Dovepress

Dovepress

236

da Volta Soas t al

membrane, lysosomal compartment, and mitochondria.

Intracellular generation o1O2

and additional ree radicals

can act as potent cytotoxic agents.

The1

O2 is the main toxic species generated by phtha-locyanines during PDT.49 The photobleaching studies

(Figure 7) are in agreement with the photobiological activity

results (Figures 35). ZnPc-loaded NPs generated1O2

more

eciently than ree ZnPc (Figure 7). The ree ZnPc suers

aggregation in water leading to a low 1O2generation. However,

encapsulation in NPs protected the ZnPc rom the aggregation

in aqueous medium. Due to slow release (Figure 2), many

ZnPc molecules can remain adsorbed on the surace o the NPs.

The adsorbed photosensitizer can be activated by red light

leading to a strong 1O2

generation. The photobleaching data

are consistent with those obtained by Zhao et al.50 The 1O2

generation was improved ater encapsulation o the lipophilic

photosensitizer (silicon phthalocyanine (SiPc)) in silica NPs.

SiPc is a lipophilic photosensitizer. It aggregates in aqueous

solution, which reduces its therapeutic ecacy and limits its

clinical use.50 Zhao et al have encapsulated SiPc using silica

NPs, which not only improves the aqueous stability but also

increases its 1O2generation and photobiological activity com-

pared with ree SiPc.50 SiPc-loaded silica NPs generated

photo-induced1O2

more eciently than ree SiPc.50

The results obtained rom in vitro phototoxicity studies

demonstrated that the phototoxicity o ZnPc against the

A549 cells occurred ater 24 h o light exposure because the

cellular damage resulting rom the photochemical reaction

is not immediately lethal. This phenomenon could be due to

an apoptotic response, which is a slow process.5 Cell death

by necrosis3 may occur on a smaller scale. Necrosis is a

drastic process o death that results in cell lysis.

ConclusionNanotechnology is bringing new perspectives to a number o

elds o human knowledge. Its applications in cancer treat-

ment by PDT are being investigated largely with excellent

results. The nanoencapsulation was suitable or the prepara-

tion o PCL NPs because the release system has nanometric

size, narrow distribution, regular shape, and sustained release.

Achieving optimal eect o PDT on tumor cells requires that

the photosensitizer becomes closely associated to the subcel-

lular organelles. Encapsulation o ZnPc in the PCL NPs

improved its stability in aqueous medium and the in vitro

phototoxicity. The photobiological activity was infuenced

by photodynamic parameters such as incubation time, lightdose, and drug concentration. Based on all the photobiological

measurements, it can be concluded that the ZnPc-loaded PCL

NPs are promising delivery systems or use in PDT.

AcknowledgmentsWe thank the nancial support o Conselho Nacional d

Desenvolvimento Cientico e Tecnolgico (CNPq) and

A B

C D

200 m 50 m

200 m 50 m

200 m 50 m

E F

Figure 6 Mopoloial valuation of t A549 lls usin lit miosop:

A) and B) lls inubatd wit PBS (ontol). C) and D) lls inubatd wit mpty

nanopatils suspnsion. E) and F) lls inubatd wit ZnP-loadd nanopatils.

(A), (C), and (E) were observed at magnication of50 (sal 200 m). (B), (I), and(F) were observed at magnication of400 (sal 50 m).Abbreviations: PBS,pospat-buffd salin; ZnP, zin ptaloyanin.

00

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

50 100

ZnPc-loaded in nanoparticles Free ZnPc Empty nanoparticles

150 200

Time (s)

Absorbancein400nm

250 300 350 400

Figure 7 Potoblain of ABMDMA by sinlt oxyn natd by napsulatd

ZnP and f ZnP. empty nanopatils w usd as ontol. T an in

ABMDMA absoption at 400 nm was masud as a funtion of t i adiation tim.

ea data point psnts t man (SD) of n = 3 dtminations.Abbreviations: ABMDMA, 9,10-antandiyl-bis(mtyln)dimaloni aid;

ZnP, zin ptaloyanin; SD, standad dviation.


