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1.-NANOTHERAPY: TARGETED DRUG DELIVERY


Despite the significant progress in the development of anticancer technology, there
is still no common cure for patients with malignant diseases. In addition, the long-
standing problem of chemotherapy is the lack of tumor-specific treatments.
Traditional chemotherapy relies on the premise that rapidly proliferating cancer
cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic
agents have very little or no specificity, which leads to systemic toxicity, causing
undesirable severe side effects such as hair loss, damages to liver, kidney, and
bone marrow.
Nowadays, drug-delivery systems with nanoparticles show a clear potential for
cancer treatments in view of advantages such as, i) the ability of targeting specific
locations in the body, ii) the ability of reducing the quantity of drug that needs to
be delivered to attain a particular concentration level in the vicinity of the target,
and iii) the ability of decreasing the concentration of the drug at non-target sites.
1

As a consequence, controlled drug delivery is one of the fastest-growing segments
of the pharmaceutical market, and in the United States alone the demand is
expected to grow nearly 9 % annually to reach more than US$ 82 billion by 2007.
2

1.1.-Preparation of nanoparticles for drug delivery.
The idea of directing a therapeutic agent to the proximity of a damaged tissue was
postulated by the Nobel Prize winner P. Ehrlich already in 1906 with the concept of
magic bullets which were compounds that would have a specific attraction to
disease-causing microorganisms. He envisioned that these magic bullets would
seek out these organisms and destroy them, avoiding other organisms and having
no side effects on healthy tissue.
3

In general, there are two possibilities of localizing a drug in the proximity of a
target (i.e., tumor): passive and active. The former implies using characteristic
properties of the tumor to locate in its proximity an encapsulated, bonded or
adsorbed drug on nanoparticles. That is by using the characteristic enhanced
permeation and retention of the vasculature of the tumor (EPR-effect) is possible to
concentrate drug-loaded nanoparticles within the tumor tissue. This is because

1
J. Ritter, A.Ebner; K. Daniel, K. Stewart. Application of high gradient magnetic separation principles to
magnetic drug targeting. J. Magn. Magn. Mater. 2004, 280, 184.
2
S. K. Sahoo, V. Labhasetwar. Nanotech approaches to delivery and imaging drug. Drug Discov. Today.
2003, 8,1112.
3
T. M. Fahmy, P. M. Fong, A. Goyal and W. M. Saltzman, Targeted for drug delivery, Mat. Today. 2005,
8, 18.
8
tumors develop a leaky vasculature as well as a poor lymphatic drainage.
4
The
majority of solid tumors exhibit a vascular pore cut-off size between 380 and 780
nm.
5
The healthy capillary vessels are permeable showing a pore size cut-off
depending on the location and kind of blood capillary. The tight-junction capillary
(blood-brain barrier), including the central nervous system has exhibited a vascular
pore cut-off size < 0.1 nm; continuous capillaries, including most tissues, such as
muscle, lung, and skin (cut-off < 6 nm); fenestrated capillaries, including kidney,
intestine, and some endocrine and exocrine glands (cut-off < 50-60 nm); and
sinusoid capillaries, including liver, spleen, and bone marrow (cut-off 100-1000
nm).
6
Therefore it is possible to synthesize drug-loaded nanoparticles with a
tailored size and with a shell that delays or avoids the action of the reticulo-
endothelial system (RES) expecting to be naturally located within a specific organ.
Other examples of passive targeting imply using specific affinity or physicochemical
properties of the tumor tissues. For instance, Kukowska-Latallo et al.
7
used the high
affinity folate receptor for the vitamin folic acid as a target for the delivery of
folate-conjugated drugs to cancer tissue. Another example of passive targeting with
nanoparticles uses pH-dependent drug release due to the tumor has a pH slightly
lower than healthy tissues. Most solid tumors have pH values of less than 7.2
8

contrary to the normal blood pH of 7.4 0.05.
9
When these pH sensitive
nanocarriers (i.e., polymers, liposomes) encounter acidic environments such as
tumor tissues, they break apart and release the molecules they contain.
10,11

Another physical property of tumor tissue is its higher temperature (hyperthermia)
compared to healthy tissues
12
and it is considered another utilization of passive

