Methods 46 (2008) 25–32

Contents lists available at ScienceDirect

Methods

journal homepage: www.elsevier.com/ locate /ymeth

Polymersomes: A new multi-functional tool for cancer diagnosis and therapy

Dalia Hope Levine a, P. Peter Ghoroghchian b, Jaclyn Freudenberg c, Geng Zhang c, Michael J. Therien d, Mark I. Greene c, Daniel A. Hammer a,b, Ramachandran Murali c,*

a Depart ment of Chem i cal and Bio mo lec u lar Engi neer ing, Uni ver sity of Penn syl va nia, Phil a del phia, PA, USAb Depart ment of Bio en gi neer ing, Uni ver sity of Penn syl va nia, Phil a del phia, PA, USAc Depart ment of Pathol ogy and Lab o ra tory of Med i cine, Uni ver sity of Penn syl va nia, 243 John Mor gan Build ing, 36th Ham il ton Walk, Phil a del phia, PA 19104-6082, USAd Depart ment of Chem is try, Duke Uni ver sity, Dur ham, NC, USA

a r t i c l e i n f o

Article history:

Accepted 22 May 2008

Available online 20 June 2008

Key words:

Polym er some

Di block copoly mer

Ves i cle

Drug deliv ery

Can cer diag no sis

Can cer ther apy

Nano par ti cle

Tar get ing

Opti cal imag ing

Near-infra red

1046-2023/$ - see front matter © 2008 Else vier Idoi:10.1016/j.ymeth.2008.05.006

* Cor re spond ing author. Fax: +1 215 898 2401.

E-mail address: mu ral i@mail.med.upenn.edu, ra

(R. Murali).

a b s t r a c t

Nano par ti cles are being devel oped as deliv ery vehi cles for ther a peu tic phar ma ceu ti cals and con trast

imag ing agents. Poly mer somes (mes o scop ic poly mer ves i cles) pos sess a num ber of attrac tive bio ma te-

rial prop er ties that make them ideal for these appli ca tions. Syn thetic con trol over block copoly mer chem-

is try enables tun able design of polym er some mate rial prop er ties. The polym er some archi tec ture, with

its large hydro philic res er voir and its thick hydro pho bic lamel lar mem brane, pro vides sig nifi cant stor age

capac ity for both water sol u ble and insol u ble sub stances (such as drugs and imag ing probes). Fur ther,

the brush-like archi tec ture of the polym er some outer shell can poten tially increase bio com pat i bil ity and

blood cir cu la tion times. A fur ther recent advance is the devel op ment of multi-func tional poly mer somes

that carry phar ma ceu ti cals and imag ing agents simul ta neously. The abil ity to con ju gate bio log i cally

active ligands to the brush sur face pro vides a fur ther means for tar geted ther apy and imag ing. Hence,

poly mer somes hold enor mous potential as nano struc tured bio ma te ri als for future in vivo drug deliv ery

and diag nos tic imag ing appli ca tions.

© 2008 Else vier Inc. All rights reserved.

1. Back ground

Nano sized car ri ers are prime can di dates for the deliv ery of

highly toxic and/or hydro pho bic ther a peu tic agents. These deliv ery

vehi cles have the potential to aug ment the phar ma co dy namic and

phar ma co ki netic pro files of drug mol e cules, thereby enhanc ing

the ther a peu tic effi cacy of the phar ma ceu ti cal agents [1]. Fur ther,

encap su lat ing the drug mol e cule in a deliv ery sys tem can increase

in vivo sta bil ity, extend its blood cir cu la tion time, and fur ther pro-

vide a means for con trol ling the release of the agent [1]. More over,

the deliv ery sys tem can alter the bio dis tri bu tion of the drug mol e-

cule by allow ing the agent to accu mu late at the tumor site, either

pas sively or actively with tar get ing [1]. In addi tion to ther a peu tic

drug deliv ery, serv ing as diag nos tics tools, nano sized car ri ers can

deliver imag ing agents to detect and non-inva sively diag nose dis-

ease.

Poly mer somes, poly mer ves i cles self-assem bled from a diverse

array of syn thetic amphi philic block copoly mers con tain ing hydro-

philic and hydro pho bic blocks [2–4], have been shown to pos sess

supe rior bio ma te rial prop er ties, includ ing greater sta bil ity and

nc. All rights reserved.

ma chan dran.mu ral i@gmail.com

stor age capa bil i ties [5–7], as well as pro longed cir cu la tion time,

as com pared to lip o somes (ves i cles derived from phos pho lip ids)

[8]. A par tic u larly attrac tive stor age fea ture, high lighted in Fig. 1, is

the large hydro pho bic core of the polym er some mem brane, which

fol lows from the mem brane-form ing amphi philic poly mers being

larger than con ven tional phos pho lip ids [9]. Fur ther, block copoly-

mer chem is tries can be tuned through poly mer syn the sis to yield

poly mer somes with diverse func tion al ity [10]. A vast major ity of

ves i cles made of syn thetic copoly mers have dense poly eth yl ene

oxide (PEO) outer shells, which affords them “stealth” like char ac-

ter that may lead to increased cir cu la tion times and in vivo bio com-

pat i bil ity [5]. Thus, although lip o somes are pres ently used in var i-

ous bio tech no log i cal and phar ma ceu ti cal appli ca tions to improve

ther a peu tic indi ces and enhance cel lu lar uptake [4], it appears that

poly mer somes can offer supe rior advan ta ges for future clin i cal

ther a peu tic and diag nos tic imag ing appli ca tions.

