Supramolecular Bioconjugates for Protein and Small Drug Delivery

DOI: 10.1002/ijch.201000022

Supramolecular Bioconjugates for Protein and Small DrugDeliveryStefano Salmaso,[a] Sara Bersani,[a] Anna Scomparin,[a] Francesca Mastrotto,[a] and Paolo Caliceti*[a]

1. Introduction

Revolutionary discoveries in life science, medicine, cellbiology, proteomics, biotechnology, and medicinal chemis-try have open unexpected therapeutic opportunities fortreatment of a wide array of diseases. The emerging classof biotech drugs promises a radical innovation in thera-peutic protocols, which includes the treatment of geneticdiseases that, up to now, have not met adequate solutions.Novel potent natural, synthetic, or semisynthetic low-mo-lecular-weight drugs have being also developed for treat-ment of diseases with poor prognosis such as cancer,cystic fibrosis, viral infections, Alzheimer�s disease, etc.

Although a number of novel pharmacologically activeproducts, either biotherapeutics or small organic agents,have demonstrated their potential efficacy, switchingthem to therapeutic agents is often complicated. Thetransformation of biotech drugs into medicines suffersfrom poor biopharmaceutical properties, low absorptionthrough biological membranes, a need for parenteral for-mulations, rapid elimination from the body, and low phys-ical, chemical, and enzymatic stability. On the other hand,medicinal chemistry is moving towards the development

of highly active molecules with limited solubility and sta-bility that consequently prevent their formulation anduse.

Modern pharmaceutical technology strives to provideproper formulation and delivery solutions for novel drugsthat can overcome the biopharmaceutical limitations totheir use.

Polymer-based nanotechnology offers challenging op-portunities for treatment of diseases by novel molecular-scale medical interventions that can be applied to pro-duce tailor made nanomedicines for improved or innova-tive therapies as well as personalized treatments.[1–4]

Polymers are key elements for fabrication of complexsupramolecular constructs, which can be designed to ach-

Abstract : Supramolecular conjugation techniques have beendeveloped to produce novel nanosized systems by assem-bling materials with diverse physicochemical and biologicalfeatures. These techniques have been adapted to obtain in-novative bioconjugates to deliver drugs with poor biophar-maceutical properties and nano-devices with potential“theranostic” activity. Supramolecular drug delivery systemsinclude polymer therapeutics such as drug–polymer biocon-jugates, and colloidal carriers such as micelles, liposomes,polyplexes, and organic and inorganic nanoparticles. Byvirtue of their wide array of chemical composition and prop-erties, polymers represent key elements for the constructionof novel supramoelcular formulations.Polymer bioconjugation is a fledged technique for fabrica-tion of protein–polymer conjugates. PEGylation, in particu-lar, produces derivatives with enhanced pharmacokinetic,immunological, and stability properties as compared to theparent protein. Over the years, new methods have been set

up to obtain site-directed polymer conjugation. In thisreview we report few grafting to and growing from PEGylationexamples for the preparation of therapeutically effective pro-tein bioconjugates.Supramolecular formulations with unique properties can bealso obtained by assembling functional polymers, targetingagents, physicochemical modifiers, and biomodulators.These systems may be designed for disease tissue disposi-tion and cell recognition/penetration. Cyclodextrins, for ex-ample, have been functionalized with polyethylene glycoland folic acid to produce tumor-targeted drug carriers. Inter-esting results have been obtained with this novel class ofdrug delivery systems. In addition, responsive polymershave been conjugated to gold nanoparticles to endow a newcolloidal platform with triggerable cell disposition proper-ties, which can be exploited either in biomedicine or diagno-sis.

Keywords: drug delivery · nanostructures · polymers · protein modifications · supramolecular chemistry

[a] S. Salmaso, S. Bersani, A. Scomparin, F. Mastrotto, P. CalicetiDepartment of Pharmaceutical Sciences, University of Padua,-Via F. Marzolo 5, 35131 Padua, Italyphone: +39 (0)49 8275695fax: +39 (0)49 8275366e-mail: [email protected]

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Review S. Salmaso et al.

ieve disease site targeting and controlled drug delivery.[5]

This heterogeneous class of macromolecules includes ma-terials with a variety of different physicochemical andbiological properties that can be properly exploited tobuild up new colloidal therapeutic systems usually classi-fied as polymer therapeutics.[6] The main advantages ofpolymeric materials in the fabrication of nanomedicinesare: multivalency, high molecular weight, controlledcharge, and hydrophobic/hydrophilic balance.

The multivalency of polymers allows for chemical ma-nipulation of the macromolecular structure, which can becustomized with drugs, targeting agents, cell penetrationenhancers, microenvironmental stimuli-sensitive moieties,and, in general, physicochemical and biological modifiers.The high molecular weight dictates the overall bioavaila-bility due to slow and prolonged absorption through bio-logical membranes, long permanence in the circulation,and accumulation in the disease sites, namely solidtumors or inflamed tissues, due to selective extravasationallowed by the specific capillary structures in those areas.Either charge or hydrophobic/hydrophilic balance influ-ence the structural conformations, which can change fromextended to coiled arrangements depending on the micro-

environmental conditions and interactions with biologicalstructures.

Typical examples of polymer-based nanomedicines are:polymer–protein bioconjugates, polymer–DNA complexes(polyplexes), and polymer–drug conjugates. Additionally,polymers are used to create new drug carrier platformssuch as micelles and nanoparticles with specific or smartbiopharmaceutical features.[7]

Stefano Salmaso obtained his degreein Pharmaceutical Chemistry and Tech-nology in 1998 and a Ph.D. in Pharma-ceutical Sciences in 2004 at Universityof Padua. He completed his scientifictraining at University of Paris Sud andNortheastern University, Boston. Hejoined the Drug Delivery researchgroup of Padua in 2004 as assistantprofessor. During his Ph.D. program,he developed cyclodextrin-based bio-conjugates as novel carriers for tumortargeting. Recently, he has been investi-gating polymer-decorated gold nanoparticles as a platform for celltargeting and brain drug delivery, and stumuli-sensitive micelles forcontrolled disease site drug release.

Sara Bersani is a Post-Doc in the DrugDelivery lab of University of Padua. Sheearned her Master’s degree in Pharma-ceutical Chemistry and Technology in2003 and a Ph.D. in Molecular Scien-ces in 2008, working in the Drug Deliv-ery group of Padua. She also worked inthe Drug Delivery lab at the School ofPharmacy of Nottingham Universityunder the supervision of Prof. CameronAlexander. Her research activities in-clude the development of macromolec-ular systems for drug delivery by inves-tigation of reactive linkers for endowing polymers with peculiar bio-modulation properties. She is co-author of 13 papers and 16 confer-ence communications.

Anna Scomparin is a Post-Doc scientistof the Drug Delivery group of Universi-ty of Padua. She graduated in Pharma-ceutical Chemistry and Technology in2006 and received her Ph.D. in Molecu-lar Sciences in 2010, working in theDrug Delivery lab of Padua and in theSackler School of Medicine at Tel AvivUniversity under the supervision of Dr.Ronit Satchi-Fainaro. She is experi-enced in bioconjugates for drug deliv-ery, polymer therapeutics, and proteinPEGylation, and is co-author of 4papers and 5 congress contributions.

Francesca Mastrotto is a Ph.D. studentworking in the Drug Delivery lab of theUniversity of Padua. She received herMaster’s degree in PharmaceuticalChemistry and Technology in 2007. Shestudied stimuli sensitive gold nanopar-ticles and other colloidal systems fordrug delivery. In 2010 she joined theSchool of Pharmacy of the University ofNottingham, where, she studied thepreparation of stimuli sensitive poly-mers under the direction of Prof. Ca-meron Alexander. She is co-author of 3papers and 4 congress contributions.

Paolo Caliceti graduated in Pharma-ceutical Chemistry and Technology in1984 and obtained his Ph.D. in Phar-maceutical Sciences in 1989 at Univer-sity of Padua. He took the Chair ofDrug Delivery at the University ofPadua in 2002. Since 1985, his main re-search interest has been protein–poly-mer conjugation, PEGylation. He devel-oped innovative bioconjugation proto-cols for site-directed protein conjuga-tion. Throughout the years he expand-ed his investigational area to novelnanosized supramolecular bioconjugates for tumor targeting ob-tained by assembling multifunctional materials (polysaccharides,temperature-sensitive polymers, and inorganic nanoparticles) withbiologically active functions (targeting agents, cell-penetrating en-hancers).