11/12


Dovepress

Dovepress

237


Fundao Carlos Chagas Filho de Amparo Pesquisa do

Estado do Rio de Janeiro (FAPERJ).

DisclosureThe authors report no confict o interest in this work.

References1. Allison RR, Downie GH, Cuenca R, Hu XH, Childs CJH, Sibata CH.Photosensitizers in clinical PDT. Photodiagnosis Photodyn Ther.

2004;1:2742.

2. Bechet D, Couleaud P, Frochot C, Viriot ML, Guillemin F, Barberi-

Heyob M. Nanoparticles as vehicles or delivery o photodynamic

therapy agents. Trends Biotechnol. 2008;26(11):612621.

3. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photody-

namic therapy: Part three Photosensitizer pharmacokinetics, biodis-

tribution, tumor localization, and modes o tumor destruction.

Photodiagnosis Photodyn Ther . 2005;2(2):91106.

4. Chatterjee DK, Fong LS, Zhang Y. Nanoparticles in photodynamic

therapy: an emerging paradigm. Adv Drug Deliv Rev. 2008;60(15):

16271637.

5. Zhou C, Shunji C, Jinsheng D, Junlin L, Jori G, Milanesi C. Apoptosis

o mouse MS-2 brosarcoma cells induced by photodynamic therapy

with Zn (II)-phthalocyanine.J Photochem Photobiol B. 1996;33(3):219223.

6. Bonnett R. Photosensitizers o the porphyrin and phthalocyanine series

or photodynamic therapy. Chem Soc Rev. 1995;26(37):1933.

7. Savolainen J, van der Linden D, Dijkhuizen N, Herek JL. Characterizing

the unctional dynamics o zinc phthalocyanine rom emtoseconds to

nanoseconds.J Photochem Photobiol A Chem . 2008;196(1):99105.

8. Zanolim AA, Volpati D, Olivati CA, Job AE, Constantino CJL. Struc-

tural and electric-optical properties o zinc phthalocyanine evaporate

in lms: temperature and thickness eects.J Phys Chem. 2010;114(28):

1229012299.

9. Ricci-Jnior E, Marchetti JM. Preparation, characterization, photocy-

totoxicity assay o PLGA nanoparticles containing zinc (II) phthalo-

cyanine or photodynamic therapy use.J Microencapsul. 2006;23(5):

523538.

10. Wainwright M, Phoenix DA, Rice L, Burrow SM, Waring J. Increasedcytotoxicity and phototoxicity in the methylene blue series via

chromophore methylation. J Photochen Photobiol B. 1997;40(3):

233239.

11. Hans ML, Lowman AM. Biodegradable nanoparticles or drug delivery

and targeting. Curr Opin Solid State Mater Sci. 2002;6(4):319327.

12. Camerin M, Magaraggia M, Soncin M, et al. The in vivo ecacy o

phthalocyaninenanoparticle conjugates or the photodynamic therapy

o amelanotic melanoma.Eur J Cancer. 2010;46(10):19101918.

13. Murthy SK. Nanoparticles in modern medicine: state o the art and

uture challenges.Int J Nanomedicine. 2007;2(2):129141.

14. De Jong WH, Borm PJ. Drug delivery and nanoparticles: applications

and hazards.Int J Nanomedicine. 2008;3(2):133149.

15. Soppimath KS, Aminabhavi TM, Kulkarni AR, Rudzinski WE.

Biodegradable polymeric nanoparticles as drug delivery devices.

J Control Release . 2001;70(12):120.16. Ungun B, Prudhomme RK, Budijon SJ, et al. Nanoabricated upcon-

version nanoparticles or photodynamic therapy. Opt Express. 2009;

17(1):8086.

17. Allison RR, Mota HC, Bagnato VS, Sibata CH. Bio-nanotechnology

and photodynamic therapystate o the art review. Photodiagnosis

Photodyn Ther . 2008;5(1):1928.

18. Konan YN, Berton M, Gurny R, Allemann E. Enhanced photodynamic

activity o meso-tetra(4-hydroxyphenyl)porphyrin by incorporation

into sub-200 nm nanoparticles. Eur J Pharm Sci. 2003;18(34):

241249.