4
M. J. Vicent and R. Duncan, Polymer conjugates: nanosized medicines for treating cancer, Trends
Biotech., 2006, 24, 39.
5
S. K. Hobbs, W.L. Monsky, F. Yuan, W.G. Roberts, L. Griffith and V.P. Torchilin, R. K. Jain, Regulation
of transport pathways in tumor vessels: role of tumor type and microenvironment, Proc. Natl. Acad. Sci.
1998, 95, 4607.
6
Y. Okuhata, Delivery of diagnostic agents for magnetic resonance imaging, Adv. Drug Delivery Rev.,
1999, 37, 121.
7
J. F. Kukowska-Latallo, K. A. Candido, Z. Y. Cao ZY, S. S. Nigavekar, I. J. Majoros, T. P. Thomas, L. P.
Balogh, M. K. Khan, J. R. Baker, Nanoparticle targeting of anticancer drug improves therapeutic
response in animal model of human epithelial cancer, Cancer Res., 2005, 65, 5317.
8
O. M. Koo, I. Rubinstein and H. Onyuksel, Role of nanotechnology in targeted drug delivery and
imaging: a concise review, Nanomed.: Nanotech. Biology Med. 2005, 1, 193.
9
G. Gaucher, M. H. Dufresne, V. P. Sant, N. Kang, D. Maysinger and J. C. Leroux, Block copolymer
micelles: preparation, characterization and application in drug delivery, J. Controll. Release. 2005, 109,
169.
10
T. Ishida, M. J. Kirchmeier, E. H. Moase, S. Zalipsky and T. M. Allen, Targeted delivery and triggered
release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells, Biochim.
biophys. acta-biomem. 2001, 1515, 144.
11
D. E. L. de Menezes, L. M. Pilarski, A. R. Belch and T. M. Allen, Selective targeting of immunoliposomal
doxorubicin against human multiple myeloma in vitro and ex vivo, Biochim. biophys. acta-biomem.
2000, 1466, 205.
12
V. P. Torchilin, Drug targeting, Europ. J. Pharmac. Sci., 2000, 11, S81.
9
targeting by using temperature-sensitive release nanoparticles.
13
The PathFinder
technology is based on the identification of naturally occurring mechanisms for the
localization of material (i.e., proteins) indifferent parts of the body and uses these
principles for the controlled production of site-specific nanoparticulate carriers.
14

On the other hand, active targeting is based on inducing external properties to the
nanoparticles to target them to specific tumor tissues and involves the use of light,
magnetism, and the specific recognition mechanisms (i.e., interaction antigen-
antibody, peptide-based targeting, DNA or RNA-based ligands, etc.) Magnetic drug
delivery is not a new therapeutic tool; in 1978 Senyei et al.
15
used an in vitro
analog of the human circulatory system to test the magnetic retention of magnetic
microspheres which consisted of adriamycin hydrochloride and ultrafine magnetite
encapsulated in an albumin matrix. From then to now, several scientific papers
have been published describing different drug-loaded magnetic nanoparticles
encapsulated in polymeric or inorganic matrices.
16,17,18
Currently, magnetic
nanoparticles are commercially used to treat tumors by hyperthermia and
thermoablation (Magforce Nanotechnologies AG, Germany) and by using the
magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix
, France, Alnis BioScience, USA). The TNT System (Triton Biosystems) consist of
polymer-coated iron oxide nanoparticles tagged with an antibody, and an external
magnetic field is used to kill diseased cells. Pre-clinical animal models test not show
any side effects in healthy tissue.
On the other hand, active targeting using the specific interaction antigen-antibody
is widely studied
19,20
and currently exist commercially a drug conjugated with an
antibody (Mylotarg) used to treat a form of bone marrow cancer (CD33 positive
acute myeloid leukemia). Several papers have been published using dendrimers,
dendritic polymers and inorganic matrices with a loaded drug and with an antibody
functionalized surface.

