polyion complex micelles entrapping cationic dendrimer porphyrin

www.elsevier.com/locate/jconrel

Journal of Controlled Release 93 (2003) 141–150

Polyion complex micelles entrapping cationic dendrimer porphyrin:

effective photosensitizer for photodynamic therapy of cancer$

Guo-Dong Zhanga,b, Atsushi Haradaa,b, Nobuhiro Nishiyamaa,d, Dong-Lin Jiangc,Hiroyuki Koyamad, Takuzo Aidac, Kazunori Kataokaa,b,*

aDepartment of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-8656, JapanbCREST, Japan Science and Technology Corporation, Japan

cDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-8656, JapandDepartment of Clinical Vascular Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-8655, Japan

Received 15 April 2003; accepted 27 May 2003

Abstract

Photosensitizers play a crucial role in the photodynamic therapy (PDT) of cancer. In this study, a third-generation aryl ether

dendrimer porphyrin with 32 primary amine groups on the periphery, [NH2CH2CH2NHCO]32DPZn, and pH-sensitive, polyion

complex micelles (PIC) composed of the porphyrin dendrimer and PEG-b-poly(aspartic acid), were evaluated as new

photosensitizers (PSs) for PDT in the Lewis Lung Carcinoma (LLC) cell line. The preliminary photophysical characteristics of

[NH2CH2CH2NHCO]32DPZn and the corresponding micelles were investigated. Electrostatic assembly resulted in a red-shift of

the Soret peak of the porphyrin core and the enhanced fluorescence. Compared to the dendrimer porphyrin

[NH2CH2CH2NHCO]32DPZn, relatively low cellular uptake of dendrimer porphyrin [NH2CH2CH2NHCO]32DPZn

incorporated in the PIC micelle was observed, yet the latter exhibited enhanced photodynamic efficacy on the LLC cell

line. Importantly, the use of PIC micelles as a delivery system reduced the dark toxicity of the cationic dendrimer porphyrin,

probably due to the biocompatible PEG shell of the micelles.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Photodynamic therapy; Photosensitizer; Dendrimer porphyrin; Polyion complex micelle; Block copolymer

0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2003.05.002

$ Presented at 11th International Symposium on Recent

Advances in Drug Delivery Systems and CRS Winter Symposium,

Salt Lake City, UT, March 3–6, 2003.

* Corresponding author. Department of Materials Science and

Engineering, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.:

+81-3-5841-7138; fax: +81-3-5841-7139.

E-mail address: [email protected] (K. Kataoka).

1. Introduction

To create new photosensitizers (PSs) or their for-

mulation plays a crucial role in the development of

photodynamic therapy (PDT). Ideal PSs should pos-

sess such characteristics as selectivity to solid tumor

tissue, and higher photocytotoxicity with lower cyto-

toxicity under dark conditions [1,2]. PDT of cancer

involves the systemic administration of photosensi-

G.-D. Zhang et al. / Journal of Controlled Release 93 (2003) 141–150142

tizers to solid tumor tissue and local illumination with

light of a specific wavelength, leading to photochem-

ical destruction of cancer cells via generation of singlet

oxygen or superoxide from molecular oxygen [3].

Since there are some drawbacks of Photofrin II, the

only PS’s applied in clinical practice, including a lack

of chemical homogeneity, skin phototoxicity, and poor

selectivity for tumor tissue, exploiting the second-

generation PSs has recently been the subject of intense

studies. However, most are hydrophobic properties,

which results in two inevitable problems: the delivery

in blood circulation and the low photophysical prop-

erties due to the aggregation of PSs, decreasing the

photo-oxidation efficacy to achieve the photodynamic

therapy.

Suitable carrier and delivery systems for PSs

should have a simple but effective strategy to realize

high selectivity, high photodynamic efficacy and

should have low side effects [4]. A variety of delivery

systems have been studied, such as polymer carriers

[5], liposomes [6,7], and bioconjugates [8–10]. On

the other hand, polymeric micelles have been inten-

sively studied to effectively deliver drugs. They are

generally more stable when compared to surfactant

micelles with a remarkably lowered critical micellar

concentration and having a slower rate of dissociation,

resulting in prolonged circulation time in vivo. More-

over, several types of polymeric micelles with PEG

shell were demonstrated to accumulate effectively and

selectively in solid tumor. For example, block copol-

ymer micelles incorporating doxorubicin are now in

phase I clinical trials [11,12]. Thereby, polymeric

micelles with a PEG shell should become a useful

vehicle to help photosensitizers accumulate into solid

tumor, achieving high photodynamic efficacy.