In aque ous solu tions, amphi philic block copoly mers can self-

assem ble into mes o scop ic struc tures (6200 nm–50 lm in diam e-

ter) [3]. The ratio of hydro philic to hydro pho bic block vol ume frac-

tion deter mines whether micelles (spher i cal, pro late, or oblate),

or ves i cles (poly mer somes) will form [2,11–13]. As a gen eral

rule, how ever, a ratio of hydro philic block to total poly mer mass

of approx i mately 635% ± 10% yields mem brane struc tures, while

copoly mers with ratios greater than 45% gen er ally form micelles;

those with ratios less than 25% form inverted micro struc tures [14].

26 D.H. Levine et al. / Methods 46 (2008) 25–32

Fig. 1. Schematic rep re sen ta tions of NIR-emis sive poly mer somes. (A) In aque ous solu tion, amphi philic di block copoly mers of poly eth yl ene ox ide-1,2 poly bu ta di ene (PEO30–

PBD46) self-assem ble into poly mer ves i cles (poly mer somes) with the hydro pho bic PBD tails ori ent ing end-to-end to form bilayer mem branes. The depicted uni la mel lar

polym er some dis plays an excised cross-sec tional slice illus trat ing the bilayer PBD mem brane (gray) con tain ing the hydro pho bic (por ph i na to)zinc(II) (PZn)-based near-IR

fluo ro phores (NIR Fs, red). (B) CAChe-gen er ated sec tional schematic of the NIR-emis sive polym er some mem brane indi cat ing the molec u lar dimen sions of: (i) the PBD com-

po nent of the bilayer (9.6 nm); (ii) the large, dis persed PZn-based NIR Fs (2.1–5.4 nm); and, (iii) a typ i cal lipo some mem brane (3–4 nm) com prised of phos pho lip ids (1-ste a-

royl-2-oleoyl-sn-Gly ce ro-3-Pho soph o-cho line—SOPC). (C) Chem i cal struc tures of NIR fluo ro phores PZn2–PZn5. [This image was repro duced from Ghorogh chian et al. [9] with

per mis sion from Copy right (2005) National Acad emy of Sci ences, USA.]

Micel lar struc tures have been used as intra cel lu lar and sys temic

deliv ery sys tems [15–18] but pres ent sig nifi cant lim i ta tions when

com pared to poly mer somes. In aque ous solu tions, they can only

encap su late hydro pho bic mol e cules unless strong bind ing or cova-

lent link ing strat e gies are incor po rated for seques ter ing aque ous-

sol u ble com po nents.

In con trast, poly mer somes can simul ta neously encap su late

hydro philic com po nents in their aque ous inte rior and hydro pho-

bic mol e cules within their thick lamel lar mem branes [10]. In addi-

tion, bio log i cally active ligands, such as anti bod ies, can be read ily

con ju gated to the exte rior brush sur face to tar get the ves i cles or

to pro vide a ther a peu tic response [19–22]. These prop er ties of

the ves i cle archi tec ture effec tively cre ate a mul ti modal plat form,

which can be used for ther a peu tic (drug deliv ery) and/or diag nos-

tic (imag ing) appli ca tions (Fig. 2).

Although ves i cles can be tar geted to spe cific sites using bio-

log i cally active ligands, the ana tom i cal and path o phys i o log i cal

abnor mal i ties of the tumor tis sue alone can be uti lized to aid in

the local ized deliv ery of mac ro mol e cules [23]. The tumor vas cu la-

ture, char ac ter ized by irreg u larly shaped, dilated, defec tive, and/or

leaky blood ves sels, dis or ga nized endo the lial cells with fene stra-

tions, as well as other abnor mal i ties, allows for the pas sive accu-

mu la tion of mac ro mol e cules at the tumor site [24]. Fur ther, due

to the poor lymphatic drain age, nano par ti cles can accu mu late and

remain at the tumor site even in the absence of a tar get ing moi-

ety [25]. This phe nom e non is known as the enhanced per me abil ity

and reten tion (EPR) effect and makes it pos si ble to achieve high

local con cen tra tions of mac ro mol e cules at the tumor site with-

out spe cific tar get ing [24]. How ever, a ques tion that has yet to be

addressed with poly mer somes is how much addi tional accu mu la-

tion is pos si ble with tar get ing.

2. Di block copoly mers form ing ves i cles and release

mech a nisms

In this sec tion, we high light some of the poly mer for mu la tions,

which have led to the for ma tion of poly mer somes, that have dem-

on strated prom ise for con trolled release of phar ma ceu ti cals.

Ini tial polym er some research by Ham mer and Di scher used

poly(eth yl ene oxide)-block-poly(eth yl eth yl ene) (PEO-b-PEE)

di block copoly mers to dem on strate the for ma tion of poly mer-

somes in aque ous solu tion, as well as to char ac ter ize the ves i cles

mate rial and phys i cal prop er ties [3]. Addi tional work in the field

has led to the syn the sis of a num ber of bio com pat i ble PEO-based

amphi philic block copoly mers that form aque ous ves i cles dis per-

sions, includ ing poly(eth yl ene oxide)-block-poly(buta di ene) (PEO-

b-PBD) [7].