13
A. Chilkoti, M. R. Dreher, D. E. Meyer and D. Raucher, Targeted drug delivery by thermally responsive
polymers, Adv. Drug Delivery Rev. 2002, 54, 613.
14
R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise of
nanomedicine, Chem. Eng. Prog. 2006, 102, 35.
15
R. DAquino, T. Harper and C. Roman-Vas, Nanobiotechnology. Fulfilling the promise of
nanomedicine, Chem. Eng. Prog. 2006, 102, 35.
16
[16] X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung and S. M. Nie, Magnetic nanoparticle design
for medical diagnosis and therapy, J. Mater. Chem. 2004, 14, 2161.
17
A. K. Gupta and M. Gupta, Synthesis and surface engineering of iron oxide nanoparticles for
biomedical applications, Biomater. 2005, 26, 3995.
18
Z. P. Xu, Q. H. Zeng, G. Q. Lu and A. B. Yu, Inorganic nanoparticles as carriers for efficient cellular
delivery, Chem. Eng. Sci. 2006, 61, 1027.
19
E. R. Gillies and J. M. J. Frechet, Dendrimers and dendritic polymers in drug delivery, Drug Discov.
Today, 2005, 10, 35.
20
X. Wu and I. Ojima, Tumor specific novel taxoid-monoclonal antibody conjugates, Current Medicnl.
Chem. 2004, 11, 429.
10
We can separate the drug-loaded nanoparticles in two groups according to their
structure, core-shell and matrix-like nanoparticles. Nanoparticles are defined
as solid, submicron-sized drug carriers that may or may not be biodegradable. The
term nanoparticle is a collective name for both nanospheres and nanocapsules.
Nanospheres have a matrix type of structure. Drugs may be absorbed at the sphere
surface or encapsulated within the particle. Nanocapsules are vesicular systems in
which the drug is confined to a cavity consisting of an inner liquid core surrounded
by a inorganic or polymeric shell. In this case the active substances are usually
dissolved in the inner core but may also be adsorbed to the capsule surface.
Nanoparticles are receiving considerable attention for the delivery of therapeutic
drugs.

1.1.1.-Core-shell based nanoparticles
Core-shell nanoparticles have recently attracted a huge scientific effort due to the
possibility of combining different properties in individual particles, based on
different compositions of the core and the shell. In addition, many interesting
technological applications can be foreseen for this kind of materials, including
analytical chemistry (chromatography), separation technology (ion exchange),
catalysis, biochemistry and medicine, etc.
21
These types of particles can be defined
by their different core and shell composition. The core often shows a useful physical
property, e.g. semiconductors, metals, magnetic oxides, encapsulated molecules,
while the shell can be useful to stabilize the core and make compatible the core and
the environment. It is also possible to change the charge, functionality or reactivity
of the surface. This is especially important for medical purposes, e.g. for drug
delivery applications.
We will focus in core-shell nanoparticles composed of a magnetic core
encapsulated in an organic (i.e., polymeric) or inorganic shell. Those nanoparticles
are based on the use of a magnet to locate them in the proximity of a tumor (active
targeting). At this location the nanoparticles can release the conjugated drug which
is associated to the magnetic core or an alternating magnetic field is used to induce
heat in the magnetic nanoparticles and produce ablation of the tumoral tissue
(hyperthermia). In all these applications the coating of the particles is a very
important issue. It helps to make the particles biocompatible, preventing
aggregation and the degradation of the metallic core, and reducing the extent of