Dendrimers, three-dimensional tree-like branched

macromolecules, possess some fascinating character-

istics: a well-defined structure, including very narrow

molecular weight distribution and three-dimensional

structures tuned by dendrimer generation and dendron

structure, and their flexibility for tailored functional

groups with high density on the periphery [13]. Thus,

studies of the biomedical application of dendrimers

are becoming more and more attractive, especially in

the field of non-viral gene vector and drug delivery

systems [14–17]. Taking into account the abovemen-

tioned advantages and the enhanced permeability and

retention effects (EPR) of macromolecular drugs [18],

we investigated the photodynamic efficacy of cationic

and anionic dendrimer porphyrin [19], 32(+)DPZn (32

quaternary ammonium groups on the periphery,

[ClMe3NCH2CH2NHCO]32DPZn) and 32(� )DPZn

(32 carboxylate groups on the periphery, [COOH]32DPZn), finding that the photodynamic efficiency of

the former is remarkably high compared to the latter

and protoporphyrin IX, a primitive but effective

photosensitizer [20,21]. It is worth noting that the

micelles from the pair of [COOH]32DPZn/PEG-block-

poly(L-lysine) show an appreciably higher stability

against sodium chloride concentration, probably due

to the formation of intermolecular hydrogen bonding

in the core. No such salt stabilization was observed for

the micelles from the pair of [ClMe3NCH2CH2

NHCO]32DPZn with poly(ethylene glycol)-b-poly(as-

partic acid) (PEG-b-P(Asp)) [22]. To make use of the

additional stability resulting from hydrogen bonding

in salt solution and explore a new photosensitizer with

a high efficacy for PDT, we synthesized the new

dendrimer porphyrin with 32 primary amine groups

on the periphery, [NH2CH2CH2NHCO]32DPZn, pos-

sessing the assembling behavior with PEG-b-P(Asp)

to form spherical PIC micelle [23]. The micelles

exhibited a high stability up to a 0.90 M (NaCl) salt

concentration and the pH-sensitive behavior to keep

their stability within the limited range of pH from 6.2

to 7.4 in physiological saline (0.15 M NaCl), suggest-

ing the potential of the pH-triggered release of the

entrapped dendrimers in the acidic pH environment

(pHf 5.0) of the intracellular endosomal compart-

ment. Both the high photodynamic efficacy of the

cationic dendrimer porphyrin 32(+)DPZn, and the

beneficial features of PIC micelle entrapping [NH2

CH2CH2NHCO]32DPZn, encourage us to investigate

the possibility of [NH2CH2CH2NHCO]32DPZn and

the corresponding micelles as new photosensitizers

for photodynamic therapy of cancer.

2. Materials and methods

2.1. Materials

h-Benzyl L-aspartate (BLA) and bis(trichloro-

methyl) carbonate (triphosgene) were purchased from

Tokyo Kasei Kogyo, Japan. a-Methoxy-N-aminopo-

ly(ethylene glycol) (Mw = 12 kg/mol) was a kind gift

G.-D. Zhang et al. / Journal of Controlled Release 93 (2003) 141–150 143

from Nippon Oil and Fats, Japan. The polymer was

precipitated in diethylether from chloroform, dried

under reduced pressure and subsequently freeze-dried

from benzene prior to use in the block copolymer

synthesis. 3,5-Dihydroxybenzyl alcohol and methyl 4-

(bromomethyl)-benzoate were purchased from

Aldrich and used without further purification. The

protoporphyrin IX (PIX, 8,13-divinyl-3,7,12,17-tetra-

methyl-21H,23H-porphine-2,18-dipropionic acid)

(Aldrich Chemical, USA) was used as a control

photosensitizer in this study.