A sig nifi cant lim i ta tion of these poly mers for in vivo ther a peu-

tics is that they are not bio de grad able and likely not fully bio com-

pat i ble. In an effort to cre ate ves i cles that degrade and release

their con tents in vivo, PEO-b-PBD poly mers have been blended

with hy dro ly sa ble block copoly mers, such as poly(eth yl ene oxide)-

block-poly(lac tic acid) (PEO-b-PLA) or poly(eth yl ene oxide)-block-

poly(cap ro lac tone) (PEO-b-PCL); these ves i cles have been shown

D.H. Levine et al. / Methods 46 (2008) 25–32 27

Fig. 2. Gen eral appli ca tion of polym er some archi tec ture in ther a peu tics. Schematic rep re sen ta tion of polym er some assem bly illus trat ing three pos si ble appli ca tions, namely

opti cal imag ing, drug deliv ery, and tar geted-ther apy.

to undergo hydro lytic deg ra da tion intra cel lu larly (in the acidic envi-

ron ment of the en do lys os o mal com part ment) lead ing to release of

the poly mer somes’ encap su lates [26–28]. Cryo-TEM images and

dynamic light scattering mea sure ments serve to dem on strate that

nano scale phase tran si tions occur in these blends as the poly es-

ter back bone of the ves i cles’ hydro lytic com po nents degrade over

time; the intact ves i cle begins to form pores, which leads to the

tran si tion to worm-like micelles and ulti mately leads to the for ma-

tion of spher i cal micelles [27]. Fur ther, it has been shown that the

release rate of encap su lates in blended poly mer somes increases

lin e arly with increas ing mole ratio of hy dro ly sa ble poly mer [26].

While these stud ies rep re sent a rea son able first step in the devel-

op ment of poly mer somes for ther apy, it is crit i cal to over come the

hur dle of in vivo tox ic ity pre sented by the resid ual PEO-b-PBD in

these struc tures.

Recently, efforts in our group have focused on the devel op-

ment of self-assem bled poly mer somes from fully-bio de grad able

syn thetic am phi philes. The abil ity to gen er ate self-assem bled,

fully-bi or e sorb able ves i cles com prised of an amphi philic di block

copoly mer con sist ing of two pre vi ously FDA-approved build ing

blocks, poly(eth yl ene oxide) (PEO) and poly(cap ro lac tone) (PCL),

has been dem on strated by Ghorogh chian and cowork ers [10].

Unlike poly mer somes formed from the blend ing of “bio-inert” and

hy dro ly sa ble block copoly mers [26], these fully-bi or e sorb able PEO-

b-PCL ves i cles undergo acid cat a lyzed hydro ly sis of their ester link-

ages and degrade with out leav ing any poten tially toxic byprod ucts

[10,29]. We have dem on strated the release of doxo ru bi cin from

these sys tems with time-con stants of 18–24 h, depend ing on pH;

in vivo test ing of these poly mer somes for deliv ery is under way.

In con trast to acid cat a lyzed hydro ly sis of the poly mer back bone,

which occurs on the order of hours to days, pH trig gered con tents

release, using block copoly mers whose sol u bil ity in aque ous solu-

tions is depen dent upon solu tion pH, can occur much more rap idly

[30]. Borc hert and col leagues gen er ated poly mer somes com prised

of poly(2-vinyl pyr i dine)-block-poly(eth yl ene oxide) (P2VP-b-PEO)

copoly mers and showed that the resul tant ves i cles dis as sem ble in

acidic solu tions and quickly and com pletely release their con tents;

this dis so lu tion is due to the pro ton ation of the P2VP block in acidic

solu tions (below pH 5) which con verts the pre vi ously hydro pho bic

block into a water sol u ble poly mer [30].

Cer ri tel li and col leagues have designed and char ac ter ized a

di block copoly mer of poly(eth yl ene gly col) (PEG) and poly(pro-

pyl ene sul fide) (PPS) with a reduc tion sen si tive disul fide link

between the two blocks (PEG-SS-PSS); they dem on strated the

abil ity of this block copoly mer to form poly mer ves i cles which

burst within a few min utes of endo cy to sis due to the reduc tive

envi ron ment in the endo some [31]. In addi tion to di block copoly-

mers, var i ous other poly meric am phi philes can form ves i cles in

aque ous solu tions. Napoli et al. syn the sized a tri block copoly mer

of poly(eth yl ene gly col)-block-poly(pro pyl ene sul fur)-block-

poly(eth yl ene gly col) (PEG-b-PPS-b-PEG) [32], which at dilute

con cen tra tions forms poly meric ves i cles [33,34]. Napoli and

col leagues then dem on strated that ves i cles com prised of this tri-

block copoly mer could be desta bi lized by the oxi da tion of the

hydro pho bic PPS block; when oxi dized, PPS is first con verted

to poly(pro pyl ene sulph ox ide) and sub se quently con verted to

poly(pro pyl ene sul fone), both of which are more hydro philic

than PPS [35]. This change in hydro pho bic ity of the “hydro pho-

bic” block alters the ratio of hydro pho bic block to total poly mer

mass, lead ing to changes in mor phol ogy of the self-assem bled

struc tures from ves i cles, to worm-like micelles, to spher i cal

micelles, and finally to uni mo lec u lar micelles [35]. These poly-

mers pres ent the prom ise of bio de grad abil ity, due to oxi da tion

of the hydro pho bic chain into small mol e cules sol utes that can

be read ily cleared [32].

Another pos si bil ity to gen er ate fully-bio de grad able ves i cles is

to uti lize poly pep tides as their com pos ite am phi philes. Ves i cles

and micelles com prised of poly pep tide block copoly mers can

mimic the shape and bio log i cal per for mance of nat u ral ves i cles

and micelles [36]. Sun et al. syn the sized var i ous di block co poly pep-

tides of poly(l-lysine)-block-poly(l-phen yl al a nine) (PLL-b-PPA)

which spon ta ne ously self-assem ble into giant ves i cles in aque ous

solu tions [36].