21
G. Kickelbick, L.M. Liz-Marzn, Core-shell nanoparticles, in Encyclopedia of Nanoscience and
Nanotechnology, H.S. Nalwa (Ed.), American Scientific Publishers, 2004, Vol. 2, pp. 199-220.
11
clearance by the reticuloendothelial system. Moreover, the outer coating surface of
the particles can be functionalized to allow the binding of drugs or biomolecules to
the system to cause a therapeutical action or to get local targeting. The magnetic
core is composed of 3d metals (iron, cobalt, iron/cobalt alloys, iron/platinum alloys,
iron/nickel alloys, and their oxides). The shell is composed of polymeric materials
(i.e., dextrane, albumin, starch, etc.) or inorganic materials (ie., silica, graphite and
gold). Inorganic, amorphous silica is biocompatible, non-toxic, and posses hydroxyl
surface groups (4.6 OH/nm
2
, though this value strongly depends on the synthesis
temperature) which provide intrinsic hydrophilicity and allow surface attachment by
covalent linkages of specific drugs or biomolecules. Also, amorphous silica is a
heat-resisting material, with a low specific gravity, high surface area and good
mechanical strength. In addition, the isoelectric point of magnetite ferrofluids is
reached at pH close to 7. Aqueous ferrofluids therefore flocculate in the pH range of
5 to 9 and are stable only under highly acidic or basic conditions. On the other
hand, the isolectric point of silica is reached at pH 2-3 and therefore silica
nanoparticles are negatively charged at the pH of the blood. As consequence,
dispersions of silica particles in biological media are stable and prevent magnetite
agglomeration. However, the magnetic nanoparticles used for MRI are based on a
magnetic core encapsulated in an organic shell (generally dextrane), for example
superparamagnetic or ferromagnetic iron oxide nanoparticles are widely used as
negative contrast agents in MRI for oral and parental administration (i.e., Feridex,
Endorem or Resovist, GastroMARK or Lumirem, Sinerem, etc). Currently,
magnetic nanoparticles are commercially used to treat tumors by hyperthermia and
thermoablation (Magforce Nanotechnologies AG, Germany) and by using the
magnetic properties of the core to activate an encapsulated prodrug (Nanoboiotix
, France, Alnis BioScience, USA). The TNT System (Triton Biosystems) consists
of polymer-coated iron oxide nanoparticles tagged with an antibody, and an
external magnetic field is used to kill diseased cells. Pre-clinical animal models test
not shown any side effects in healthy tissue when using silica or carbon
encapsulated iron and iron oxide nanoparticles.
-Silica coated nanoparticles
A versatile method for the preparation of core-shell nanomaterials is the
growth of the shell material on pre-existing cores by chemical methods. This has
been used often for the deposition of silica shells on various nanoparticles, including
metallic,
22
semiconductor,
23
or magnetic
24,25
materials. A schematic view of the

22
L.M. Liz-Marzn, P. Mulvaney, The assembly of coated nanocrystals, J. Phys. Chem. B 2003, 107,
7312-7326.
12
synthetic steps followed for silica encapsulation of magnetic nanoparticles in
solution is shown in the figure below, comprising coprecipitation of Fe(II) and
Fe(III) hydroxides, slow deposition of sodium silicate for thin silica shells, and shell
growth in ethanol.



For example, it has been recently demonstrated
26
that semiconductor quantum dots
can be assembled on the surface of silica-coated magnetic nanoparticles, so that
single particles containing magnetic and luminescent functionalities can be obtained
through relatively simple chemical processes.











23
M.A. Correa-Duarte, M. Giersig, L.M. Liz-Marzn, Stabilization of CdS Semiconductor Nanoparticles
Against Photodegradation by a Silica Coating Procedure, Chem. Phys. Lett. 1998, 286, 497-501.
24
M.A. Correa-Duarte, M. Giersig, N.A. Kotov, L.M. Liz-Marzn, Control of Packing Order of Self-
Assembled Monolayers of Magnetite Nanoparticles with and without SiO2 Coating by Microwave
Irradiation, Langmuir 1998, 14, 6430-6435.
25
Y. H. Deng, C. C. Wang, J. H. Hu, W. L. Yang and S. K. Fu, Investigation of formation of silica-coated
magnetite nanoparticles via solgel approach, Colloids Surf. A: Physicochem. Eng. Aspects, 262 (2005)
(1-3), pp. 87-93.
26
V. Salgueirio-Maceira, M.A. Correa-Duarte, M. Spasova, L.M. Liz-Marzn, M. Farle, Composite Silica
Spheres with Magnetic and Luminescent Functionalities, Adv. Funct. Mater. 2006, 16, 509-514.

EFTEM
Green SiO 2
Red Fe
EFTEM (708 eV)
Fe L 3 Edge
EFTEM (22 eV)
SiO2 Plasmon


HRTEM images and EFTEM color
map showing the Iron (red)
atomic distribution (in this case
forming the Fe
3
O
4
nanoparticles),
and the silicon (green)
distribution, as part of the shells.
The silica coating was provided by
the sol-gel method. (M. Arruebo,
R. Fernndez-Pacheco et al.
publishing)
13

The assembly of nanoparticles on the surface of silica particles can be
achieved trough a technique based on the alternate deposition of oppositely
charged species,
27,28
using polyelectrolytes as molecular cement, and can be
implemented for the incorporation of drugs or other biomolecules within the
nanoparticles. Additionally, the nature of the polyelectrolytes can be tailored, so
that they are sensitive to external stimuli, such as pH or temperature, to trigger
drug encapsulation and release. Combination of these techniques can be useful to
fabricate multifunctional nanoparticles including a magnetic core (for external
manipulation), a luminescent, intermediate shell (for labelling) and an external
polyelectrolyte shell (for drug encapsulation). The various steps can be
schematically represented as shown in the figure below.
A B C A B C