2.2. Synthesis of poly(ethylene glycol)-poly(a,b-aspartic acid) block copolymer

Poly(ethylene glycol)-poly(a,b-aspartic acid) blockcopolymer [PEG-P(Asp)] was prepared by a previ-

ously reported procedure [24]. Briefly, PEG-b-P(Asp)

was synthesized by alkali hydrolysis of benzyl groups

of the side chain of the poly(ethylene glycol)-poly(b-benzyl-L-aspartate) block copolymer (PEG-b-PBLA,

Mw/Mn = 1.07), which was synthesized by the ring-

opening polymerization of BLA-NCA initiated with

the terminal primary amino group of a-methoxy-x-

amino poly(ethylene glycol) (Mn = 1.2193� 103,

DP= 275) under an argon atmosphere in DMF [22].

From the 1H NMR spectrum in D2O, the polymeri-

zation degree of the poly(aspartic acid) segment was

determined to be 28.

2.3. Synthesis of [CF3CONHCH2CH2NHCO]32DPZn

Triethylamine(2.28� 10� 3 mol) was added to tri-

fluroacetyl-1,2-ethylenediamine hydrochloride

(1.14� 10� 3 mol) in 3 ml of anhydrous DMF, stir-

red for 30 min at room temperature, and filtered to

remove the side product, triethylamine hydrochloride.

A mixture of [CO2H]32DPZn (2.54� 10� 6 mol), N-

trifluoroacetyl-ethylene-1,2-diamine (1.14� 10� 3

mol), DCC (4.88� 10� 3 mol), HOBt (3.25� 10� 3

mol) in DMF (8 ml) was stirred at room temperature

under Ar for 7 days. The solution was dialyzed against

water, then dried in vacuum and purified by column

chromatography, gradually eluting with increas-

ing methanol to 20% methanol/CHCl3, giving

[CF3CONHCH2CH2NHCO]32DPZn, yield (32%).

MALDI-TOF-MS (dithranol matrix): M/z = 12448.6

(calcd. 12448.3); 1H NMR (DMSO-d6, 25 jC):

d= 9.48 (s, 32H, NHCOCF3), 8.88 (s, 8H, pyrrole-h),8.57 (s, 32H, NHCO), 7.81, 7.33 (both d, 128H, Ar-o-

H, Ar-p-H), 7.48 (s, 8H, Ar-o-H), 7.14 (s, 4H, Ar-p-H),

6.76 (d, 16H, Ar-o-H), 6.61 (d, 32H, Ar-o-H), 6.55 (s,

8H, Ar-p-H), 6.51 (s, 16H, Ar-p-H), 5.17 (s, 16H,

OCH2), 4.96 (s, 64H, OCH2), 4.91 (s, 32H, OCH2).

IR mmax (KBr Pellet): 1717.3 cm� 1 (CF3CONH-),

1630.0 cm� 1 (-CONH-).

2.4. Synthesis of [NH2CH2CH2NHCO]32DPZn

The resulting dendrimer [CF3CONHCH2CH2NH-

CO]32DPZn was dissolved in 13 ml methanol (105

mg K2CO3 and 0.9 ml water), refluxed for 3 h,

dialyzed and lyophilized to provide the title den-

drimer, yield (53%). 1H NMR (DMSO-d6, 25 jC):d= 8.81 (s, 8H, pyrrole-h), 8.50 (s, 32H, NHCO),

7.81, 7.33 (both d, 128H, Ar-o-H, Ar-p-H), 7.48 (s,

8H, Ar-o-H),7.14 (s, 4H, Ar-p-H), 6.76 (d, 16H, Ar-o-

H), 6.61 (d, 32H, Ar-o-H), 6.55 (s, 8H, Ar-p-H), 6.51

(s, 16H, Ar-p-H), 5.17 (s, 16H, OCH2), 4.96 (s, 64H,

OCH2), 4.91 (s, 32H, OCH2). IR mmax (KBr Pellet):

1630.0 cm� 1 (-CONH-).