3. Ther a peu tic appli ca tions of poly mer somes

Cur rently, many com pounds with toxic side effects and/or low

bio avail abil ity hold extraor di nary prom ise as potential ther a peu-

tic agents. How ever, lim ited bio avail abil ity of hydro pho bic com-

pounds and/or toxic side effects of these mol e cules can ren der

their ther a peu tic value inef fec tive. Fur ther, the abil ity of the ther a-

peu tic agents to reach the tar get site can be lim ited by the body’s

clear ance. Thus, the devel op ment of a poly meric deliv ery vehi cle

with spe cifi cally tuned phar ma co ki net ics, which can encap su late

and release highly toxic ther a peu tic agents for con cen trated local

deliv ery, should greatly increase ther a peu tic effi cacy.

28 D.H. Levine et al. / Methods 46 (2008) 25–32

Fig. 3. Kinet ics of doxo ru bi cin loaded poly mer somes. (A) Cumu la tive in situ release

of doxo ru bi cin, loaded within 200 nm diam e ter PEO(2 K)-b-PCL(12 K)-based poly-

mer somes, under var i ous phys i o log i cal con di tions (pH 5.5 and 7.4; T = 37 °C) as mea-

sured fluo ro met ri cally over 14 days. N = 4 sam ples at each data point; indi vid ual

data points for each sam ple var ied by less than 10% of the value dis played at each

time inter val. (B) Release rates of DOX (Vdox) from 200 nm diam e ter PEO(2 K)-b-

PCL(12 K)-based poly mer somes vs. time. Dot ted and solid lines rep re sent expo nen-

tial fits obtained by regres sion anal y sis (R2 = 0.99 for each curve), and the dis played

equa tions cor re spond to the respec tive release regimes (a, b, b9). (C) Schematic illus-

trat ing dif fer ing regimes of DOX release via (a) intrin sic drug per me ation through

intact ves i cle mem branes vs. (b and b9) release pre dom i nantly by PCL matrix deg ra-

da tion. [This image was repro duced from Ghorogh chian et al. [10] with per mis sion

from Copy right (2006) Amer i can Chem i cal Soci ety.]

Doxo ru bi cin (DOX) is an amphi pathic anti-neo plas tic agent

that shows much prom ise in can cer ther apy, both alone and in

con junc tion with anti bod ies and pep tides [37]. One of the major

lim i ta tions asso ci ated with admin is tra tion of this che mo ther a peu-

tic agent is car diac myo cyte tox ic ity [38]. How ever, uti liz ing drug

car ri ers to deliver doxo ru bi cin can alle vi ate some of the asso ci ated

cardio-tox ic ity by alter ing the phar ma co dis tri bu tion of the drug,

thereby reduc ing the drug con cen tra tion in the heart [38]. Deliv-

ery of doxo ru bi cin in lip o somes has been shown to extend the cir-

cu la tion time and alter the phar ma co dy nam ics of doxo ru bi cin in

such a way as to decrease its tox ic ity while still main tain ing its anti-

can cer activ ity [38]. Using active load ing meth ods orig i nally devel-

oped for lip o somes, doxo ru bi cin can be effi ciently loaded into the

aque ous cen ter [10,26,39] of poly mer ves i cles.

Ghorogh chian et al. have suc cess fully loaded doxo ru bi cin into

the hydro philic res er voir of PEO-b-PCL poly mer somes [10] by using

an ammo nium sul fate gra di ent [40–42]. Using doxo ru bi cin, as a

model sys tem, the mech a nism by which PEO-b-PCL based poly mer-

somes release a phys i o log i cally rel e vant en caps u lant was assessed

under var i ous con di tions (pH 7.4 and pH 5.5 at 37 °C) [10]. In-situ

doxo ru bi cin release from poly mer somes was mon i tored spec tro-

flu oro met ri cal ly (kex 480 nm, kem 590 nm) over 14 days. While the

kinet ics of release var ied at the two pHs, an ini tial burst release

phase (approx. 20% of the ini tial pay load within the first 8 h) was

observed at both pH con di tions fol lowed by a more con trolled,

pH-depen dent release over the fol low ing sev eral days [10]. At a

pH of 7.4, kinetic release stud ies sug gest that the drug mol e cules

ini tially escape the polym er some through pas sive dif fu sion of the

drug across the intact PCL mem brane (days 1–4) and sub se quently

through hydro lytic matrix deg ra da tion of PCL (days 5–14) [10] (Fig.

3). At a pH of 5, how ever, it appears that the dom i nant mech a nism

of release at both short and long times is acid cat a lyzed hydro ly sis

of the PCL mem brane [10].

More over, Levine and col leagues have recently dem on strated

the ther a peu tic potential of doxo ru bi cin loaded PEO-b-PCL poly-

mer somes in vivo; drug loaded bi or e sorb able poly mer somes were

admin is tered to xe no trans plant ed (T6-17 cells) tumor-bear ing

mice and their capa bil ity to retard tumor growth was assessed

using such met rics as tumor size and body weight (Levine, unpub-

lished data). As dem on strated in Fig. 4, doxo ru bi cin loaded PEO-

b-PCL poly mer somes were able to retard tumor growth in a live

ani mal on a par with the com mer cially avail able agent DOX IL®

(a clin i cally admin is tered lipo so mal for mu la tion of doxo ru bi cin)

(Levine, unpub lished data). Fur ther, mouse weights remained

within ±1.5 g of the ini tial weight, for all treat ment groups through-

out the study (Levine, unpub lished data).