-Carbon coated iron nanoparticles
Carbon coated iron and iron oxide nanoparticles are suitable for biomedical
applications.
29
The inert carbon encapsulation, provide a way to make
biocompatible, functionalize and also gives the possibility to adsorb and desorb
therapeutical agent as doxorrubicine.
30
These nanoparticles are obtained by two
procedures: by the discharge arc method designed by Krtschmer-Huffman in

27
F. Caruso, R. A. Caruso, H. Mhwald, Nanoengineering of inorganic and hybrid hollow spheres by
colloidal templating, Science 1998, 282, 1111-1114.
28
V. Salgueirio-Maceira, F. Caruso, L.M. Liz-Marzn, Coated Colloids with Tailored Optical Properties,
J. Phys. Chem. B 2003, 107, 10990-10994.
29
Kuznetsov, A. et al , J. Mag. Mag. Mat. 194, 22 (1999).
30
De Teresa J.M., Marquina C., Algarabel P.A., Morelln L,.Fernandez-Pacheco R. Ibarra M.R.
Interantional Journal of Nanotechnology (2005)
Schematic view of the synthesis of multifunctional nanoparticles. First, magnetic
cores are encapsulated in a silica shell, onto which luminescent quantum dots are
assembled. Subsequently, a second silica shell is deposited and
polyelectrolyte/biomolecule multilayers are deposited.
14
1990,
31
or by high energy ball mill grinding.
32
The Krtschmer method uses a
cylindrical chamber, in which there are two graphite electrodes: a stationary anode
containing 10 microns starting iron powders, and a moveable graphite cathode. An
arc is produced between the graphite electrodes in a helium atmosphere. The
graphite electrode is sublimed and builds up a deposit on the inner surface of the
chamber. In the material collected from this deposit we found: carbon
nanostructures, amorphous carbon and iron and iron oxide nanoparticles
encapsulated in graphitic layers. High energy grinding is performed in a ball mill. A
suspension of iron micrometric powders and graphite powders in ethanol is
grounded for several hours to obtain a viscous solution that is dried up to obtain
the final product as fine iron carbon powders.













-Gold coated iron nanoparticles
In many cases gold could be the ideal coating material because of its well-
known optical properties,
33
easy chemical functionalization with thiolated organic
molecules
34
and high stability of the gold-coated nanoparticles in solutions of
physiological pH. Recently, gold-coated nanoparticles of about 60 nm in size with

31
Kratschmer W. et al., Nature 347, 354 (1990)
32
Tapolsky, G. et al, Eur. Cells and Materials Vol. 3 Suppl. 2, 12 (2002).
33
T.A. Taton et al. Science 289, 1757 (2000)
34
M. Brongesma, Nat. Mater. 2, 296 (2003)

HRTEM images and EFTEM
color map showing the iron
(green) atomic distribution (in
this case forming the Fe
3
O
4

nanoparticles), and the
carbon (red) distribution, as
part of the shells. (R.
Fernandez- Pacheco et al.)
15
an iron oxide core of 9 nm have been synthesized by a wet-chemical method in two
steps.
35
However, metallic iron is expected to be a better core magnetic material
than iron oxides for drug delivery and biosensor applications because it is
magnetically softer and its saturation magnetization is about a factor two larger
than that of iron oxides.
Unfortunately, successful preparation of Fe/Au core-shell nanostructures has
been a lasting scientific challenge. Several works have reported the synthesis of
non-well structural characterized and/or non-long term stabilized nanoparticles,
until a novel method combining wet chemistry for synthesis of the Fe cores and
laser irradiation of Fe nanoparticles and Au powder in liquid medium has been very
recently proposed
36
.
In this CONSOLIDER project we aim to study the synthesis of gold-coated
metallic and oxidized iron nanoparticles and their feasibility for drug-delivery
applications. Au/(-Fe
2
O
3
, Fe
3
O
4
) core-shell nanoparticles will be prepared by
reduction of Au
3+
onto the Fe oxide surfaces of previously synthesized nanoparticles
(co-precipitation and further oxidation) using a modification of iterative
hydroxylamine seeding procedure
37
. We intend to get a good control of the Au shell
thickness, since this layer is responsible of the resulting optical properties, total size
and colloidal stability of the core-shell nanoparticles.
Gold-coated metallic iron nanoparticles will be synthesized following a two
steps method. First, Fe cores will be obtained by thermal decomposition of iron
pentacarbonyl in the presence of oleic acid
38
which acts as a surfactant, and further
particle precipitation by addition of ethanol. In the second step, we will explore two
techniques to achieve gold coating of the iron cores.
In the first method, Fe and Au nanoparticles will be dispersed together in a
water-based solution, which will be irradiated with a pulsed laser beam in order to
promote the selective fusion of the gold nanoparticles and ulterior condensation
onto the Fe cores, as described in reference
36
.
The second method is based on sonochemistry, which is an alternative
technique that can be used for the production of coated particles
39
. Power
ultrasound effects provoke chemical changes due to cavitation phenomena
involving the formation, growth and implosive collapse of bubbles in liquids.