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Supramolecular Bioconjugates for Protein and Small Drug Delivery

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2. Protein–Polymer Bioconjugation

Proteins represent a revolutionary class of therapeuticagents that have opened up interesting new perspectivesin the treatment of a large array of diseases.[8] In recentyears, an increasing number of new protein drugs and bio-similars has been enriching the pharmaceutical arsenal asa consequence of the comprehension of their structure/functional mechanism relationship and the establishmentof fledged biotechnology techniques for production.[9]

However, although the extraordinary therapeutic poten-tial of these macromolecules makes them attractive drugs,their exploitation in medicine is often limited by theirpoor physicochemical, immunological, and biopharma-ceutical properties.[10,11]

Due to their fragile structures, proteins easily undergophysical, chemical or enzymatic inactivation during ma-nipulation, formulation, storage, and delivery. Their hy-drophilic character and large size significantly limit theirpermeation through biological membranes. Their systemicbioavalibility is usually low because of their rapid elimi-nation from the bloodstream, which takes place by sever-al mechanisms including glomerular ultrafiltration, liverup-take, degradation, or inactivation. Finally, proteins areintrinsically immunogenic and antigenic molecules thatcan be rapidly cleared from the body as well as inactivat-ed or converted into toxic products by the immunosys-tem.[11, 12]

So far, several formulations have been explored toameliorate the biopharmaceutical profile of proteins andprovide for suitable delivery, such as micro and nanopar-ticle encapsulation and polymer conjugation.[11,13]

Polymer attachment, in particular, has been demon-strated to succeed in producing effective biopharmaceu-tics with enhanced therapeutic performance as comparedto the parent molecules.

2.1. Grafted-to-Protein PEGylation

Polymer–protein conjugation is usually referred to as PE-Gylation, since monomethoxy-polyethylene glycol (PEG)is the polymer of choice for preparation of this class ofconjugates. Despite polyvinylpyrrolidone, acryloylmor-pholine, dextrans, acrylates, and a few other natural, syn-thetic, and semisyntehtic polymers that have been investi-gated so far to obtain protein–polymer bioconjugates,namely, PEG has been demonstrated to possess the phar-maceutical requisites for parenteral administration.[11] Byvirtue of its hydrophilicity, low immunogenicity, and anti-genicity, high local and systemic biocompatibility, and sta-bility, PEG has been approved by FDA, EMEA, and themain worldwide regulatory agencies for injectable formu-lations.

The main effects of PEG conjugation with proteins aresize enlargement and structure masking, which result inslow glomerular ultafiltration and prolonged permanence

in the bloodstream.[13] Additionally, the hydrated polymershell prevents the approach of proteolytic enzymes, andlimits the recognition from the immune system, thus en-dowing products with low immunogenicity and antigenici-ty.[14] Accordingly, PEGylated proteins are less susceptibleto inactivation and elimination, and entail lower risk oftoxic effects due to immunoreactions, compared to nativemolecules. The masking of charges, hydrophobic regions,glycosylic functions, or protein regions involved in specif-ic or unspecific clearance mechanisms, such as cell up-take and processing, is reflected also in prolonged perma-nence in the bloodstream.[15] Nevertheless, PEGylationcan reduce the biological activity of proteins as a conse-quence of inactivation due to polymer-induced functionalfolding, or chemical and physical masking of the activesite that hampers the protein interaction with substratesor receptors.

Throughout the years, particular efforts have beendedicated to setting up efficient and selective conjugationstrategies, which bestow derivatives with suitable physico-chemical and biopharmaceutical properties, high biologi-cal activity, and well-defined chemical structure as re-quired by the regulatory agencies.

The first PEGylated proteins were produced byrandom conjugation of short polymer chains, mainly5 kDa PEG, to the protein surface. Because of the lowmolecular weight of the polymer, extensive conjugationwas required to produce derivatives with enhanced bio-pharmaceutical and immunological properties as com-pared to the native counterparts. Extensive PEGylationwas achieved by multiple polymer attachment to theamino groups exposed on the protein surface, since thesereactive functional groups are abundantly represented inthe protein structure.

A typical example of extensive protein PEGylation isoffered by superoxide dismutase (SOD), which was modi-fied with 5 kDa PEG.[16] SOD is an enzyme with interest-ing therapeutic applications for the treatment of superox-ide ion overproduction diseases such as inflammation, in-fection, reaction to organ transplantation, etc. Despitepromising preclinical results using SOD therapeutic appli-cation, its rapid elimination from the circulation and highimmunogenicity have strongly limited the pharmaceuticalexploitation of this orphan molecule. PEGylation wasfound to prolong the blood circulation of SOD after in-travenous administration to rats, resulting in 25 hoursblood half life while the native protein disappeared fromthe bloodstream in few minutes. The polymer conjugationalso increased the protein bioavailability after subcutane-ous and intramuscular administration, and the immuno-genic and antigenic character of this protein dramaticallydecreased. Despite the high number of polymer chains at-tached to the protein surface, bioconjugation was foundto induce only a limited decrease of the enzymatic activi-ty of this protein, and the bioconjugate was found thera-peutically effective in different pre-clinical trials.[17] Simi-

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lar results were obtained with other enzymes involved inbiotransformation of small substrates that freely dock tothe active site sliding through the polymer cloud. Accord-ing to this evidence, random and extensive polymer con-jugation has been successfully applied to produce the firstgeneration of PEGylated products introduced in themarket in the 1990 s, Oncaspar� (asparaginase–PEG) andAdagen� (adenosine deaminase–PEG).

Studies carried out by protein modification with a vari-able number of 5 kDa PEG chains underlined the effectof the PEGylation degree, number of polymer chains perprotein molecule, on the pharmacokinetic, immunologi-cal, physicochemical, and biological features of the conju-gates.[12] These properties are strongly affected by theoverall polymer mass of the construct and the architec-ture of the polymer affecting the protein surface. Thus,conjugation of few linear or branched high-molecular-weight polymers has been explored as an alternative ap-proach to extensive conjugation of small linear PEGs.

The effect of polymer size and shape on the physico-chemical, biopharmaceutical, and immunological proper-ties of PEGylated proteins was investigated using avidinas a protein model. Avidin is a protein of interest inimmuno-pretargeting protocols associated with anticancerdrugs or radioisotopes. To reduce the avidin immunoge-nicity and prolong its permanence in the bloodstream, theprotein was conjugated with four 5-kDa linear, 10-kDabranched, or 20-kDa branched PEG chains, which wereattached to 10 % of the available protein amino groups.[18]

The study showed that, in general, PEGylation reducesthe renal clearance and the liver up-take while it pro-motes the bioconjugate accumulation in solid tumors bythe enhanced permeation and retention (EPR) effect.[19]

A detailed analysis of the results showed that the phar-macokinetic, immunological, and biological properties ofthe conjugates were strictly related to the polymer massassociated to the protein. The PEG molecular weight in-crease prolongs the bioconjugate permanence in the cir-culation (Figure 1) and favors the tumor disposition. Theattachment of four 20-kDa PEG chains suppressed com-pletely the avidin immunogenicity and antigenicity, whilethe attachment of four 10-kDa or 5-kDa PEG chains re-duced the protein immunogenicity by about 85 % and65%, respectively. According to these results, the PEGy-lated forms of avidin obtained with 10- and 20-kDa PEGare considered promising products for therapeutic appli-cations.

Studies reported in the literature show that the perma-nence in the bloodstream and the immunological behav-ior of bioconjugates depends on the size and shape of thepolymer. As the polymer mass on the protein surface in-creases, regardless whether it is achieved by multiplePEG chain conjugation or by using high-molecular-weightpolymers, the immuogenicity decreases and the perma-nence time in the circulation increases.[20] Since biologicalactivity has been found to decrease as the polymer mass

increases, the balance between polymer and protein massis particularly relevant to produce derivatives with en-hanced biopharmaceutical and immunological propertiesthat simultaneously maintain high activity.[13,15] This con-sideration is of particular significance in the case of cyto-kines and antibodies that operate by interacting withhigh-molecular-weight receptors.