2.5. Preparation of PIC micelles

Given amounts of [NH2CH2CH2NHCO]32DPZn

and PEG-b-poly(Asp) were separately dissolved in

NaH2PO4 (10 mM, pH 3.0 by adding 0.01 M HCl)

and Na2HPO4 (10 mM) solution to prepare the stock

solutions, then mixed in a stoichiometric ratio, fol-

lowed by dialysis against 10 mM PBS until the pH of

the micelle solution was 7.4.

2.6. Methods

The 1H NMR spectra were obtained in DMSO-d6on a JEOL type GSX-270 spectrometer operating at

270 MHz. Matrix-assisted laser desorption ionization

time-of-flight mass spectroscopy (MALDI-TOF-MS)

was performed with an Applied Biosystems model

Voyager-DE STR TOF mass spectrometer using 3-

indolacrylic acid or dithranol as the matrix. Fluores-

cence measurement was performed by JASCO spec-

trofluorometer FP-6500 (Tokyo, Japan). UV–VIS

spectroscopy was performed using a JASCO V-550

(Tokyo, Japan). The light-scattering measurements

were performed on a Photal dynamic laser scattering

Fig. 1. Chemical structure of related porphyrin dendrimers and the

synthesis route. (1) DCC, HOBt, CF3CONHCH2CH2NH2. (2)

K2CO3, methanol.


DLS-7000DL spectrometer (Otsuka Electronics)

equipped with an argon laser (k0 = 488 nm).

2.7. Quantitative analysis of cellular uptake of

dendrimer and micelles by LLC cells

Quantification of the amount of the dendrimer or

micelles associated with Lewis Lung Carcinoma

(LLC) cells at 37 jC was performed by utilizing the

fluorescence of the dendrimer at 609 nm (excitation at

432 nm). Following exposure to dendrimer or

micelles for 2, 6, and 12 h, the cells were washed

three times with sterile PBS, harvested and then

dissolved in 5% SDS solution prior to fluorescence

measurement (n = 3).

2.8. Photodynamic efficacy and dark toxicity of

photosensitizers in vitro

The cytotoxicity of each photosensitizers in vitro

was assessed against LLC cells. In a dark room,

different concentrations of photosensitizers in Dulbec-

co’s modified Eagle’s medium (DMEM+10% fetal

bovine serum (FBS)) were added to cells in 96-well

culture plates (n = 4). After a defined incubation time

(4 and 12 h) at 37 jC, the photosensitizers were

removed, and then plates were photoirradiated for

10 min with a broadband visible light using a Xenon

lamp (150 W) equipped with a filter passing light of

400–700 nm fluence energy: 180 kJ/cm2. The viabil-

ity of photoirradiated and non-photoirradiated cells

was evaluated using mitochondrial respiration via the

3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium

bromide cleavage assay (MTT assay) following incu-

bation for 24 h after photoirradiation or removing the

photosensitizers by washing in the case of the dark

toxicity investigation.

3. Results and discussion

3.1. Synthesis of dendrimer porphyrin and prepara-

tion of micelles

We have described the synthesis of a new den-

drimer porphyrin bearing 32 primary amine groups on

the periphery by amidation of the corresponding

carboxylic acid porphyrin dendrimer with mono-pro-

tected ethylenediamine, followed by quantitative

deprotection (Fig. 1) [23]. The perfection of den-

drimer porphyrin after amidation of [COOH]32DPZn

was confirmed by MALDI-TOF-MS investigation,

shown in Fig. 2, and was achieved by two means:

exhausted reaction of carboxylic acid on the periphery

of the dendrimer porphyrin by N-trifluoro-acetylethy-

lenediamine more than 10 times equivalent of carbox-

ylic acid; effective purification method, including

column chromatography (SiO2) and preparative

HPLC using THF as eluent.

The stoichiometric mixing of solutions of the

resulting dendrimer and PEG-b-P(Asp) resulted in

spontaneously formation of water-soluble PIC

micelles, driven by electrostatic interactions [24,25].

The resulting micelles are spherical, with a diameter

of ca. 55 nm, and a narrow size distribution (unim-

odal, A2/G2 < 0.09), based on dynamic light scattering

Fig. 2. MALDI-TOF-MS spectrum of the porphyrin dendrimer [CF3CONHCH2CH2NH]32DPZn, matrix: dithranol.


