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									                        Update                      TRENDS in Biotechnology   Vol.24 No.4 April 2006



                        Research Focus


Nanoparticles as targeting ligands
K.K. Jain
Jain PharmaBiotech, Blaesiring 7, CH-4057 Basel, Switzerland



A recently published technique enables the attachment                           authors conjugated 146 different small molecules to
of small molecules to nanoparticles for improved                                nanoparticles in an array format. More than 30 screening
targeting of nanomaterials. This new methodology has                            procedures were performed and, on average, 60 small
been compared with some of the other nanoparticle-                              molecules were attached per nanoparticle. Screening this
based techniques for target discovery and found to be                           library against different cell lines led to the discovery of a
more versatile and specific. This approach has potential                         series of nanoparticles with specificity for endothelial
for high-throughput drug discovery, improved drug                               cells, activated human macrophages or pancreatic cancer
delivery and linking of diagnostics to therapeutics for                         cells. This multivalent approach might facilitate the
the development of personalized medicines.                                      development of functional nanomaterials for applications
                                                                                such as differentiating between cell lines, detecting
There is a great need for the discovery of new drugs and                        distinct cellular states and targeting specific cell types.
for the development of targeted rational therapies. The                            The approach of Weissleder et al. [1], a union between
specific targeting of cells and receptors has assumed                            small molecule chemistry and nanotechnology, has the
importance in screening for drug targets, diagnostics,                          potential to be developed for use with a wide range of
drug delivery and, more importantly, in linking diagnos-                        nanomaterials for biomedical applications. One such
tics to therapeutics. Although many targets are being                           application is target screening in high-throughput drug
discovered through the application of genomic and                               discovery. This technique can enable small-molecule
proteomic technologies, the efficiency of screening and                          modification of nanoparticles to impart desirable biologi-
validation processes needs to be increased. Approaches                          cal properties both for the in vivo visualization of targets
based on the use of nanoparticles have been used, recently,                     and for the delivery of therapeutics, thus enabling
for target discovery and are now taking on a key role.                          diagnostics and therapeutics to be combined. The tech-
Given the inherent nanoscale functional components of                           nique might also facilitate toxicity and pharmacokinetic
living cells, nanotechnologies appear to be among the most                      studies. In addition to the flexibility and specificity of this
promising for use in biomedical applications. For this                          approach, the most important advantage is the potential
reason, nanobiotechnologies are being applied in biologi-                       to develop disease-specific nanoparticles without prior
cal research, molecular diagnostics, drug discovery, drug                       knowledge of the target, which will answer one of the big
delivery and, ultimately, in the development                                    limitations in drug discovery.
of nanomedicines.
   Studies to improve the targeting properties of nano-
particles will be valuable in these approaches and for the                      Other nanotechnology-based approaches for cell
development of targeted therapeutics. This article reviews                      targeting
a recent study to improve the targeting of nanoparticles                        Several other research groups have investigated different
through small-molecule attachment and considers other                           approaches for targeting nanoparticles to cells: these
approaches to improve nanoparticle targeting.                                   range from the use of tumour-homing molecules through
                                                                                to exploitation of the specificity of the avidin–biotin
                                                                                complex. Several such examples are discussed in greater
Small molecules attached to nanoparticles                                       detail below. The article from Weissleder and colleagues
Weissleder and colleagues investigated if the multivalent                       [1] also gives examples of nanotechnologies that have been
attachment of small molecules to nanoparticles can be                           used for cell targeting and point out some of their
used to increase specific binding affinity and to reveal new                      limitations. Furthermore, a recent review on the role of
biological properties of such nanomaterials [1]. The                            nanobiotechnology in drug discovery evaluated other
nanoparticle used in this study was a monocrystalline                           nanotechnologies [2], and some examples are listed in
magnetic particle with a core diameter of 3 nm. The                             Table 1.
particle was covered with a layer of dextran, which was                            In one successful approach for targeting nanoparticles,
cross-linked with epichlorohydrin and aminated by                               a lipoprotein-based nanoplatform, generated by conjugat-
reaction with ammonia; the resulting overall volume was                         ing tumour-homing molecules (folic acid and peptide) to
38 nm. The authors describe the parallel synthesis of a                         the protein components of naturally occurring lipopro-
library comprising magnetofluorescent nanoparticles                              teins, reroutes them from their normal lipoprotein
decorated with different synthetic small molecules: the                         receptors to other selected cancer-associated receptors
   Corresponding author: Jain, K.K. (jain@pharmabiotech.ch).                    [3]. Multiple copies of these targeting moieties, or a
   Available online 20 February 2006                                            variety of different targeting moieties, can be attached to
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144                     Update                     TRENDS in Biotechnology     Vol.24 No.4 April 2006