Aimed at obtaining protein derivatives with enhancedbiopharmaceutical properties without compromising theirbiological activity, bioconjugation protocols have been de-veloped either by active site protection or site-selectivepolymer attachment.

Heterogeneous phase site protection during PEGyla-tion was investigated using urokinase and trypsin as pro-tein models.[21, 22] The protein PEGylation was carried outin the presence of a SephadexTM resin functionalized withbenzamidine, an enzyme inhibitor that allowed for pro-tein reversible attachment to the resin and simultaneouslyprotected the active site from polymer modification. Thepolymer conjugation yielded extensively modified pro-teins, which maintained a high activity degree towards themacromolecular substrates.

Active site protection has been successfully pursued byusing large soluble or insoluble inhibitors that preventpolymer attachment in the vicinity of the protein activesite. Biotinylated-PEG (Scheme 1) was synthesized toprotect the avidin recognition site from polymer attach-ment during the PEGylation reaction.[23] The use of theprotection agent during conjugation of high-molecular-weight polymer chains (linear 10-kDa and branched 20-kDa PEG) preserved the avidin recognition properties asthe conjugates maintained 70–80% of the protein�s abilityto interact with biotinylated antibodies. These bioconju-gates displayed remarkable increase in bloodstream per-manence after intravenous injection (Figure 1) and com-

Figure 1. Plasmatic half-life of 5-, 10-, and 20-kDa PEG modifiedavidin after intravenous injection to mice. Avidin modified with 4PEG chains (*), avidin modified with about 16 PEG chains(*)(ref. [18]).




plete suppression of the avidin immunogencity and anti-genicity. In contrast, the products obtained without activesite protection and with the protein modified with linear5-kDa PEG obtained in the presence of the protectiveagent displayed 20–40% of the native biorecognitionproperties. The PEGylation of Lys amino groups locatedin the close vicinity of the biotin recognition region wasthe possible reason for the low activity of the conjugatesobtained without the active site protecting agent. The lowactivity of the derivative obtained with 5-kDa PEG in thepresence of the protecting agent was probably due to thesmall size of the activated polymer, which can easily ap-proach the recognition site and react with the Lys hereindespite the presence of the macromolecular protectingagent.

In recent years, several techniques have been proposedto achieve tailor-made PEGylated derivatives with de-fined structural composition and high biological activity.Site-selective PEGylation can be obtained by polymer an-choring to selected and rare amino acids along the pro-tein sequence or by peculiar conjugation conditions. Neu-lasta�, the rh-GCSF–PEG derivative available on themarket and used as a supporting drug in myelosuppres-sive chemotherapy, is a typical example of site-selective

modified protein prepared under reductive conditionswith end terminal aldehyde functionalized linear 20-kDaPEG.[24] The terminal amino group conjugation was ob-tained by exploiting the lower pKa of this amino group(7.6–8.0) as compared to the Lys amino group (9.3–10.2).

Cystein conjugation represents one of the most inter-esting and versatile techniques for selective protein PE-Gylation. Cys residues are in fact seldom represented inprotein structures and can be also artificially introducedinto the protein by site-directed mutagenesis of the pri-mary sequence, by changing aminoacids that are not di-rectly involved in the biological activity. When nativelypresent, only one Cys is usually available for conjugation,as the thiol groups are engaged in disulphide bonds. Inorder to exploit Cys for polymer anchoring, several ap-proaches for thiol conjugation have been set up and vari-ous PEGylated proteins have been produced, namely an-tibody fragments and engineered proteins.[25,26] Unfortu-nately, Cys conjugation is often difficult because thisamino acid, when available in the reduced form, is oftenlocated inside deep hydrophobic pockets, which stericallyforbid the protein dimerization through disulphide bond-ing. Cys inaccessibility prevents the penetration of largehydrated molecules like PEG, making their conjugationdifficult. Protein denaturation under controlled condi-tions, temporarily exposing the thiol function for polymerconjugation, has been demonstrated inadequate to obtainactive proteins. In fact, the unfolding/refolding processand the polymer conjugation can yield mismatched pro-tein structure and induce conformational alterations. Thismay result in decreased activity and stability, thus prohib-iting its formulation and therapeutic application. In orderto achieve efficient PEGylation of buried aminoacids,namely Cys, without structural protein alteration or harshconjugation conditions required by the use of commercialactivated PEG, a novel end-hydrophobized PEG hasbeen synthesized and investigated using rh-G-CSF.[27]

The linear 20-kDa PEG was end-functionalized with adi-amidoalkyl [–NHCO–(CH2)11–NHCO(CH2)5–] spacerand activated with a maleimide group (PEG–C18-Mal) formodification of the crypted 17Cys residue of rh-G-CSF(Scheme 2). Aiming at facilitating the PEG docking tothe aminoacid under physiological conditions, the hydro-phobic spacer arm was selected on the basis of the struc-tural properties of the pocket hosting the free Cys of rh-G-CSF. A comparative study between the end-hydro-phobized PEG and the commercial maleimide-activatedpolymer showed that the former efficiently conjugated tothe Cys group of the protein. The end-hydrophobizedPEG yielded about 60% rh-G-CSF conjugation in 250 h,while the commercial PEG yielded less than 20% proteinconjugation. The serial addition of the end-hydrophob-ized PEG and the temperature increase resulted in fasterpolymer conjugation. This result was explained with thehigh PEG–C18-Mal stability, as this polymer can form mi-cellar assemblies that chase the maleimide group into the

Scheme 1. Synthesis of biotnylated branchched 20 kDa PEG(ref. [22]).




hydrophobic core of the system and preserve the polymerreactivity toward thiol groups. The commercial PEG–Mal, which does not assemble into micelles, is insteadmore susceptible to maleimide inactivation. The rh-G-CSF–PEG derivative obtained with the end-hydrophob-ized polymer was found to be stable under stressing shearforce conditions, while the derivative obtained with thecommercial PEG underwent rapid denaturation and ag-gregation. These results supported the hypothesis that thepenetration of the hydrophobic spacer of PEG–C18-Malinto the protein bundle does not provoke dramatic struc-tural alterations, thus preserving its conformational integ-rity, a paramount requisite for the protein stability andactivity. The biological studies carried out by NFS-60 celltreatment with the native and hydrophobized PEG-modi-fied rh-GCSF demonstrated that the polymer conjugationslightly decreases the cytokine activity, probably becauseof the steric hindrance of the high-molecular-weight poly-mer. Nevertheless, the bioconjugate showed similar bioac-tivity to Neulasta�.

More sophisticated approaches to achieve site-specificPEGylation involve the use of protein engineering proce-dures, tag-based technologies, or enzyme-mediated PE-Gylation.[28,29] Transglutaminase and carboxypeptidase,for example, have been exploited to PEGylate glutaminesand terminal carboxylic moieties exposed on the proteinsurface, respectively.[30,31] These methods have been foundto be highly selective since specific aminoacids on theprotein structure are recognized by the enzymes. Glyco-proteins can also be selectively oxidized at the level ofthe glycosylic residues and then PEGylated with PEG–

hydrazide (PEG–Hz). Unfortunately, most of recombi-nant proteins are of bacterial origin and, therefore, theydo not present oligosaccharides constitutively in theirstructure. Recently, Neose Technologies disclosed a fasci-nating PEGylation procedure, GlycoPEGylation, whichinvolves the selective glycosylation of serines and threo-nines in proteins expressed without glycosydic units in Es-cherichia coli, followed by the conjugation of sialic acidderivatized PEG to the introduced N-acetyl galactosa-mine (GalNAc) residues by sialyltransferase.[32]

A novel PEGylation protocol which involves the chem-ical insertion of glycosyl functions into the protein struc-ture, followed by PEGylation of the oligosaccharide tag,has been set up.[33] Maltosyl- and lactosyl-based linkershave been synthesized for chemical conjugation (glyca-tion) to hidden protein sites and provide for polymermultiple attachment. Galactosyl-glucono-CO-NH-(CH2)12-NH-CO-(CH2)2-maleimide and maltosyl-glucono-CO-NH-(CH2)12-NH-CO-(CH2)2-maleimide (Scheme 3),were conjugated to the thiol groups of 34Cys of humanserum albumin (HSA) and 17Cys of rh-G-CSF locatedinto a hydrophobic cleft. The small glycosyl linker at-tached to the Cys was activated by selective oxidationwith periodate, which switches vicinal diols into aldheydegroups, and then conjugated with PEG–Hz. Such activat-ed PEG, in fact, can react with the aldehyde moieties toform reversible hydrazone bonds.[34,35] After 50 h reaction,the degree of conversion to PEGylated species was over90% for both the glycated proteins, while less than 30 %PEGylation was obtained with the commercial PEG–mal-eimide. The combination of site-specific PEGylation with

Scheme 2. Synthesis of maleimide activated end hydrophobizedlinear 20 kDa PEG (PEG-C18-Mal) (ref. [26]).