Pac lit axel (taxol) (TAX), an anti can cer agent, whose ther a peu tic

effi cacy is lim ited by its poor aque ous sol u bil ity [43] is cur rently

admin is tered in a mix ture of Crem o phor EL (poly oxye thy lat ed cas-

tor oil) and dehy drated eth a nol [44] to increase bio avail abil ity. Sys-

temic admin is tra tion of taxol is asso ci ated with sev eral neg a tive

side effects in patients includ ing dysp nea, hypo ten sion, bron cho-

spasm, urti caria, and ery them a tous rashes [44]. In addi tion, the for-

mu la tion agent (Crem o phor EL) used to sol u bi lize the hydro pho bic

taxol is believed to be respon si ble for induc ing the hyper sen si tiv-

ity reac tions observed in patients [44]. As a result, var i ous aque-

ous for mu la tions of taxol have been exam ined to decrease toxic

side effects and increase water sol u bil ity. Li et al. dem on strated

the abil ity to load taxol into the hydro pho bic bilayer of PEO-b-PBD

poly mer ves i cles and thus increase the water sol u bil ity of this drug

while main tain ing its cyto toxic prop er ties [45].

Com bi na tion ther apy involves the admin is tra tion of dif fer ent

clas ses of che mo ther a peu tics to a patient in order to treat their dis-

ease; this approach has been shown to be gen er ally effec tive and

many can cer treat ment regimes employ such multi-drug ther apy.

A com bi na tion regime of DOX and TAX has been shown clin i cally to

retard tumor growth more effec tively in com par i son to the admin is-

tra tion of a sin gle agent alone [46]. A rea son able hypoth e sis is that

the syn er gis tic effect of these two drugs would be increased when

both drugs are admin is tered in the same deliv ery vehi cle, as this

would ensure deliv ery of the drug mol e cules in pre scribed ratios

to a given tar get at the same time; Ahmed et al. dem on strated the

abil ity to co-encap su late DOX and TAX into poly mer ves i cles and

showed the increased syn er gis tic effect when DOX and TAX are in

the same polym er some [27,28]. PEG-b-PLA/PEG-b-PBD blended

poly mer ves i cles were loaded with DOX in their hydro philic res er-

voir and TAX in their hydro pho bic bilayer, and were admin is tered

in vivo; the results dem on strate a higher max i mum tol er ated dose

D.H. Levine et al. / Methods 46 (2008) 25–32 29

Fig. 4. Anti-tumor effects of doxo ru bi cin loaded polym er some in mice. Mice were inoc u lated with tumor cells on day 0, were admin is tered drug (free dox, dox loaded polym-

er some, or DOX IL) or PBS on day 7, and sac ri ficed on day 16. Images of tumor bear ing mice admin is tered PBS (A) and DOX poly mer somes at the cul mi na tion of the study, day

16. (B); (C–E) Aver age tumor Vol ume vs. Time, Tumor vol umes of the 5 mice per group aver aged. Error bars are reported as stan dard error.

(MTD), as well as increased tumor shrink age and main te nance,

when both agents are admin is tered in ves i cles rather than as free

drugs [27]. Since there are a wide vari ety of both hydro philic and

hydro pho bic phar ma ceu ti cals, this par a digm is gen er ally appli ca-

ble to cre at ing other polym er some-for mu la tions for com bi na tion

ther apy. Ulti mately, as men tioned before, fur ther work to com bine

these phar ma ceu ti cals within a safe and fully-bio de grad able for-

mu la tion is nec es sary.

In addi tion to small mol e cules, pep tides, pro teins, and nucleic

acids have been encap su lated in block copoly mer assem blies. Lee et

al. suc cess fully encap su lated myo glo bin, hemo glo bin, and albu min in

PEO-b-PBD based poly mer ves i cles at vary ing degrees of encap su la-

tion effi ciency [5]. Ari fin and Palmer fur ther dem on strated that bovine

hemo glo bin (Hb) could be encap su lated inside PEO-b-PBD poly mer

ves i cles with oxy gen affin i ties sim i lar to those of human red blood

cells; they dem on strated that these “poly mer somes-enca pus lat ed

hemo glo bin” (PEH) dis per sions could store and trans port Hb and

poten tially act as in vivo oxy gen ther a peu tics [47]. The abil ity to encap-

su late pro teins within poly mer somes pro vides a prom ise for future

pro tein ther a pies, which are cur rently fac ing deliv ery obsta cles.

4. Diag nos tic appli ca tions of poly mer somes

The abil ity to non-inva sively image nano par ti cles in vivo is a

major advan tage in deter min ing their bio dis tri bu tion and devel-

op ing these deliv ery vehi cles for both ther a peu tic and diag nos tic

appli ca tions. Bio dis tri bu tion stud ies with poly mer somes, in par tic-

u lar, would be greatly aided by the encap su la tion of an imag ing

agent in the ves i cles; this would enable non-inva sive mon i tor ing

of the loca tion of ves i cles dur ing drug deliv ery with out the need

to sac ri fice the ani mal. Although nano par ti cles have been used

with a spec trum of dif fer ent imag ing modal i ties includ ing PET

[48,49] and MRI [50–52], here we will focus on poly mer somes that

encap su late fluo res cent agents for opti cal imag ing. Because light

scattering decreases with increas ing wave length, and hemo glo bin

and water absorp tion spec tra have their nadir in the near infra red

(NIR) spec tral region, much work has been focused on devel op ing

NIR con trast agents for in vivo imag ing stud ies [9]. To this end,

Ghorogh chian et al. have suc cess fully loaded por phy rin-based near

infra red fluo ro phores (NIR Fs) into the hydro pho bic bilayer mem-

branes of PEO-b-PBD [9,10,53,54], PEO-b-PCL [54], PEO-b-PEE [54],

and poly(eth yl ene oxide)-block-poly(meth ylc ap ro lac tone) (PEO-b-

PmCL) [54] poly mer somes.