Table 1. Examples of use of nanoparticles for specific targeting of cells and receptors
Nanoparticle                     Characteristics                                       Applications                                          Refs
Gold                             Are prepared easily, have low toxicity and can be     This technology might enable tracking of a single     [8]
nanoparticles                    attached, readily, to molecules of biological         molecule of a drug in a cell or other biological
                                 interest. The laser light used to visualize the       samples.
                                 particles is a wavelength that causes only
                                 minimal damage to most biological tissues.
Quantum dots                     Nanoscale crystals of semiconductor material          Quantum dots can be used for high-throughput          [9]
                                 that glow or fluoresce when excited by a light         cell-based studies with the advantage of multi-
                                 source such as a laser.                               plexing
                                                                                       (i.e. multiple leads can be tested simultaneously).
Dendrimers                       Three-dimensional, nanoscale core-shell struc-        Can be conjugated to different biofunctional          [10]
                                 tures. Polyvalent dendrimers interact simul-          moieties, such as folic acid, using cDNA oligo-
                                 taneously with multiple drug targets.                 nucleotides to produce clustered molecules,
                                                                                       which target cancer cells that overexpress the
                                                                                       high affinity folate receptor.
Nanobodies                       The smallest available intact antigen-binding         Potential of a new generation of antibody-based       [11]
                                 fragments harbouring the full antigen-binding         therapeutics in addition to diagnostics for
                                 capacity of the naturally occurring heavy-chain       diseases such as cancer.
                                 antibodies.
Lipoparticles                    Enable integral membrane proteins to be solu-         For optimal lead selection and optimization.          Developed
                                 bilized but retain their intact structural confor-                                                          by Integral
                                 mation, which is essential during assay                                                                     Molecular,
                                 development.                                                                                                Inc.
Magneto-                         Magnetic and fluorescent.                              In vivo imaging rapid screening.                      [1]
fluorescent nanoparticles