Scheme 3. Synthesis of malimide activated glycosyl linkers(ref. [32]).




releasable polymer conjugation may have numerous ad-vantages in the development of therapeutic bioconjugates,since it combines the beneficial attributes of proteinhyper-PEGylation with the prodrug concept. Therefore,the activity of the protein resulting from PEG detach-ment is recovered and the detached PEG can be rapidlyeliminated from the body via glomerular ultrafiltration.

2.2. Growing From Protein PEGylation

The alternative option to attach pre-formed polymers toa protein, known as grafting to method described above,is the growing from approach that refers to straightfor-ward polymer growth on a protein surface.

The first benefit of the growing from technique is theease of purification of the bioconjugates from the mono-mers, while the separation of polymer bioconjugates fromthe unreacted polymer in the grafting to method is usuallydifficult due to the close hydrodynamic size of the macro-molecules. Additionally, the growing from approachallows the preparation of a wide array of bioconjugateswith potentially “tunable” properties. These bioconju-gates can be prepared from a single protein precursorusing block co-polymers wherein different segments ofthe polymer chain confer specific properties such as pHor other microenvironmental stimuli responsiveness,tightly defined hydrophilic/hydrophobic character, cellmembrane association, cell recognition or protease inhibi-tion.

Although the growing from technique offers several ad-vantages over grafting to, the polymer growth from a pro-tein in a controllable manner, such as living anionic poly-merization, is poorly compatible with retaining proteinstructure and activity.

Recently, controlled radical polymerization techniqueshave been set up to grow polymers with highly definedchemical composition, structure, and molecular weightfrom a wide variety of commercially available mono-mers.[36–39] Comb-like polymer analogues of PEG can beproduced by radical polymerization techniques startingfrom methacrylate monomers bearing oligo(ethyleneglycol) side-chains. PEG analogues (polyPEG) developedby WEP (Warwick, UK) have been demonstrated to bean interesting alternative to replace PEG in classicalgrafting to protein conjugation as well as in other biomed-ical applications.[40,41] Radical polymerization techniquesalso offer the possibility to generate polymer–proteinconjugates by growing PEG-analogue polymers such asPEGmethacrylate (PEGMA) from proteins functional-ized with proper initiators. Post-polymerization attach-ment of poly(PEGmethacrylates) (pPEGMA) to peptidesand proteins have been reported recently. Nevertheless,studies exploring the feasibility of using proteins as mac-roinitiators to grow directly polymers are still required.[42]

Aimed at setting up a suitable protocol to producetherapeutically active polymer–protein bioconjugates ac-cording to the growing from concept, the pPEGMAgrowth on the recombinant human growth hormone (rh-GH) by atom transfer radical polymerization (ATRP) hasbeen explored (Figure 2).[43] ATRP is an advantageoustechnique for generating polymers with a very wide rangeof molar masses with good control. This inherently flexi-ble approach allows for the introduction of many differ-ent monomers, each of which might contribute to a de-sired property or activity. Importantly, ATRP can operateunder mild conditions in aqueous solution, which is im-portant to prevent protein denaturation and inactivation.

rh-GH was transformed into a macroinitiator by aminogroup conjugation with an a,w-heterobifunctional ATRP

Figure 2. PEGMA polymerisation on protein surface: (a) protein; (b) succinimidyl activated initiator; (c) microinitiator (initiotor conjugatedprotein); (d) PEGMA; (e) protein–PEGMA (ref. [42]).




initiator via an ester activated tetraethyleneglycol spacer,and the resulting multi-point activated protein was usedfor the polymer growth. The SDS-PAGE, iodine staining,gel permeation chromatography and titrations showedthat the macroinitiator completely converted to polymer–protein conjugate. All the initiator domains on the pro-tein surface were involved in the polymerization process,indicating the robustness of the polymer growth method-ology. The complete Lys conjugation demonstrated thatthe initiator termini, regardless of their environmentaldifferences, were free to react equally and that chaingrowth occurred smoothly from all the sites. The polymerchains grown from the protein possessed similar mass(about 20 kDa) with narrow polydispersity (Mw/Mn =1.2), indicating that the polymerization process yielded ahomogeneous product, which is an important requisitefrom a regulatory viewpoint.

The resulting rh-GH–pPEGMA derivative was foundto possess similar physicochemical properties to the cor-responding rh-GH–PEG counterpart obtained by classicalgrafting to PEGylation. The PEGMA conjugation signifi-cantly reduced the rh-GH aggregation and precipitationfrom solutions. Similarly to conventional PEG, PEGMAchains have in fact been reported to form dense hydro-philic surface layers that prevent aggregation phenom-ena.[44] The PEGMA conjugation increased the rh-GH re-sistance to pepsin degradation, indicating that the poly-mer on the protein surface reduced access of the proteas-es to the protein-sensitive sequences, confirming thesteric shielding of the conjugated polymer chains. Sincesteric shielding often results in active site masking, whichcompromises the activity of the protein, in vivo compara-tive studies have been performed to examine the biologi-cal performance of the polymer-modified hormone.[45] Hy-pophysectomized female rats were subcutaneously treatedwith native rh-GH or rh-GH–PEGMA according to twodifferent treatment protocols: 1) daily administration of40 mg hormone equivalent dose; 2) 120 mg hormoneequivalent dose in a dosing regime scheduled at 3 daysbetween the injections. Daily administration of 40 mgequivalent doses of rh-GH and rh-GH–PEGMA over 6days resulted in similar weight gain profiles while thehigher dose (120 mg at day 0 and day 3) resulted in a sig-nificant weight gain enhancement of the animals treatedwith the biconjugate compared to the animals treatedwith the native protein. These results indicate that thePEGMA bioconjugation endows a derivative with en-hanced and sustained bioactivity. These results are ingood agreement with those reported for rh-GH analoguesobtained by site-specific 20-kDa PEG mono-conjugationand multiple 5-kDa PEG attachment, although the invivo activity of the PEG derivatives decreased dependingon PEG size and shape.[46–48]

Interestingly, the PEGMA-modified rh-GH showed en-hanced activity compared to both the native rh-GH overthe same dosing regime and the previously reported

mono-PEGylated analogue, despite the more extensivederivatization on multiple amino groups residues for therh-GH–PEGMA. These results are supportive of ATRP-polymer conjugation as an alternative technique to conju-gate polymer to therapeutic proteins. ATRP polymeri-zation can be carried out under controlled conditions toobtain protein derivatives with controlled physicochemi-cal properties, enhanced stability, and pharmacodynamicproperties compared to the native counterparts. As com-pared to classical grafted to methods, the polymeric partof a bioconjugate attached via ATRP might also containheterogeneous but defined blocks that further enhancethe activity or introduce some peculiar properties or re-sponsive activity. PEGMA analogues are more easilymodified via co-polymerization with components sensitiveto pH, temperature, redox potential, or other microenvir-onmental stimuli, enabling the activity of the protein tobe modulated by the attached polymer. Although theyare still unexplored, stimuli responsive polymer–proteinconjugates may offer interesting opportunities for thera-peutic applications, because they can provide for selectiveon/off switching activity. Furthermore, PEGMA polymerscreated by ATRP can be easily functionalized with a vari-ety of bioactive agents such as targeting moieties and cellpenetrating peptides that can yield new “smart” bioconju-gates.