Stud ies using PEO-b-PBD poly mer somes have shown that

por phy rin-based NIR Fs, when encap su lated in poly mer somes,

are able to gen er ate a sig nal with enough inten sity to pen e trate

through 1 cm of a solid tumor [9]. Fur ther, when these NIR-emis-

sive nano poly mer somes are injected into the tail-vein of mice,

the bio dis tri bu tion of the nano par ti cles can be tracked in vivo via

non-inva sive NIR fluo res cence-based opti cal imag ing [55]. Fig. 5

dem on strates the abil ity to track PEO-b-PBD poly mer ves i cles in

tumor bear ing mice over 12 h (Levine, unpub lished data). Com bin-

ing drug deliv ery with imag ing will allow for the con tin u ous non-

inva sive mon i tor ing of drug-loaded nano poly mer somes in vivo,

obvi at ing the need to sac ri fice ani mals at each time point to deter-

mine basic phar ma co ki netic and bio dis tri bu tion pro files, thereby

greatly reduc ing ani mal load.

In addi tion to devel op ing drug deliv ery appli ca tions, NIR-emis sive

poly mer somes have also been shown to be use ful for ex vivo cel lu lar

label ing and in vivo cel lu lar track ing. Den dritic cells (DCs) play an

impor tant role in the immune response and have shown potent anti-

can cer activ ity lead ing to DC-based vac cines research [56]. Cur rent

pro gress in DC-based vac cines has been, how ever, lim ited by var i ous

fac tors [56], some of which could be over come by the devel op ment

of imag ing meth ods for in vivo DC track ing [19]. Chris tian et al. have

dem on strated the abil ity to label DCs ex vivo with poly mer somes

encap su lat ing por phy rin-based NIR Fs; the TAT pep tide, as will be

dis cussed in greater detail below, was con ju gated to these NIR-emis-

30 D.H. Levine et al. / Methods 46 (2008) 25–32

Fig. 5. Tumor imag ing by NIR-em mi sive polym er some. Fluo res cence images obtained using eXplore Op tix instru ment of the same mouse taken prior to admin is tra tion of

NIR-emis sive poly mer somes, and at 4, 8, and 12 h post tail-vein injec tion. (A) Prone position, (B) supine position (kex = 785 nm, kem = 830–900 nm). The arrows in the prone

and supine posi tions sug gest loca tion of organs. In the supine position, the arrow sug gests the fluo res cence ema nat ing from the lower portion of the mouse body is from the

tumor; it may also be ema nat ing from the gut of the mouse due to break down of food.

sive polym er some to facil i tate effi cient uptake of poly mer ves i cles

by DCs [19]. Chris tian and col leagues deter mined that DC sur face-

asso ci ated poly mer somes shed over the first 24–48 h; but, poly mer

ves i cles that were fully inter nal ized by the DCs remained sta bly incor-

po rated over 3 days [19]. They fur ther showed that the NIR-emis sive-

polym er some-labeled DCs, when admin is tered into the foot pad of

mice, traffi c to the near est lymph node (pop li teal lymph node) and

could be tracked in vivo via opti cal imag ing over 33 days [57]. They

fur ther showed that den dritic cells are seques tered in the liver when

the cells are deliv ered intra ve nously [42], indi cat ing that the mode

of den dritic cell deliv ery will be crit i cal for the effec tive ness of can-

cer immu no ther apy. These results sug gest that poly mer ves i cles

can be employed for cell track ing in lon gi tu di nal stud ies and could

thus assist in the fur ther devel op ment of cell-based vac cines. Over-

all, the results in this sec tion dem on strate that the load ing of imag-

ing agents, such as por phy rin-based NIR Fs, into the polym er some

bilayer cre ates soft mat ter opti cal imag ing agents suit able for in vitro

diag no sis and deep-tis sue imag ing, non-inva sive bio dis tri bu tion and

phar ma co ki netic stud ies, as well as in vivo cel lu lar track ing.

An alter na tive imag ing modal ity that can be used to image

poly mer ves i cles is diag nos tic ultra sound. Zhou et al. prepared air-

encap su lated poly mer somes via lyoph i li za tion and rehy dra tion of

pre vi ously formed poly mer ves i cles [58]. The poly mer bub bles were

imaged using a Pie Med i cal Scan ner 350 and were visu al ized as

bright spots, val i dat ing the acous tic activ ity of air-encap su lated poly-

mer somes [58]. These results show that poly mer ves i cles hold prom-

ise in the realm of ultra sound imag ing as well as opti cal imag ing.