the same nanoparticle. Such a diverse set of tumour-                                  The choice of the nanoparticle will depend on the
homing molecules can be used to create a variety of                               intended application. For example, although the imaging
conjugated lipoproteins as multifunctional, biocompatible                         ability of quantum dots (nanocrystals of semiconductor
nanoplatforms with a broad application to both cancer                             material that glow or fluoresce when excited by a light) is
imaging and treatment.                                                            being used with some success in developing diagnostics
   Some attempts have been made to modify the surface                             linked to therapeutics in cancer, some limitations for their
of nanoparticles for improving the attachment of                                  use in drug discovery studies have to be resolved, namely
molecules but the full potential of this approach has                             toxicity, size variation, agglomeration, potential for
not been realized. Methods of protein chemistry can be                            multiple drug attachment to a single quantum dot and
used for the effective attachment of drug-targeting                               blinking (i.e. photoluminescence intermittency). Research
ligands to the surface of protein-based nanoparticles.                            is in progress to address some of these issues.
For example, previous work from the group of                                          Lastly, when investigating interactions between nano-
Weissleder showed that surface modification of nano-                               particles and their targets it is necessary to consider the
particles by attachment of membrane-translocating,                                whole picture. Polyvalent biological processes, such as
sequence-based peptides can alter nanoparticle trans-                             carbohydrate–protein interactions, occur frequently in
port through monolayers and might facilitate drug                                 recognition events on cellular membranes, and it is
delivery and high-permeability nanoparticle-based                                 difficult to inhibit disease-relevant processes with mono-
therapeutics [4].                                                                 valent oligosaccharides. Multivalent drug design is,
   In a different approach, the specificity of the avidin–                         therefore, a desirable goal and antiviral and anti-
biotin interaction was used to attach molecules to the                            inflammatory agents that are several orders of magnitude
surface of nanoparticles and to confer targeting specificity.                      more potent than monovalent agents have been produced.
Gelatin nanoparticles were used for the attachment of                             Glycodendrimers fulfil single molecule entity criteria
biotinylated anti-CD3 antibodies by avidin–biotin                                 and self-assemble to form non-covalent nanoparticles,
complex formation to provide a carrier system for specific                         which function as polyvalent ligands that efficiently
drug targeting to T-lymphocytes [5].                                              inhibit polyvalent processes both in vitro and in vivo [7].
   Nobs and co-workers [6] reported an interesting                                It is also possible that non-covalent polyvalent
methodology for the surface modification of poly(lactic                            ligands are optimized in size and shape in the presence
acid) (PLA) nanoparticles. PLA nanoparticles can be                               of natural polyvalent receptors. If this can be confirmed,
modified by the covalent attachment of tris(2-carbox-                              it might provide control of a broad variety of
yethyl)-phosphine hydrochloride, resulting in the substi-                         polyvalent interactions.
tution of carboxylic acid groups with a high number of
activated sulfhydryl moieties on the surface of PLA                               Concluding remarks
nanoparticles. This approach enables a significant num-                            Small molecules form the backbone of modern thera-
ber of thiol functional groups to be covalently bound to the                      peutics because they exert their effect primarily by
particles without the problem of undesired interactions.                          targeting cellular elements or proteins that are essential
There have been no recent developments in the use of this                         for the progression of disease. However, recognition and
approach for drug discovery, although PLA nanoparticles                           validation of new drug leads is a time-consuming and
are recognized as drug delivery vehicles.                                         difficult task. The combination of small molecules with
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                        Update                      TRENDS in Biotechnology   Vol.24 No.4 April 2006                                                       145



nanoparticles has been shown to facilitate this process and                      5 Balthasar, S. et al. (2005) Preparation and characterisation of
several technologies using this approach have been briefly                          antibody modified gelatin nanoparticles as drug carrier system for
                                                                                   uptake in lymphocytes. Biomaterials 26, 2723–2732
reviewed with mention of their advantages and limi-                              6 Nobs, L. et al. (2003) Surface modification of poly(lactic acid)
tations. Some of the technologies are suitable for drug                            nanoparticles by covalent attachment of thiol groups by means of
delivery but not so useful for targeting ligands. By                               three methods. Int. J. Pharm. 250, 327–337
modifying the surface of nanoparticles it is possible to                         7 Thoma, G. et al. (2005) Non-covalent polyvalent ligands by self-
improve the attachment of small molecules, thus enabling                           assembly of small glycodendrimers: a novel concept for the inhibition
                                                                                   of polyvalent carbohydrate–protein interactions in vitro and in vivo.
nanotechnology to have a role in refining the process of
                                                                                   Chemistry 12, 99–117
drug discovery and facilitating the development of                               8 Farrer, R.A. et al. (2005) Highly efficient multiphoton-absorption-
personalized medicines in the future.                                              induced luminescence from gold nanoparticles. Nano Lett. 5,
                                                                                   1139–1142
References                                                                       9 Ozkan, M. (2004) Quantum dots and other nanoparticles: what can
 1 Weissleder, R. et al. (2005) Cell-specific targeting of nanoparticles by         they offer to drug discovery? Drug Discov. Today 9, 1065–1071
   multivalent attachment of small molecules. Nat. Biotechnol. 23,              10 Choi, Y. and Baker, J.R. (2005) Targeting cancer cells with DNA-
   1418–1423                                                                       assembled dendrimers: a mix and match strategy for cancer. Cell Cycle
 2 Jain, K.K. (2005) The role of nanobiotechnology in drug discovery.              4, 669–671
   Drug Discov. Today 10, 1435–1442                                             11 Revets, H. et al. (2005) Nanobodies as novel agents for cancer therapy.
 3 Zheng, G. et al. (2005) Rerouting lipoprotein nanoparticles to selected         Expert Opin. Biol. Ther. 5, 111–124
   alternate receptors for the targeted delivery of cancer diagnostic and
   therapeutic agents. Proc. Natl. Acad. Sci. U. S. A. 102, 17757–17762
 4 Koch, A.M. et al. (2005) Transport of surface-modified nanoparticles          0167-7799/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved.
   through cell monolayers. ChemBioChem 6, 337–345                              doi:10.1016/j.tibtech.2006.02.004