3. Supramolecular Carriers for Small DrugDelivery

To date, supramolecular carriers have been investigatedas colloidal systems for selective drug delivery and re-lease into the disease target. According to the magicbullet concept formulated by Erlich, the structural designproposed by Ringsdorf,[49] and the EPR mechanism dis-covered by Maeda,[50] a variety of colloidal formulationshave been developed. These systems are obtained byphysical or chemical combinations of materials with dif-ferent physicochemical and biological properties thatallow for high drug payload, disease tissue target recogni-tion, and disposition either by passive or active mecha-nism, cellular or subcellular compartment localization,and drug release under microenvironmental physiopatho-logical conditions. The specific targeting of the therapeut-ically active substance to a certain diseased body region isdeemed the ideal treatment for any disease, as it resultsin high drug efficacy and reduced side effects.[51] In partic-ular, drug targeting is a key approach in anticancer che-motherapy, which is generally associated with severe sideeffects due to systemic or random distribution of mole-cules with a narrow therapeutic index.[52,53] Selective andcontrolled drug release exploits the specific target recog-nition and the peculiar disease site conditions, namely,temperature, pH, redox potential, enzyme composition.




3.1. Targeted Cyclodextrins

In the last forty years, natural cyclodextrins (CDs) andtheir semisynthetic derivatives have been developed asfunctional pharmaceutical excipients for the formulationand delivery of various drugs. CDs can form inclusioncomplexes by hosting hydrophobic drugs into the thoroidcavity thus improving their water solubility, dissolutionrate, chemical stability, and bioavailability.[54,55] Further-more, due to their physicochemical characteristics,namely hydrophobic/hydrophilic features and multivalen-cy, CDs have been incorporated into supramolecular car-riers for active or controlled drug delivery. Based onproper chemical protocols, the CD hydroxyl groups canbe conjugated with various moieties to produce multi-functional targetable nanocarriers, the delivery efficiencyof which depends on both the nature of the conjugatedtargeting moieties and the stability constant of the inclu-sion complex.[56]

Novel CD-based drug delivery systems for tumor tar-geting have been recently developed by a few researchgroups.[57–63] Anticancer drugs are in fact “ideal” mole-cules for CD formulation as they are usually insolubleand chemically instable. Thus, their inclusion into CDscan enhance their poor physicochemical properties. Onthe other hand, CDs do not possess site-specific recogni-tion properties per se, but can be switched from “inert”to “active” carriers by functionalization with targetingagents that selectively recognize specific receptors andantigens exposed on the cancer cell surfaces. Cell-specificexpression of defined membrane-associated targets is thefoundation of the selective drug delivery concept. Thepivotal role of active tumor targeting was highlighted interms of cytosolic access rather than simple tumor accu-mulation.[64] Therefore, tumor-directed CDs should be de-signed in order to accumulate in the diseased tissue, rec-ognize the cell receptors via the targeting moiety, andthen penetrate into the cytosolic compartment. Moreover,the drug/CD system must guarantee stability of the com-plex until the carrier reaches the cytosol, where the drugis released.

A simple CD-based carrier for active tumor targetingwas obtained by oligosaccharide functionalization withfolic acid, an essential vitamin involved in the biosynthet-ic cycle of purines and pyrimidines. Folic acid is taken upby normal cells via a low-affinity (KD ~ 1–5 mM) mem-brane-associated protein that transports the reducedfolate into the cytosol where it remains anchored to thecell membrane. This transmembrane biotransporter isunable to translocate folate conjugates.[65] However, manymalignant cells develop a high affinity (KD ~ 100 pM)transport system (folate receptor) that can internalizeeither folate or folated conjugates in the oxidized form.Upon receptor binding, the folate/folate receptor complexis taken up by cells according to a mechanism known as“potocytosis” and migrates into the cytosol.[66] Since the

folate receptor is overexpressed by many types of tumorcells, it is considered an efficient gate for anticancer drugdelivery.[67] Folate-based carriers are reported in the liter-ature, such as radionuclide deferoxamine complexesaimed at radiopharmaceutical imaging, liposome encapsu-lating drugs, oligonucluotides, and cytotoxins.[68–71]

When folic acid is used as targeting agent, the conjuga-tion to the supramolecular carrier must be carried out inorder to maintain its recognition properties. Studies dem-onstrated that the chemical derivatization of the gluta-mate g-carboxyl group does not alter the affinity of folicacid for its receptor.[72] Folic acid activation with hydroxy-succinimide takes place preferentially at the level of theg-carboxyl group with respect to the a-carboxyl group, asthe latter is sterically hindered.

In order to exploit the inclusion properties of b-CD todeliver actively hydrophobic drugs to tumor tissues, solu-ble and targetable CD based supramolecular systemswere designed. It was hypothesized that once the drug-loaded b-CDs reach the cancer cells, they would be takenup and release the drug as a consequence of environmen-tal changes. Indeed, the pH-induced ionization of thedrugmay take place in the lysosomes and promote the diffu-sion from the cyclodextrin cavity.

According to the hypothesis, a chemical protocol wasset up to conjugate folic acid to the oligosaccahridethrough a short PEG spacer (Figure 3).[73] PEG was usedbecause of its high solubility and flexibility. PEG�s highhydrophilicity can counterbalance the hydrophobic char-acter of folic acid thus endowing soluble conjugates whilethe flexibility of the chain can facilitate the folate dockingto the membrane receptor. The preparation of the mono-substituted bCD–PEG–folate (CD–PEG–FA) was per-formed by coupling a 0.7 kDa diamino-PEG chain to 6�-monotosylated-b-cyclodextrin. The resulting CD–PEG–NH2 was reacted with an excess of succinimidyl ester acti-vated folic acid to obtain CD–PEG–FA.

The PEG conjugation increased the CD solubility ofabout three times. The ability of the CD–PEG–FA conju-gate to form inclusion complexes was investigated usingestradiol and chlorambucil as model drugs.[74] Inclusioninto CD–PEG–FA increased the b-estradiol solubility byabout 540 times as compared to drugs in plain buffer andabout 9 times with respect to b-estradiol formulation with

Figure 3. Folic-acid–PEG conjugated bCD: CD–PEG–FA (ref. [71]).




plain bCDs. Conceivably, the increased b-estradiol solu-bility in the presence of CD–PEG–FA was ascribed to thehigh solubility of the carrier itself. However, the carriercomponents, namely PEG and folic acid, had a detrimen-tal effect on the CD inclusion properties. The modifica-tion of the hepta-glucopyranose ring with hydrophilicPEG chains and the presence of folic acid, which com-petes for the cyclodextrin cavity, provoked a 5-fold de-crease of the bCD/estradiol inclusion constant (Kc). Insilico studies supported the hypothesis that b-estradiol as-sociates with CD–PEG–FA with a 2 : 1 drug : carrier stoi-chiometry, while a 1 :1 complex was calculated for plainbCD.

A study performed with chlorambucil, an anticancerdrug that undergoes rapid degradation in aqueous solu-tion, showed that the inclusion into the novel CD–PEG–FA carrier enhances the molecule stability, although theprotective effect was remarkably lower than that obtainedwith bCDs.

bCDs have intrinsic hemotoxic effect upon parenteraladministration, since they can extract cholesterol andphospholipids from the blood cells, which provokes theirdisruption. Furthermore, bCDs/cholesterol crystals depos-it in the kidneys with consequent nefrotoxicity.[75] Forthese reasons, bCDs have not been approved for paren-teral formulations. CD–PEG–FA displayed negligible he-molytic properties (15 % hemolytic effect) as comparedto the parent bCDs (100 % hemolytic effect) even at veryhigh concentration (20 mM). This was ascribed to the re-duced extraction of blood cell components.

BIAcore analysis and cell culture investigations showedthat the CD–PEG–FA bioconjugate displayed affinity forthe folate receptor.[76] The BIAcore studies carried outusing a folate binding protein (FBP) immobilized chipshowed that CD–PEG–FA interacts selectively with theFBP, although the affinity was 400-fold lower than that offree folic acid reported in literature for (Ka = 50 pM).[77]

The affinity reduction was partly ascribed to the co-pres-ence of 20% derivative obtained by a-carboxyl folic acidconjugation, which is an essential functional group for thereceptor recognition. A study carried out using folate re-ceptor overexpressing KB cells showed that the CD–PEG–FA up-take is inhibited by the presence of [3H]-folic acid in the cell culture medium, confirming that thebioconjugate recognizes selectively the folate receptor onthe biological model. The CD�PEG�FA maintained7 % folic acid affinity for the cellular folate receptor.