35
J.L. Lyon et al., Nano Letters 4, 719 (2004)
36
J. Zhang et al., J. Phys. Chem. B 110, 7122 (2006)
37
K. J. Brown et al., J. Chem. Mater. 12, 306 (2000)
38
D. Farrell et al., J. Phys. Chem. B 107, 11022 (2003)
39
V.G. Pol, A. Gedanken and J. Caldern-Moreno, Chem. Mater. 15, 1111 (2003)
16
Sonication of a solution containing a gold precursor in the presence of
(metallic/oxidized) iron cores provides an alternative means of trapping gold
nanometer seeds, which produce active supported heterogeneous catalysis giving
rise to the formation of core-shell nanoparticles.
A careful structural characterization of the obtained core-shell
nanostructures will be carried out to determine the quality and continuity of the
resulting gold coatings which ensure the long-term stability of the iron core.

Work Plan 1.1.1
-Optimization of core-shell nanoparticles with high magnetic response
Until now, silica and carbon coated magnetic nanomaterials have been mostly
restricted to iron oxides, since these present a better chemical compatibility with
the silica shell. The magnetic response of such core-shell nanoparticles can be
improved by using other magnetic materials, such as metals (Co, Ni) or alloys
(CoPt
3
, FePt) in the case of silica. The formulation of such novel core-shell materials
is expected within the duration of the project.
-Development of novel multifunctional core-shell nanoparticles
The process outlined above for the incorporation of multiple functionalities in a
single particle can be extended to other materials. For instance, magnetic cores can
be combined with metallic shells, which can be continuous or assemblies of smaller
nanoparticles. In either case, the morphology can be tailored for high absorption
coefficients in the visible or NIR.
-Drug encapsulation in multifunctional nanoparticles
For the described multifunctional nanoparticles, drug encapsulation is envisaged via
wrapping with polyelectrolyte multilayers. As mentioned above, such multilayers
can be designed so that they are sensitive toward pH or temperature, and this can
be used to trigger encapsulation and release.