(DLS) measurement (Fig. 3). Interestingly, these

spherical micelles have two distinguishing features.

First, the micelles possessed extraordinary stability in

salt (NaCl) solution up to 0.90 M. Second, there is a

strict pH-dependent stability of the micelles, that is,

Fig. 3. DLS histogram of PICmicelles of [NH2CH2CH2NH]32DPZn/

PEG-b-P(Asp). At 37 jC, 150 mM NaCl, detection angle 90j.

they can remain stable in the solutions with a pH

ranging from 6.2 to 7.4, whereas a sharp structural

transition took place if the pH was beyond this region.

These two features are related to the globular structure

of the dendrimer and the unique interactions between

the rigid macroion and the poly(aspartic acid) segment

of the block copolymer [23]. The high stability of the

micelles, under physiological conditions (pH 7.4, 0.15

M NaCl) provides the possibility to avoid the leakage

of cargo from the micellar nanocarrier before they

selectively accumulate in the solid tumor tissue.

However, upon accumulating in a solid tumor, where

the local pH should be significantly lower than that of

normal tissue, or entering the acidic intracellular

endosomal compartment (f pH 5.0) after endocyto-

sis [26], the micelles may be easily broken down in

such complex circumstances, due to the disturbance

from a variety of charged biomacromolecules as well

as the acid-sensitive characteristics, releasing the

encapsulated dendrimer porphyrins and providing a

unique photosensitizing effect. Overall, both the high

stability in salt solution and the acid-responsive be-

havior of the micelle system suggested that the


micelles encapsulated porphyrin dendrimer have the

high potential for tumor environment-sensitive deliv-

ery of system photosensitizers for PDT.

3.2. Photophysical characteristics of dendrimer and

the micelles

For preliminary evaluation of photosensitizers, the

UV–VIS absorption and fluorescence emitting behav-

ior of the dendrimer porphyrin and the corresponding

micelles were investigated (Fig. 4). Clearly, there are

intermolecular aggregates of dendrimer porphyrin

[NH2CH2CH2NHCO]32DPZn, as indicated by dual

Soret peaks (448.5 and 433 nm) appearing in Fig. 4A.

Fig. 4. UV–VIS absorption (Panel A) and fluorescence emission

(Panel B) spectra of [NH2CH2CH2NH]32DPZn and the correspond-

ing micelles in PBS solutions (pH 7.4). D, dendrimer porphyrin; M,

micelles. In Panel A, dendrimer porphyrin equivalent concentration:

12 AM. In Panel B, dendrimer porphyrin equivalent concentration

[a] = 12 AM, [b] = 6 AM.

On the other hand the electrostatic assembly between

the cationic surface of dendrimer porphyrin and

anionic segments of PEG-b-P(ASP) exhibited the

ability to prevent the interactions of the dendrimer

porphyrins and diminish the aggregates, since the

dual Soret peaks were substituted by a sole peak

(433 nm). Moreover, the red shift (2 nm) of the Soret

peak (433 nm) of the dendrimer porphyrin, entrapped

in the core of PIC micelles, is characteristic of

electrostatic assembly of charged porphyrins and

oppositely charged compounds [27,28]. From Fig.

4B, the intensity of fluorescence of micelle solution

emitting at 610 nm was improved, compared to

[NH2CH2CH2NHCO]32DPZn under the same condi-

tions, resulting from the microstructure of the polyion

complex micelles. First, the assembly between the

porphyrin dendrimer and the poly(aspartic acid)

segment effectively eliminated the aggregates of

dendrimer porphyrin, as shown in the UV–VIS spec-

trum. Additionally, in the core of micelles, the higher

microviscosity significantly refrained the internal mo-

lecular motion of the dendrimer porphyrin, leading to

the inhibition of radiationless decay [29]. We will

investigate the fluorescence anisotropy and lifetime to

get detailed information of dendrimer porphyrin

entrapped in the core of PIC micelles.