Since the interaction with the folic acid receptor is re-ported to promote cell uptake of folated nanocarriers viapotocytosis, selective drug delivery to folate receptoroverexpressing cells was investigated. The KB cell incuba-tion with rhodamine-B loaded CD�PEG�FA resultedin 19 % higher rhodamine up-take than the rhodamine B/bCD formulation, while neither formulation yielded sig-nificant fluorophore up-take by MCF7 cells, a humanbreast cancer cell line that lacks the folate receptor, as a

control.[78] Confocal laser-scanning microscopy visualizedthat CD�PEG�FA loaded with rhodamine-B wasspread into the cytosol of KB cells, associated with tubu-lar endosomal structures in the perinuclear region. Earlyendosomes were formed and large cytosolic fluorescentspots were observed over time, indicating that the endo-somes move and localize in proximity to the Golgi appa-ratus.

A second generation of CD-based carriers was devel-oped by expanding the hydrophobic cavity of the hepta-glucopyranose of bCD ring to a bouquet-like structure inorder to enhance the drug hosting cavity.[79] The new car-rier was obtained by a three-step procedure: 1. Thehepta-glucopyranose hydroxyls were reacted with excessof hexa-methylene diisocyanate to obtain CD–(C6-NCO)5; 2. The distal isocyanate groups of CD–(C6-NCO)5 were conjugated to 0.7 kDa diamino-PEG toobtain CD–(C6-PEG-NH2); 3. CD–(C6-PEG-NH2)5 werefinally functionalized with succinimidyl ester activatedfolic acid to obtain CD–(C6-PEG)5-FA. The final conju-gate contained a mean of five hexa-alkyl-PEG functionsand one folic acid per CD moiety (Scheme 4).

A study carried out using b-estradiol and curcumin aspoorly soluble (11 mM and 30 nM water solubility, respec-tively) and instable drug molecules showed that the newsupramolecular carrier has high affinity for this class ofcompounds. CD–(C6-PEG)5–FA increased the b-estradiolsolubility by about 320 times and the inclusion constantwas 12975 M–1, which is in the same range of that ob-served for the first generation cyclodextrin derivative(CD–PEG–FA).[73]

Promising results were obtained with curcumin, a natu-ral diaryl-heptanoid extracted from Curcuma longa L.This molecule has several biological activities includinganti-angiogenic, anti-inflammatory, and cytotoxic effectson many cancer cells. Its large array of activities, togetherwith low toxicity, make this molecule a potential thera-peutic agent of great interest.[80] Nevertheless, the phar-maceutical exploitation of curcumin suffers from its poorsolubility and stability in water. CD–(C6-PEG)5–FA in-creased the curcumin solubility of 1.16 � 105 times andthe dissociation constant was calculated to be ~1 mM,which is one of the highest ever reported for semi-syn-thetic CDs. The affinity data obtained with curcuminshowed that the cyclodextrin cavity expansion with hexa-alkyl functions promotes the affinity for small, linear mol-ecules. In fact, the linear structure allows drugs to pene-trate deeper into the hepta-glucopyranose cavity and betightly surrounded by the newly introduced alkyl chains.On the other hand, the inclusion of larger non linear mol-ecules like b-estradiol is sterically hampered.

Curcumin inclusion in the CD–(C6-PEG)5–FA carrierincreased the drug stability at pH 6.5 and 7.2, which simu-lated the tumor interstitial compartment and the plasma,respectively. Furthermore, CD–(C6-PEG)5–FA was muchmore efficient than bCD in preserving the drug from deg-




radation, in agreement with the different inclusion con-stants assessed (Figure 4). The curcumin-loaded CD–(C6-PEG)5–FA displayed 21 mM ED50 towards KB cells, while58 mM ED50 was obtained with curcumin loaded into un-folated CD–(C6-PEG-NH2)5. The MCF7 treatment withcurcumin-loaded folated and unfolated carrier yieldedover 60mM ED50. These results confirmed that the carriercan selectively deliver the drug to folate receptor overex-pressing cells.

3.2. Thermosensitive Gold Nanoparticles for Tumor Targeting

Nanotechnology has emerged recently as the science thatinvestigates and develops objects in the nanometer scaleby combining components with diverse biopharmaceutical

features and activities. Nanosized multifunctional systemscan be designed in order to endow objects with specificbio-recognition features and surface properties, therapeu-tic and diagnostic properties, and controlled drug re-lease.[81,82]

Multifunctional nano-objects, metal based nanoparti-cles, nano-shells, nano-cages, and nano-rods have attract-ed increasing attention for a variety of electrochemical,mechanical, optical, and analytical applications. Recently,they have been investigated for biomedical applications,where many needs remain still unmet.[83–89] The idealnanosized platforms can be manipulated by conjugatingsmart polymers, triggerable materials, and biological li-gands that endow them with peculiar features.

Gold nanoparticles are versatile colloidal systems thatcombine large surface-to-volume ratio and optical/ther-mal properties. The physical response to “surface plas-mon absorption” (SPA) can be tuned by modulating thesize, composition architecture, and shape. Furthermore,these nanoparticles offer the advantage of being easilysurface-modified with thiol-bearing polymers, linkers, tar-geting agents, and biomolecules endowing nanosystemswith new and desirable physicochemical and biologicalproperties.[90–93] Recent studies demonstrated that quan-tum dots (CdS), core shell nanoparticles of defined com-position (Fe3O4@SiO2@CdTe), and gold nanoparticles(AuNP) coated with thermosensitive poly-N-isopropyla-crylamide (PNIPAm) homopolymer display new optical,magnetic, and thermal properties.[94—101] The decoration ofgold nanoparticles with temperature-responsive polymercan endow nanosystems with triggerable hydrophilic/hy-drophobic character. Temperature-sensitive polymers can,in fact, reversibly change from extended hydrophilic state

Scheme 4. Synthesis procedure for preparation of the CD-(C6-PEG)5-FA carrier ref. [77] .

Figure 4. Curcumin hydrolysis profile in phosphate buffer 0.1 MpH 7.2 at 30 8C (dotted columns), in the presence of cyclodextrins(dashed columns), or CD–(C6-PEG)5–FA (grey columns) (ref. [71]).




to collapsed hydrophobic state by bulk temperature in-creasing/decreasing.

According to their features and high biocompatibility,surface decorated gold colloidal systems have been inves-tigated as a platform for drug and gene delivery, biodiag-nosis, and other biomedical applications.[102–106] Cationizedgold nanoparticles protected with mixed composite mon-olayers have been exploited for gene delivery. When usedin mammalian cell transfection, an increase of about 8-fold in oligonucleotide delivery efficiency was observedas compared to more conventional polyethylenimine-based systems.[107] Complex gold-based devices have beendesigned for detecting prostate cancer antigen (PSA) atthe attomolar scale. Oligonucleotide/anti-PSA monoclo-nal antibody-derivatized gold nanoparticles combinedwith anti-PSA polyclonal antibody decorated magneticparticles have been developed to enhance the sensitivityto blood PSA screening by a PCR protocol used to ampli-fy the bio-bar code of the gold nanoparticles.[108] Addi-tional biomedical applications exploit the physical re-sponse of gold nanoparticles to laser irradiation. Heat re-lease was exploited for ex vivo purging of bone marrowcancer cells by irradiation of gold nanoparticles after anti-body-mediated biorecognition and for in vivo “thermalablative therapy”.[83,84,109–111]

Cell interaction and up-take is an important requisitefor the biomedical applications of gold nanosystems. De-tailed studies have highlighted the correlation betweencell penetration efficiency and particles size, shape, andchemical functionalization.[112–115] These properties may bemodulated to achieve active targeting to a peculiar bodydistrict such as the brain.[116] Nanoparticle decoration withstimulus-sensitive polymers can also bestow cell-specificbiological properties on nanoparticles.