19. Konan YN, Chevallier J, Gurny R, Allemann E. Encapsulation o

p-THPP into nanoparticles: cellular uptake, subcellular localization and

eect o serum on photodynamic activity. Photochem Photobiol.

2003;77(6):638644.

20. Gomes AJ, Lunardi LO, Marchetti JM, Lunardi CN, Tedesco AC.

Photobiological and ultrastructural studies o nanoparticles o

poly(lactic-co-glycolic acid)-containing bacteriochlorophyll-a as a

photosensitizer useul or PDT treatment.Drug Deliv. 2005;12(3):

159164.

21. Konan-Kouakou YN, Boch R, Gurny R, Allemann E. In vitro and

in vivo activities o verteporn-loaded nanoparticles.J Control Release.

2005;103(1):8391.

22. Tang W, Xu H, Kopelman R, Philbert MA. Photodynamic characteriza-

tion and in vitro application o methylene blue-containing nanoparticle

platorms.Photochem Photobiol. 2005;81(2):242249.

23. Zeisser-Labouebe M, Lange N, Gurny R, Delie F. Hypericin-loaded

nanoparticles or the photodynamic treatment o ovarian cancer.Int J

Pharm. 2006;326(12):174181.

24. Allemann E, Rousseau J, Brasseur N, Kudrevich SV, Lewis K, van

Lier JE. Photodynamic therapy o tumours with hexadecafuoro zinc

phthalocyanine ormulated in PEG-coated poly(lactic acid) nano-

particles.Int J Cancer. 1996;66(6):821824.

25. Nair LS, Laurencin CT. Biodegradable polymers as biomaterials.Prog

Polym Sci. 2007;32(89):762798.

26. Sinha VR, Bansal K, Kaushik R, Kumria R, Trehan A. Poly- -caprolactone microspheres and nanosperes: an overview.Int J Pharm.2004;278(1):123.

27. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanopar-

ticles based drug delivery systems.Colloids Surf B Biointerfaces. 2010;

75(1):118.

28. verger ML, Fluckiger L, Kim YI, Homan M, Maincent P. Preparation

and characterization o nanoparticles containing an antihypertensive

agent.Eur J Pharm Biopharm. 1998;46(2):137143.

29. Ferranti V, Marchais H, Chabenat C, Orecchioni AM, Laont O.

Primidone-loaded poly--caprolactone nanocapsules: incorporationeciency and in vitro release proles. Int J Pharm. 1999;193(1):

107111.

30. Kim SY, Lee YM. Taxol-loaded block copolymer nanospheres com-

posed o methoxy poly(ethylene glycol) and poly(epsilon-caprolactone)

as novel anticancer drug carriers. Biomaterials. 2001;22(13):

16971704.31. Zili Z, Sar S, Fessi H. Preparation and characterization o poly--

caprolactone nanoparticles containing griseoulvin.Int J Pharm. 2005;

294(12):261267.

32. Shenoy DB, Amiji MM. Poly(ethylene-oxide)-modiied poly(-caprolactone) nanoparticles or targeted delivery o tamoxien in breast

cancer.Int J Pharm. 2005;293(12):261270.

33. Blouza IL, Charcosset C, Sar S, Fessi H. Preparation and characteriza-

tion o spironolactone-loaded nanocapsules or pediatric use. Int J

Pharm. 2006;325(12):124131.

34. Prabu P, Chaudhari AA, Dharmaraj N, Khil MS, Park SY, Kim HY.

Preparation, characterization, in-vitro drug release and cellular uptake

o poly(caprolactone) grated dextran copolymeric nanoparticles

loaded with anticancer drug. J Biomed Mater Res. 2009;90(4):

11281136.

35. Zheng D, Li X, Xu H, Lu X, Hu Y, Fan W. Study on docetaxel-loadednanoparticles with high antitumor ecacy against malignant melanoma.

Acta Biochim Biophys Sin . 2009;41(7):578587.

36. Costa P, Sousa Lobo JM. Modeling and comparison o dissolution

proles. Eur J Pharm Sci. 2001;13(2):123133.