3.3. Cellular uptake of dendrimer porphyrin and the

micelles in the LLC cell line

On the basis of the fluorescence of the dendrimer

porphyrin, the time-dependent cellular uptake of

dendrimer polyphyrin and its micelles by LLC cells

at different temperatures (4 and 37 jC) was investi-

gated based on their fluorescence (Fig. 5). In the

case of the dendrimer porphyrin alone, the initial

cellular association reached a relatively high amount

within 2 h, which is independent on the temperature,

and then increased linearly with the incubation time

at 37 jC, and the same phenomenon was also

observed in the case of 32(+)DPZn [19]. The

micelles exhibited different behavior of cellular up-

take, where the initial uptake amount is very low,

and then increasing gradually with prolonged time.

During the observing time, the uptake of dendrimer

porphyrin alone was about three times greater than

that of the corresponding micelles. Similarly, the

cellular uptake of both dendrimer porphyrin and its

Fig. 5. Uptake of [NH2CH2CH2NH]32DPZn and the micelles by

LLC cells as a function of time at 37 and 4 jC. Concentration of

dendrimer porphyrin equivalent in medium: 12 AM, (a) at 37 jCin the presence of dendrimer porphyrin, (b) at 4 jC, with

dendrimer porphyrin, (c) at 37 jC with micelle, (d) at 4 jC with

micelle.


micelles is greatly diminished at 4 jC, showing the

process to be energy-dependent, suggesting that

endocytosis might be the major internalization pat-

tern. Regarding the internalization of the dendrimer

porphyrins, both 32(+)DPZn and 32(� )DPZn, en-

docytosis is the major pattern of internalization [19],

since both dendrimers internalized and finally

appeared to localize in endosomal compartments,

due to their colocalization with Tex-Red dextran, a

fluid-phase endocytic marker. As to cellular internal-

izations of polymeric micelles, recently, Luo et al.

[30] first gave direct evidence that the micelles,

consisting of rhodamine-labeled PEO45-b-PCL23

block copolymer, can be internalized into P19 cells

by endocytosis, and Kabanov’s group [31] reported

that Pluronic copolymer micelles can also be inter-

nalized by an endocytotic pathway. The preliminary

results with PIC micelles entrapping dendrimer por-

phyrins support the abovementioned findings of

endocytotic pathway of polymeric micelles. Addi-

tionally, the initially rapid uptake of dendrimer

porphyrin seems to have resulted from the electro-

static interaction of the positive surface of dendrimer

and the plasma membrane, since the plasma mem-

brane of mammalian cells possesses a negative

charge and tumor cells have a more negative net

charge than normal cells resulting from overexpres-

sion of polysialic acid residues [32].

3.4. In vitro photodynamic effect and dark toxicity of

dendrimer and the micelles

The viability of LLC cells upon photoirradiation

was evaluated by MTT assay and determined as a

function of concentration and exposure time with the

photosensitizers, PIX, [NH2CH2CH2NHCO]32DPZn,

and the micelles. In this assay, photosensitizers were

incubated with LLC cells for a definite period

(4, 12 h), and then fully washed with PBS to remove

non-associated photosensitizers prior to photoirra-

diation. The cellular viability upon photoirradiation

indicates the photodynamic effect in vitro. Fig. 6

illustrates the photodynamic effect on the LLC cell

line, in a function of the photosensitizer concentration

and incubating time with photosensitizers, and

the photodynamic effects of different photosensitizers

are summarized in Table 1, in the form of the IC50,

defined as the concentration of photosensitizer

at which 50% of tumor cells survive after pho-

toirradiation, calculated from Fig. 6(A,B). At 37

jC dendrimer porphyrin incorporated in micelles

had 147 and 60 times higher photodynamic effi-

cacy than PIX after 4 or 12 h incubation, respec-

tively. Moreover, [NH2CH2CH2NHCO]32DPZn

produced the lower photodynamic effect about one

order of magnitude compared to 32(+)DPZn [19].

When the IC50 doses of the dendrimer porphyrin

[NH2CH2CH2NHCO]32DPZn and the micelles were

normalized to the uptake amount of porphyrin den-

drimer equivalent, it was found that the micelles

delivery system showed 42 and 35 times higher

normalized photodynamic efficacy than dendrimer

porphyrin alone after 4 and 12 h incubation, respec-

tively. Apparently, the photodynamic efficacy of den-

drimer porphyrin [NH2CH2CH2NHCO]32DPZn and

the PIC micelle delivery system is not only determined

by their cellular uptake, since the uptake amount of

dendrimer porphyrin alone was about three times

greater than that of the corresponding micelles.