5. Polym er some sur face mod i fi ca tions for deliv ery and ther apy

Bio log i cally-active mol e cules con ju gated to the sur faces of

poly mer somes can be used to direct these nano par ti cles to sites

of dis ease and inflam ma tion. Mod i fy ing poly mer ves i cles with bio-

log i cal ligands enables tar get ing of upreg u lated recep tors and mol-

e cules on affected cells in vitro and in vivo, thereby enhanc ing the

nano par ti cles’ EPR effect and fur ther mit i gat ing the potential toxic

side effects of sys temic deliv ery. Addi tion ally, che mo ther a peu tics,

when used in con junc tion with molec u lar tar get ing agents, can

D.H. Levine et al. / Methods 46 (2008) 25–32 31

have a syn er gis tic effect [59]. In addi tion to ther a peu tic appli ca-

tions, over the past two decades the use of anti can cer anti bod ies

against molec u lar tar gets has been devel oped for tumor imag ing

appli ca tions [37]. Poly mer ves i cles can be directed to spe cific sites

in vivo by con ju ga tion of tar get ing moi e ties to the end group of

their hydro philic poly mer block (usu ally PEO). It is impor tant to

rec og nize that the con ju ga tion of ligands to the polym er some

sur faces can alter the com pos ite poly mer am phi philes’ hydro-

philic-block-to-total-mass ratio lead ing to a change in struc tural

mor phol ogy (e.g. from ves i cles to micelles).

Using a mod u lar bio tin–avi din chem is try, Lin and col leagues

func tion al ized poly mer ves i cles with anti-ICAM-1 anti body to

tar get ICAM-1 (inter cel lu lar adhe sion mol e cule-1) [21], a mol e-

cule that is upreg u lated on endo the lial cells dur ing inflam ma tion.

Using micro pi pette aspi ra tion, they mea sured the adhe sive ness

of these func tion al ized poly mer ves i cles to ICAM-1 immo bi lized

on the sur face of poly sty rene beads and deter mined that the adhe-

sion strength is lin e arly pro por tional to the sur face den sity of the

anti-ICAM-1 mol e cules on the polym er some [21]. This find ing is

in con trast to their ear lier adhe sion exper i ments car ried out with

func tion al ized bio tin yl a ted poly mer somes and avi din coated

beads [22], sug gest ing that the adhe sive ness of func tion al ized ves-

i cles is not only depen dent on sur face den sity, but also upon the

pre sen ta tion/ori en ta tion of the tar get ing mol e cules on the ves i cle

sur face [21].

Addi tion ally, si al yl lewisx (sLex), a selec tin ligand, has been

con ju gated to poly mer ves i cles using sim i lar bio tin–avi din mod-

u lar chem is try as pre vi ously described (Ham mer et al., in press,

Far a day-dis cus sions 139). In addi tion to ICAM-1 mol e cules, selec-

tins are also upreg u lated at sites of inflam ma tion [21]. In an effort

to cre ate “leu ko poly mer somes,” i.e. poly mer somes that mim ics

the adhe sive prop er ties of leu ko cytes, dual func tion al ized ves i-

cles of sLex and anti-ICAM-1 have been made by the Ham mer

lab. These inves ti ga tors were able to mea sure firm and roll ing

adhe sion of anti-ICAM-1-, sLex-, and anti-ICAM-1/sLex con ju-

gated poly mer somes under flow along ICAM-1, P-selec tin, and

ICAM-1/P-selec tin coated sur faces, respec tively, at venous shear

rates. It is believed that dual func tion al ized leu ko poly mer somes

will be able to serve as tar get ing agents to bring both ther a peu-

tics (drugs) and diag nos tics (imag ing agents) to sites of inflam-

ma tion [21].

Meng and co-work ers func tion al ized poly mer somes com prised

of PEG-block-poly(ester) and PEG-block-poly(car bon ate) di block

copoly mers with anti-human IgG (a-HIgG) or anti-human serum

albu min (a-HSA) [6]. a-HIgG and a-HSA were either con ju gated to

the polym er some through cova lent attach ment to car boxyl groups

on the ves i cle sur face or by attach ment to pro tein G, which was

cova lently attached to the polym er some sur face via the car boxyl

groups; using imag ing sur face plas mon res o nance (iSPR), they

deter mined that immo bi li za tion of anti bod ies on the ves i cle sur-

face through pro tein G is pre ferred for tar get ing [6]. iSPR was fur-

ther used to dem on strate the potential of anti body func tion al ized

ves i cles for tar get ing anti gens [6].

In addi tion to tar get ing, these bio log i cally active ligands can

aid in cel lu lar uptake [60]. As pre vi ously men tioned, Chris tian et

al. dem on strated that the highly cat ionic HIV-derived TAT pep-

tide, when cou pled to NIR-emis sive poly mer somes, enhances

cel lu lar deliv ery of poly mer ves i cles to den dritic cells while mod-

er ately affect ing cell via bil ity [19]. Intra cel lu lar uptake of poly-

mer somes was depen dent upon their con cen tra tion and incu ba-

tion time in solu tion; via bil ity was affected by these fac tors as

well [19].