37. Barzegar-Jalali M, Adibkia K, Valizadeh H, et al. Kinetic analysis o

drug release rom nanoparticles. J Pharm Pharm Sci. 2008;11(1):

167177.

38. Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G. A continuous

tumor-cell line rom a human lung carcinoma with properties o

type II alveolar epithelial cells.Int J Cancer. 1976;17(1):6270.


12/12

International Journal of Nanomedicine

Publish your work in this journal

Submit your manuscript here:ttp://www.dovpss.om/intnational-jounal-of-nanomdiin-jounal

The International Journal o Nanomedicine is an international, peer-reviewed journal ocusing on the application o nanotechnology indiagnostics, therapeutics, and drug delivery systems throughout the

biomedical eld. This journal is indexed on PubMed Central, MedLine,CAS, SciSearch, Current Contents/Clinical Medicine, Journal

Citation Reports/Science Edition, EMBase, Scopus and the ElsevierBibliographic databases. The manuscript management system iscompletely online and includes a very quick and air peer-reviewsystem, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes rom published authors.


D

Dovepress

Dovepress

238

da Volta Soas t al

39. Mosmann T. Rapid colorimetric assay or cellular growth and survival:

application to prolieration and cytotoxicity assays.J Immunol Methods.

1983;65(12):5563.

40. Veiga VF, Nimrichter L, Teixeira CA, et al. Exposure o human

leukemic cells to direct electric current: generation o toxic compounds

inducing cell death by dierent mechanisms. Cell Biochem Biophys.

2005;42(1):6174.

41. Lindig BA, Rodgers MAJ, Schaap AP. Determination o the lietime

o singlet oxygen in D2using 9,10-anthracenedipropionic acid, a water-

soluble probe.J Am Chem Soc . 1980;102(17):55905593.

42. Alexis F, Rhee JW, Richie JP, Radovic-Moreno AF, Langer R,

Farokhzad OC. New rontiers in nanotechnology or cancer treatment.

Urol Oncol. 2008;26(1):7485.

43. Kong G, Braun RD, Dewhirst MW. Hyperthermia enables tumor-specic

nanoparticle delivery: eect o particle size. Cancer Res. 2000;

60(16):44404445.

44. Tanaka N, Imai K, Okimoto K, et al. Development o novel sustained-

release system, disintegration-controlled matrix tablet (DCMT) with

solid dispersion granules o nilvadipine. J Control Release. 2005;

108(23):386395.

45. Hixson AW, Crowell JH. A simple model based on rst order kinetics

to explain release o highly water. Ind Eng Chem Res. 1931;23:

923931.

46. Baker RW, Lonsdale HS. Controlled Release of Biologically Active

Agents. Tanquary AC, Lacey RE, editors. New York, NY: Plenum

Press; 1974.

47. Higuchi T. Mechanism o sustained-action medication: theoretical

analysis o rate o release o solid drugs dispersed in solid matrices.

J Pharm Sci. 1963;52:11451149.

48. Isele U, Schieweck K, Kessler R, van Hoogevest P, Capraro HG.

Pharmacokinetics and body distribution o liposomal zinc phthalocya-

nine in tumor-bearing mice: infuence o aggregation state, particle size,

and composition.J Pharm Sci. 1995;84(2):166173.

49. Allen CM, Sharman WM, van Lier JE. Current status o phthalocyanines

in the photodynamic therapy o cancer. J Porphyr Phthalocyanines.

2001;5:161169.

50. Zhao B, Yin JJ, Bilski PJ, Chignell CF, Roberts JE, He Y-Y. Enhanced

photodynamic ecacy towards melanoma cells by encapsulation o

Pc4 in silica nanoparticles. Toxicol Appl Pharmacol. 2009;241(2):

163172.
http://www.dovepress.com/international-journal-of-nanomedicine-journalhttp://www.dovepress.com/testimonials.phphttp://www.dovepress.com/testimonials.phphttp://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/http://www.dovepress.com/testimonials.phphttp://www.dovepress.com/testimonials.phphttp://www.dovepress.com/international-journal-of-nanomedicine-journal

ijn 15860 nano structured delivery system for zinc phthalocyanine prep 012411[1]

Documents