The ribosome as a drug target
         ¨
Erik C. Bottger
Institut fur Medizinische Mikrobiologie, Universitat Zurich, Gloriastrasse 30/32, CH-8006, Switzerland
          ¨                                       ¨   ¨



The elucidation of the crystal structure of the ribosome                        drug target. The existing drugs inhibit the ribosome by
and its subunits has dramatically increased our under-                          binding to relatively few sites; therefore, a considerable
standing of this organelle and the molecular interactions                       hurdle is to identify hitherto unexploited functional sites
that determine its functional capabilities. Two recent                          of the ribosome for use as drug targets.
publications, one on the structure of the bacterial                                                             ˚
                                                                                   The recent report on a 3.5A resolution structure of the
                  ˚
ribosome at 3.5 A resolution and one on the identifi-                            Escherichia coli 70S ribosome [5] is an important and
cation of functionally relevant sites within the small                          welcome addition to the various structures already
subunit rRNA, illustrate the importance of interdisci-                          available [6–10]. Furthermore, a genetic strategy to
plinary approaches in exploiting the ribosome as a drug                         identify functionally relevant sites in rRNA [11] provides
target.                                                                         an important means to highlight potential novel drug-
                                                                                target sites. In this latter study, a random mutant library
The ribosome is a particularly versatile target for drug                        of 16S rDNA was used to select for deleterious mutations
development [1–3]. Although highly conserved among                              in this molecule that interfere with cell growth. The
living organisms, subtle differences enable the develop-                        authors estimate that approximately one-third of all
ment of compounds endowed with a surprising degree of                           possible deleterious mutations in 16S rRNA have been
selectivity [4]. Indeed, many of the compounds that are                         covered by their study. Several of the mutations identified
used in clinical medicine for the treatment of bacterial                        this way mapped to sites targeted by known antibiotics,
infections (e.g. macrolides, ketolides, lincosamides, oxazo-                    thus validating the procedure; other mutations coincided
lidinones, aminoglycosides and tetracyclines) specifically                       with known functional sites in the ribosome involved in,
inhibit the bacterial as opposed to the eukaryotic                              for example, tRNA binding, accuracy control, translation
ribosome. In general, antibiotics target the ribosome at                        initiation, translocation and subunit association. Inter-
locations of functional relevance. The increasing incidence                     estingly, a significant portion of deleterious mutations
of antibiotic resistance and the toxicity associated with                       affected ribosomal sites of no clear function; furthermore,
some of the available compounds constitutes a formidable                        the majority of deleterious mutations involved nucleotides
challenge for further exploitation of the ribosome as a                         that have a high degree of conservation among evolution-
                                                                                ary domains. Although this supports the identification of
                           ¨
   Corresponding author: Bottger, E.C. (boettger@immv.unizh.ch).
   Available online 21 February 2006                                            these regions as sites of functional importance, it also
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