According to the results of UV–VIS investigation,

[NH2CH2CH2NHCO]32DPZn demonstrates the ten-

dency to aggregate in physiological conditions, and

usually aggregate damages the photodynamic effica-

cy by lowering the yield of singlet oxygen [33,34].

Fig. 6. The photocytotoxicity against LLC cells after 4 h incubation

with PIX (square), [NH2CH2CH2NH]32DPZn (triangle) and its

micelles (circle) (Panel A) and 12 h incubation with photosensitizer

(Panel B), and the dark toxicity of PIX, [NH2CH2CH2NH]32DPZn

and its micelles after 12 h incubation (Panel C).

Table 1

The photodynamic effect (IC50, AM) of PIX, [NH2CH2CH2NHCO]32DPZn and the micelles in the LLC cell line

(h) PIX DPZn Micelle

4 4.26 0.403 0.0289

12 1.67 0.327 0.0275


Importantly, the IC50 values of current micelles were

basically the same order of magnitude as that of

micelles entrapping [COOH]32DPZn [35], whereas

[NH2CH2CH2NHCO]32DPZn produced the higher

photodynamic efficacy about one order of magni-

tude compared to [COOH]32DPZn [35]. Thereby,

we may hypothesize that the similar micelles

brought about the same subcellular fate of den-

drimer porphyrins despite opposite charges on their

surface. Interestingly, Hamblin et al. [36] recently

demonstrated that pegylation of chlorin-e6 may

increase the efficacy of photodynamic therapy, since

pegylation led to more mitochondrial localization

and mitochondria might be one of the most effec-

tive targets in photodynamic cell death, based on

the findings of the mitochondrial localization of

many photosensitizers and their early responses to

light activation [37,38].

Low dark toxicity is one of the important criteria

for assessing the usefulness of photosensitizers, since

the major side effects in clinical PDT result from the

dark toxicity of photosensitizer to normal tissue. From

Fig. 6(C), PIC micelles entrapping [NH2CH2CH2

NHCO]32DPZn was basically non-toxic at the con-

centration used, yet the dendrimer porphyrin alone

showed dark toxicity with approximately 85% cell

viability observed at 33 AM of [NH2CH2CH2NH-

CO]32DPZn. The very low toxicity of PIC micelles

might result from the good biocompatibility and low

toxicity of poly(ethylene glycol) [39]. Importantly,

PIC micelles with poly(ethylene glycol) as hydrophil-

ic shell, possess the ability of selectively accumulat-

ing in the solid tumor tissue and long circulation in

vivo. Furthermore, those micelles usually are nano-

scaled particles, with narrowly distributed size in the

range of several tens of nanometers. Therefore, the

efficient photodynamic efficacy of PIC micelles

entrapping dendrimer porphyrin, together with the

abovementioned advantages, make this type of pho-

tosensitizer formulation potentially very useful for

clinical application.


4. Conclusion

In conclusion, nanocarriers consisting of polyion

complex micelles, composed of PEG-b-P(Asp)

and the nano-scaled dendrimer porphyrin

[NH2CH2CH2NHCO]32DPZn, could effectively de-

liver dendrimer porphyrin photosensitizers into

cancer cells and produce enhanced photodynamic

efficacy, over cationic dendrimer porphyrin and

PIX. Importantly, the PIC micelle formulation

exhibited no dark toxicity within the concentration

used. It is expected that this promising nanocarrier

of PIC micelles can be practical for encapsulation

of other ionic photosensitizers, especially for

phthalocyanine derivatives, typical second-genera-

tion photosensitizers.

Acknowledgements

This work was supported by Core Research for

Evolutional Science and Technology (CREST), Japan

Science and Technology (JST), and by a Grant-in-aid

for Scientific Research, as well as by Special

Coordination Funds for Promoting Science and

Technology, Ministry of Education, Culture, Sports,

Science and Technology, Japan (MEXT).

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