We have recently attempted to con ju gate small anti-HER2/

neu pep ti dom i met ics, designed by Mu ral i and cowork ers [59],

to poly mer somes in order to fur ther develop these nano par ti cles

for both clin i cal breast can cer diag no sis (NIR-emis sive poly mer-

somes) and ther apy (e.g. with and with out doxo ru bi cin incor po ra-

tion). In com par i son to nor mal epi the lial tis sues, over-expres sion

of the HER2 pro tein, a mem ber of the epi der mal growth fac tor

(EGFR) or HER fam ily, has been seen in approx i mately 30% of

breast, ovar ian, and colon can cers [37,59]. A fam ily of anti-HER2/

neu pep tides (AH NPs) designed by Mu ral i et al. has a potency

on par with that of the full-length mono clo nal anti body (Her cep-

tin®; Genen tech, San Fran cisco, CA) and dem on strates bio chem-

i cal and bio log i cal prop er ties pre dic tive of clin i cal ther a peu tic

response [59]. It has been dem on strated that AHNP pre vents

tumor growth of trans formed T6-17 cells, in which HER2/neu is

over-expressed, in vivo and in vitro [59]. How ever, the rel a tively

short half-life of pep tides and pro teins in vivo is one chal lenge

that still remains to be over come when using such agents for

ther a peu tic appli ca tions [61]. To over come the chal lenge of rapid

clear ance, “stealth” or “ste ri cally sta bi lized” nano par ti cles, such

as pegy lat ed lip o somes, have been employed to deliver pep tides

[62]. Thus, link ing AHNP to a nano par ti cle sur face can greatly

improve the phar ma co ki net ics of the small pep tide and allow for

tar get ing as well as improved ther a peu tic effi cacy.

Ghorogh chian et al. observed changes in polym er some mor-

phol ogy from ves i cles to micelles post-con ju ga tion of the AHNP

pep tides to PEO-b-PBD ves i cles [55]. Ves i cles, as well as small

spher i cal micelles, not pres ent in aque ous sus pen sions of the func-

tion al ized and un func tion al ized di block copoly mer with out pep-

tide, were observed in the polym er some sus pen sion post AHNP

con ju ga tion [55]. Since these micelles were not seen in cryo TEM

images of the pure or un func tion al ized poly mer, it is prob a ble

that they are com prised of pep tide-con ju gated poly mer; fur ther-

more, it is hypoth e sized that the ves i cles in the sus pen sion con-

sist of poly mer not con ju gated to AHNP [55]. Pep tide-con ju gated

ves i cle gen er a tion with less hydro pho bic AHNP fam ily mem bers

were also attempted and again resulted in phase sep a ra tion of the

di block copoly mer-pep tide “tri block” from the di blocks [55]. Our

inter pre ta tion of these results is that the under ly ing poly mer mate-

rial needs to be rede signed to accom mo date pep tides and preserve

vesic u lar struc ture in order to develop AHNP poly mer somes fit for

clin i cal diag nos tic and ther a peu tic appli ca tions.

6. Future direc tions

Poly mer somes are new and valu able tools for both dis ease diag-

no sis and ther apy. Our view is that the enhanced sta bil ity and tun-

abil ity of poly mer somes will ulti mately lead to the devel op ment of

effec tive car ri ers for in vivo drug deliv ery, molec u lar imag ing, and

cel lu lar mim icry that extend well beyond what has thus far been

achieved with phospho lipid ves i cles.

In drug deliv ery, the potential to co-encap su late two drug mol-

e cules in the same polym er some enables com bi na tion ther a pies

and elim i nates the need to indi vid u ally admin is ter two sep a rate

drug for mu la tions. As such, polym er some may not only be more

effec tive in treat ing recur rent, resis tant, or resid ual tumors, but

may also be more con ve nient for patient admin is tra tion and treat-

ment tol er ance. It is also pos si ble to make sep a rate polym er some-

for mu la tions, each with dif fer ent drugs or with dif fer ent dos ing

that deliver drugs in a sequence, as needed for the par tic u lar type

of dis ease that is being treated. Addi tion ally, local iz ing ther a peu-

tics to the site of intent, either through pas sive accu mu la tion

(EPR effect) or with tar get ing ligands, can enable admin is tra tion

of higher doses of drug while min i miz ing the toxic side effects of

sys temic deliv ery. Fur ther, the abil ity to image poly mer ves i cles

dur ing deliv ery will offer numer ous advan ta ges for under stand ing

the mech a nisms of ther apy as well as effi ciently design ing drug

deliv ery reg i mens in small ani mal mod els. Aside from the dem on-

stra tion of the activ ity of multi-modal poly mer somes with exist-

ing block copoly mers, we believe that fur ther devel op ments in

32 D.H. Levine et al. / Methods 46 (2008) 25–32

poly mer design will extend the appli ca bil ity of poly mer somes to

dif fer ent drugs and imag ing modal i ties.

In addi tion to tar geted ther a peu tic drug deliv ery, tar get ing

ligands can be used to direct diag nos tic agents to tumors sites,

assist ing in in vivo diag nos tic imag ing. Air-encap su lated poly meric

ves i cles facil i tate nan od i ag nos tics using ultra sound. Fur ther, the

encap su la tion of both por phy rin-based near-infra red fluo ro phores

and air into the same ves i cle should yield a multi-modal polym-

er some, where both ultra sound and opti cal imag ing can be per-

formed con cur rently thereby enhanc ing tumor imag ing. Finally,

we see prom ise for simul ta neous clin i cal diag nos tic imag ing and

in vivo ther a peu tic drug deliv ery with the cor rect poly mer for mu-

la tions.

Acknowl edg ments

This work is funded, in part, under a grant through the Health

Research For mula Funds, Com mon wealth of Penn syl va nia to

R.M., M.J.T. and D.A.H. The Depart ment spe cifi cally dis claims

respon si bil ity for any anal y ses, inter pre ta tions, or con clu sions.

This work is also sup ported, in part, under grants from the National

Insti tutes of Health (EB003457-01 and CA115229-01A1) to D.A.H.

and M.J.T. The authors thank N.A. Chris tian, and G. Rob bins for shar-

ing unpub lished results and/or recently sub mit ted data.

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