1.1.2.-Matrix-like nanoparticles
-Nanoparticles based on polymeric matrices
Natural occurring polymers and synthetic polymeric (dendrimers, dendritic
polymers and micelles) are used to deliver an adsorbed or encapsulated drug. Their
advantages are their biodegradability in many cases and their well-known
chemistry. These advantages have made that different research groups and
17
pharmaceutical companies have invested all their efforts in developing drug-
conjugated nanoparticles for drug delivery. Hence, interdisciplinary research at the
interface of polymer chemistry and the biomedical sciences has produced the first
polymer-based nanomedicines for the diagnosis and treatment of cancer. These
water-soluble hybrid constructs, designed for intravenous administration, fall into
two main categories: polymerprotein conjugates or polymerdrug conjugates.
Polymer conjugation to proteins reduces immunogenicity, prolongs plasma half-life
and enhances protein stability. Polymerdrug conjugation promotes tumor targeting
through the enhanced permeability and retention (EPR) effect and, at the cellular
level following endocytic capture, allows lysosomotropic drug delivery. The
successful clinical application of polymerprotein conjugates (PEGylated enzymes
and cytokines) and promising results arising from clinical trials with polymer-bound
chemotherapy (i.e., doxorubicin, paclitaxel, camptothecins) has provided a firm
foundation for more sophisticated second-generation constructs that deliver the
newly emerging target-directed anticancer agents (e.g. modulators of the cell cycle,
signal transduction inhibitors and antiangiogenic drugs) in addition to polymerdrug
combinations (i.e., endocrine- and chemo-therapy). First-generation technologies
include antibodydrug conjugates, for example Mylotarg
(http://www.pharmacist.com/pdf/mylotarg.pdf), and also several polymer
conjugates carrying either low-molecular-weight drugs or proteins.
Besides these drug-polymer conjugates, biodegradable polymers such as polyesters
and polyanhydrides and their copolymers with hydrophilic segments (e.g. PEO,
PPO) have been efficiently used for the nanoencapsulation and delivery of drugs.
These polymers have the specific advantage of being degraded by a hydrolitic
mechanism which leads to the erosion of the nanomatrix and subsequent delivery
of the associated drug. Moreover, these polymers have a long safety record as
shown by the fact that they are major components of several marketed
formulations (Lupon depot, Decapeptil, Gliadel..among others). Recently, the
attention has been directed to the use of copolymers of poly(lactic acid/glycolic
acid) with PEG. This is due to the ability of these copolymers to organize forming
PEG-coated PLGA nanoparticles. This PEG coating is critical for drug targeting
purposes since it provides the nanoparticle with long-circulating properties in the
blood stream as well as targeting capabilities (PEG can be linked to peptide or
antibody molecules).



18
-Polipeptide matrix nanoparticles
Polypeptide nanoparticles will be exploited to entrap drugs and obtain a controlled
release. Moreover, the entrapped drug will be protected from degradation.
Two kind of polypeptide nanoparticles will be explored: polypeptide dendrimers and
SAP-based nanoparticles. In the first group, the covalent assembly of the multiple
peptide chains will provide a matrix nanoparticle, with the ability to entrap the
drug, either covalently or non-covalently. In the case of SAP-based nanoparticles,
the assembly of different copies will be achieved due to the non-covalent self-
assembly properties of sweet-arrow peptide (SAP).

i) Polypeptide dendrimers
Dendrimers are nanoparticles that are creating great interest. Dendrimers
are synthetic, highly branched, monodispersed macromolecules. As their molecular
size increases, they adopt a spherical shape that has a vacant inner core that can
encapsulate drug molecules, and a highly functionalized surface that can be
derivatized with a ligand. Biocompatible dendrimers are obvious candidates for drug
delivery applications.
A particularly interesting class of biocompatible dendrimers is obtained when
peptides are the base of the dendrimeric structrure. Polypeptide dendrimers have
been previously described,
39
and the pioneering work of the group of J. Tam has
demonstrated the application of lysine dendrimers as immunogens.
40
In the present
project we plan to focus our attention on polyproline dendrimers a particular class
of polypeptide dendrimers. Proline is singular among the 20 genetically coded
amino acids in that it contains both a cyclic backbone as well as a secondary, as
opposed to primary, -amino group. These structural features impart unique
stereochemical properties to proline. Polyproline oligomers exist in two distinct
conformations. In organic solvents, they adopt a conformation known as polyproline
I, a right-handed helix in which all peptide bonds are cis-oriented ( = 0 ).
41
In
aqueous solvents, they adopt the conformation known as polyproline II, a left-
handed helix in which all peptide bonds are trans-oriented ( = 180 ).
42
The
transition from polyproline I to polyproline II implies a considerable increase in the
long dimension of the helix that changes from 1.9 to 3.1 per residue.

39
L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Royo, F. Albericio. Chem. Rev., 2005, 105, 1663-
1681.
40
Nardelli, B.; Lu, Y. A.; Shiu, D. R.; Profy, A. T.; Tam, J. P. Immunology 1992, 148, 914-920.
41
Traub, W.; Shmueli, U. Nature 1963, 198, 1165-1166.
42
Cowan, P. M.; McGavin, S. Nature 1955, 176, 501-503
19


This unique conformational plasticity of polyproline chains allows to modulate the
dendrimer properties by changing the length of the branches in response to the
environment. In this way, drugs could be trapped in organic solvents, where
polyproline chains adopt a polyproline I conformation, and released under
physiological conditions in which polyproline spacers form more extended
polyproline II helices. A more classical alternative to this non-covalent drug
entrapment could be attachment by covalent bond formation with the building
blocks of the dendrimer.
One of the characteristics of dendrimers is the facility to modulate its properties by
modification of the dendrimer surface.
43
This surface modification would be used
either for covalent attachment of the drug or, even more interesting, to
functionalize the dendrimer surface with vector peptide sequences able to target
the nanovector to a given tissue, and especially to a tumoural tissue (see point
1.2.). The biocompatibility of peptide structures and the possibility of cellular