Thermosensitive polymer-decorated gold nanoparticleshave been investigated as cell up-take “switchable” nano-systems for biomedical and diagnostic applications.[117]

The thermoresponsive thiol-terminating poly-N-isopropy-lacrylamide-co-acrylamide polymer (PNIPAm-co-Am)that exhibits a low critical solution temperature (LCST)at 37 8C was conjugated to 18 nm gold nanoparticles ob-tained in water by laser ablation synthesis in liquid solu-tion (LASiS).[118] LASiS allows for production of colloidalgold particles without the stabilizing agents that mayhave negative impact on the overall toxicity of the formu-lation. Below the LCST the polymer displays extendedconformation as a consequence of water molecule coordi-nation, while above the LCST the water molecules are re-leased and the polymer collapses into a coiled structure.Therefore, at temperature slightly above the physiologictemperature the polymer was expected to display hydro-phobic character and collapse with a sharp conversiontemperature window. Since it has been reported that hy-drophobic polymers pass through cell membranes moreeasily than hydrophilic ones, it is conceivable that abovethe LCST these materials can promote the cell penetra-

tion of a decorated nanosystem. Accordingly, a polymerwith switching behavior can endow nanosystems with astealth feature below its LCST and cell penetration prop-erties above the LCST, and can be used to obtain micro-environmentally stimulus-responsive nanosystems that ex-ploit the thermal abnormalities in several disease sites,namely solid tumors and area of inflammations(Figure 5). In particular, neo-angiogenesis of canceroustissues is related to tissue hyperthermia, with the meantemperature in malignant cancer tissues being 1.5 8Chigher as compared to normal tissue.[119]

The thiol-terminating PNIPAm-co-Am conjugation tothe 18 nm gold nanoparticles yielded about 3800 polymerchains per particle, conveying high stability to the colloi-dal dispersion that otherwise underwent rapid salt-in-duced aggregation. Furthermore, the polymer-decoratedparticles displayed reversible aggregation into 70-nm-di-ameter clusters above 37 8C and in the presence of saltsor serum proteins. At 40 8C, strong aggregation of thepolymer-functionalized particles was accompanied byplasmon absorption profile changes, indicating that thepolymer decoration confers new physical/optical proper-ties to the nanoparticles surface, which may be advanta-geous to biomedical applications.

In vitro studies carried out by MCF7 cell incubationwith naked and PNIPAm-co-Am-SH functionalizedAuNP at 34 and 40 8C, showed that above the LCST thedecorated particles interact strongly with the cells. About6000 naked AuNP were up-taken per cell, regardless ofthe incubation temperature. The functionalized nanopar-ticles showed negligible cell uptake at 34 8C (140 nanopar-ticles/cell) when the polymer was in the hydrophilic ex-

Figure 5. Temperature-controlled cell uptake of gold nanoparticles.(a-b) Surface decoration of gold nanoparticles with thermosensitivepolymer. (b-c) Reversible polymer transition induced by tempera-ture increase. (d) aspecific cell entry of naked nanoparticles. (e)stealth behavior of PNIPAm-co-Am-SH decorated gold nanoparti-cles at temperature below the polymer LCST. (f) temperature trig-gered cell uptake of cell uptake of PNIPAm-co-Am-SH decoratedgold nanoparticles at temperature above the polymer LCST (Repro-duced by permission of The Royal Society of Chemistry http://dx.doi.org/10.1039/b816603j) (ref. [71]).




tended conformation, while about 12000 particles/cellwere detected in the cells after incubation at 40 8C whenthe polymer chains were in a hydrophobic globule state.Transmission electronic microscopy showed diffusedsingle naked AuNP spread into the cytoplasmic compart-ment, occasionally associated with nuclear membrane,while cell incubation with PNIPAm-co-Am-SH decoratedAuNP at 40 8C resulted in a few large clusters (200–400 nm diameter) localized in the cytosol. These resultsindicate that raising the temperature above the LCSTsupports particle hydrophobic surface and aggregation,which favors the cell up-take. It has been also speculatedthat hydrophobic surfaces can effectively adsorb proteinsof the serum that may favor cell up-take.[120]

These results show that thermally controlled “smart”nanocarriers can be easily designed by assembling goldnanoparticles and thermosensitive polymers. These sys-tems can target those sites, namely solid tumors and in-flamed tissues, where local temperatures can be highercompared to normal tissue. Additionally, by virtue of theoptical/thermal properties of colloidal gold, these systemscan be irradiated, producing selective cell killing by im-proving the thermal ablation therapy.

4. Conclusions

This review highlights the relevance of supramolecularbioconjugation for production of novel therapeutic sys-tems, either drug bioconjugates or nanosized carriers.Based on a multidisciplinary approach, the research is ex-ploring sophisticated structures obtained by assemblingmolecules with different physicochemical and biologicalproperties that can display specific functions.

The experience gained by the study of new nanosizedformulations, together with a strong knowledge of thebiological mechanisms involved in disease evolution andthe physicochemical and biological properties of materi-als, may lead to the fabrication of effective and exploita-ble supramolecular therapeutic systems that are expectedto represent the new frontier in health science.

However, despite much progress in bioconjugationchemistry approaching solutions to the challenges ofnanomedicine, the development of effective therapeuticformulations is still an unfinished task. The developmentof nanomedicines with pre-defined therapeutic perfor-mance requires complex structures that may be difficultto set up and characterize. Furthermore, these systemsoften display unpredictable in vivo behaviors that makethem ineffective.

In conclusion, it is interesting to note that the advancesin drug delivery follow the “fashion law”, starting fromhaut couture and moving to prÞt a porter. Similarly, thedesign of complicated drug delivery architectures gener-ates simple colloidal products such as PEGylated proteins(namely Neulasta�, Pegasys�, PegIntron�, Cimzia� etc.),

liposomes (Myocet�, Daunoxome�, Doxyl�, Ambisome�),and polymer bioconjugates (Abraxane� and Opaxio�)that can be easily managed in therapeutic treatments. Thedevelopment of more sophisticated multifunctional drugdelivery systems relies on parallel progress in manufactur-ing and analytical technologies that will allow for fabrica-tion of high quality products with affordable costs.

References

[1] A. Patil, I. M. Shaikh, V. J. Kadam, K. R. Jadhav, Curr.Nanosci. 2009, 5, 141 –153.

[2] P. Debbage, Curr. Pharm. Des. 2009, 15, 153–172.[3] F. Greco, M. J. Vicent, Front. Biosci. 2008, 13, 2744–2756.[4] K. Riehemann, S. W. Schneider, T. A. Luger, B. Godin, M.

Ferrari, H. Fuchs, Angew. Chem. 2009, 121, 886–913;Angew. Chem. Int. Ed. 2009, 48, 872–897.

[5] J. J. Khandare, P. Chandna, Y. Wang, V. P. Pozharov, T.Minko, J. Pharmacol. Exp. Ther. 2006, 317, 929–937.

[6] K. Miller, R. Satchi-Fainaro, in Wiley Encyclopedia ofChemical Biology (Ed: Tadhg P. Begley),Wiley-Inter-science, Hoboken, 2009, vol. 3, pp. 783 –799.

[7] R. Duncan, H. Ringsdorf, R. Satchi-Fainaro, J. Drug Tar-geting 2006, 14, 337–341.

[8] P. Rishabh, A.V. Singh, P. Awanish, T. Poonam, S. K. Ma-jumdar, L. K. Nath, Research J. Pharm. Technol. 2009, 2,228–233.

[9] D. T. Molowa, R. Mazanet, Biotechnol. Annu. Rev. 2003, 9,285–302.

[10] G. Orive, R. M. Hern�ndez, R. A. Gasc�n, A. Dom�nguez-Gil, L. J. Pedraz, Curr. Opin. Biotechnol. 2003, 14, 659 –664.

[11] S. Salmaso, S. Bersani, A. Semenzato, P. Caliceti, J. Nano-sci. Nanotechnol. 2006, 6, 2736–2753.

[12] P. Caliceti, F. M. Veronese, Adv. Drug Delivery Rev. 2003,55, 1261 –1277.

[13] U. Bilati, E. Allemann, E. Doelker, Drug Delivery Tech-nol. 2005, 5, 42 –47.

[14] P. Caliceti, O. Schiavon, F. M. Veronese, BioconjugateChem. 2001, 12, 515–522.

[15] F. M. Veronese, P. Caliceti in Cancer Drug Discovery andDevelopment: Tumour Targeting in Cancer Therapy (Ed.:M. Page). Humana Press, New York, 2005. pp. 411–429.

[16] F. M. Veronese, P. Caliceti, A. Pastorino, O. Schiavon, L.Sartore, L. Banci, L. Monsu Scolaro, J. Controlled Release1989, 10, 145–154.