43
Lee, J. W.; Ko, Y. H.; Park, S. H.; Yamaguchi, K.; Kim, K. Angew. Chem., Int. Ed. 2001, 40, 746-749
polyproline I polyproline III
20
internalization of polyproline structures
44,45
are additional potential benefits of this
approach.

ii) SAP- based nanoparticles
Sweet Arrow Peptide (SAP), VRLPPPVRLPPVRLPP, is an amphipathic Pro-rich
cell-penetrating peptide, i.e., it has the special ability to cross the cell membrane.
Because of its amhiphathic character, SAP was expected to self-assemble. In
aqueous media SAP adopts polyproline II structure, with hydrophobic residues
pointing to one face of the helix and hydrophilic ones facing to the opposite (see
figure). The self-assembling properties of SAP were proven by circular dichroism
(CD) and transmission electron microscopy (TEM). TEM micrographs of the replicas
showed fibrils of 16 (3) nm width and variable length.


In the present project, nanoparticulate molecular materials of SAP will be obtained
by top down methods. In order to covalently attach the drug, a percentage of the
arginines of the SAP sequence, (VRLPPP)
3
, will be replaced by lysine or serine in
order to bind carboxylic containing-drugs to the SAP-based nanoparticles through
amide or ester bonds, respectively.

44
L. Crespo, G. Sanclimens, B. Montaner, R. Prez-Toms, M. Royo, M. Pons and F. Albericio, E. Giralt.
Peptide Dendrimers Based on Polyproline Helices. J. Am. Chem. Soc., 2002, 124, 8876-8883.
V R L P P P V R L P P P V R L P P P
21
Apart from preventing the drug degradation in the biological media and to
offer a controlled release, SAP-based nanoparticles will have the unique ability to
internalize the entrapped drug inside the cell.
46,47,48

Workplan 1.1.2
a) Synthesis of polyproline dendrimers with reactive functional groups at the
surface.
b) Covalent attachment of antitumoral drugs to polyproline dendrimers. We will
focus on doxorubicin (as a proof of concept) and on antiangiogenic drugs.
c) Non-covalent entrapment of antitumoral drugs in polyproline dendrimers. We will
exploit the conformacional plasticity of the polyproline branches in response to
changes in solvents polarity. The effect of the size of the drug will be also studied.
Again, we will focus on antiangiogenic drugs.
d)Synthesis of SAP-based nanoparticles. Monomeric SAP will be prepared using well
established solid-phase peptide synthesis methodologies. SAP-based nanoparticles
will be obtained by top down methods (use of critical superfluids) from the
nanofibrilar structures formed in highly concentrated SAP aqueous solutions.
e)Synthesis of functionalized SAP-based nanoparticles. Lysine- and serine-
containing SAP monomers will be prepared by solid-phase synthesis. Attachment of
antiangiogenic drugs will be done in solution by reaction with the amino- and the
hydroxy- groups of lysine and serine side-chains, respectively. Then, drug-loaded
nanoparticles will be prepared as described above.
In an alternative procedure that will also be explored, the drug will be attached in
solution to preformed lysine- or serine-containing SAP-based nanoparticles.

45
Farrera-Sinfreu, Josep; Giralt, Ernest; Castel, Susanna; Albericio, Fernando; Royo, Miriam. Cell-
Penetrating cis-g-Amino-L-Proline-Derived Peptides. J. Am. Chem. Soc., 2005, 127, 9459-9468.
46
S. Pujals, J. Fernndez-Carneado, C. Lpez-Iglesias, M. J. Kogan and E. Giralt, BBA-Biomembranes,
2006 , in press.
47
[9 J. Fernandez-Carneado, M. J. Kogan, S. Pujals, and E. Giralt, Biopolymers 2004, 76 196-203.
48
10 J. Fernandez-Carneado, M. J. Kogan, S. Castel, and E. Giralt, Angewandte Chemie, Int. Ed. 2004,
43,1811-1814.

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