[17] F. M. Veronese, P. Caliceti, O. Schiavon, M. Sergi, Adv.Drug Delivery Rev. 2002, 54, 587–606.

[18] P. Caliceti, M. Chinol, M. Roldo, F. M. Veronese, A. Se-menzato, S. Salmaso, G. Paganelli, J. Controlled Release2002, 83, 97–108.

[19] Y. Matsumura, H. Maeda, Cancer Res. 1986, 46, 6387 –6392.

[20] S. Bowen, N. Tare, T. Inoue, M. Yamasaki, M. Okabe, I.Horii, J. F. Eliason, Exp. Hematol. 1999, 27, 425–432.

[21] P. Caliceti, M. Morpurgo, O. Schiavon, C. Monfardini,F. M. Veronese, J. Bioact. Compat. Polym. 1994, 9, 252 –266.

[22] P. Caliceti, O. Schiavon, L. Sartore, C. Monfardini, F. M.Veronese, J. Bioact. Compat. Polym. 1993, 8, 41–50.

[23] P. Caliceti, M. Chinol, G. Paganelli, S. Salmaso, S. Bersani,A. Semenzato, Biochim. Biophys. Acta 2005, 1726, 57–66.




[24] D. M. Piedmonte, M. J. Treuheit, Adv. Drug Delivery Rev.2008, 60, 50–58.

[25] M. J. Roberts, M. D. Bentley, J. M. Harris, Adv. Drug De-livery Rev. 2002, 54, 459 –476.

[26] S. Brocchini, A. Godwin, S. Balan, J. Choi, M. Zloh, S.Shaunak, Adv. Drug Delivery Rev. 2008, 60, 3–12.

[27] S. Salmaso, S. Bersani, A. Scomparin, F. Mastrotto, R.Scherpfer, G. Tonon, P. Caliceti, Bioconjugate Chem. 2009,20, 1179 –1185.

[28] A. Tirat, F. Freuler, T. Stettler, L. M. Mayr, L. E. Leder,Int. J. Biol. Macromol. 2006, 39, 66–76.

[29] A. Deiters, T. A. Cropp, D. Summerer, M. Mukherji, P. G.Schultz, Bioorg. Med. Chem. Lett. 2004, 14, 5743–5745.

[30] H. Sato, Adv. Drug Delivery Rev. 2002, 54, 487 –504.[31] B. Peschke, M. Zundel, S. Bak, T. R. Clausen, N. Blume,

A. Pedersen, F. Zaragoza, K. Madsen, Bioorg. Med. Chem.2007, 15, 4382–4395.

[32] S. DeFrees, Z. G. Wang, R. Xing, A. E. Scott, J. Wang, D.Zopf, D. L. Gouty, E. R. Sjoberg, K. Panneerselvam,E. C. M. Brinkman-Van der Linden, R. J. Bayer, M. A.Tarp, H. Clausen, Glycobiology 2006, 16, 833–843.

[33] S. Salmaso, A. Semenzato, S. Bersani, F. Mastrotto, A.Scomparin, P. Caliceti, Eur. Polym. J. 2008, 44, 1378 –1389.

[34] T. P. King, S. W. Zhao, T. Lam, Biochemistry 1986, 25,5774 –5779.

[35] S. M. Sawant, J. P. Hurley, S. Salmaso, A. Kale, E. Tolche-va, T. S. Levchenko, V. P. Torchilin, Bioconjugate Chem.2006, 17, 943–949.

[36] W. A. Braunecker, K. Matyjaszewski, Prog. Polym. Sci.2007, 32, 93–146.

[37] T. Noda, A. J. Grice, M. E. Levere, D. M. Haddleton, Eur.Phys. J. D 2007, 43, 2321–2330.

[38] X. Bories-Azeau, S. P. Armes, H. J. W. Van Den Haak,Macromolecules 2004, 37, 2348–2352.

[39] C. D. Vo, J. Rosselgong, S. P. Armes, N. C. Billingham,Macromolecules 2007, 40, 7119–7125.

[40] S. M. Ryan, X. X. Wang, G. Mantovani, C. T. Sayers, D. M.Haddleton, D. J. Brayden, J. Controlled Release 2008, 135,51–59.

[41] J. Nicolas, G. Mantovani, D. M. Haddleton, Macromol.Rapid Commun. 2007, 28, 1083–1111.

[42] B. S. Lele, H. Murata, K. Matyjaszewski, A. J. Russell, Bio-macromolecules 2005, 6, 3380 –3387.

[43] J. P. Magnusson, S. Bersani, S. Salmaso, C. Alexander, P.Caliceti, Bioconjugate Chem. 2010, 21, 671–678.

[44] O. G. Schramm, G. M. Pavlov, H. P. van Erp, M. A. R.Meier, R. Hoogenboom, U. S. Schubert, Macromolecules2009, 42, 1808–1816.

[45] S. Pradhananga, I. Wilkinson, R. J. M Ross, J. Mol. Endo-crinol. 2002, 29, 11–14.

[46] G. N. Cox, M. S. Rosendahl, E. A. Chlipala, D. J. Smith,S. J. Carlson, D. H. Doherty, Endocrinology 2007, 148,1590 –1597.

[47] R. Clark, K. Olson, G. Fuh, M. Marian, D. Mortensen, G.Teshima, S. Chang, H. Chu, V. Mukku, E. Canova-Davis,T. Somers, M. Cronin, M. Winkler, J. A. Wells, J. Biol.Chem. 1996, 271, 21969 –21977.

[48] B. Peschke, M. Zundel, S. Bak, T. R. Clausen, N. Blume,A. Pedersen, F. Zaragoza, K. Madsen, Bioorg. Med. Chem.2007, 15, 4382–4395.

[49] H. Ringsdorf, J. Polym. Sci. Part C 1975, 51, 135–153.[50] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Con-

trolled Release 2000, 65, 271–284.[51] B. Witkop, Proc. Am. Philos. Soc. 1999, 143, 540 –557.

[52] J. D. Byrne, T. Betancourt, L. Brannon-Peppas, Adv. DrugDelivery Rev. 2008, 60, 1615 –1626.

[53] Y. H. Bae, J. Controlled Release 2009, 133, 2–3.[54] T. Loftsson, D. DuchÞne, Int. J. Pharm. 2007, 329, 1–11.[55] F. Hirayama, K. Uekama, Adv. Drug Delivery Rev. 1999,

36, 125-141.[56] H. Gonzalez, S. J. Hwang, M. E. Davis, Bioconjugate

Chem. 1999, 10, 1068 –1074.[57] C. Bellocq, S. H. Pun, G. S. Jensen, M. E. Davis, Bioconju-

gate Chemistry, 2003,14, 1122-1132.[58] S. H. Pun, F. Tack, N. C. Bellocq, J. Cheng, B. H. Grubbs,

G. S. Jensen, M. E. Davis, M. Brewster, M. Janicot, B. Jans-sens, W. Floren, A. Bakker, Cancer Biol. Ther. 2004, 3,641-650.

[59] S. Hu-Lieskovan, J. D. Heidel, D. W. Bartlett, M. E Davis,T. J. Triche, Cancer Res. 2005, 65, 8984–8992.

[60] D. W. Bartlett, H. Su, I. J. Hildebrandt, W. A. Weber,M. E. Davis, Proc. Natl. Acad. Sci. USA USA 2007, 104,15549 –15554.

[61] J. D. Heidel, Z. Yu, J. Yi-Ching Liu, S. M. Rele, Y. Liang,R. K. Zeidan, D. J. Kornbrust, M. E. Davis, Proc. Natl.Acad. Sci. USA 2007, 104, 5715 –5721.

[62] N. Schaschke, I. Assfalg-Machleidt, W. Machleidt, T. Las-sleben, C. P. Sommerhoff, L. Moroder, Bioorg. Med.Chem. Lett. 2000, 10, 677–680.

[63] J. Defaye, J. M. Garcia-Fernandez, C. Ortiz Mellet, Ann.Pharmac. FranÅaises 2007, 65, 33–49.

[64] D. B. Kirpotin, D. C. Drummond, Y. Shao, M. R. Shalaby,K. Hong, U. B. Nielsen, J. D. Marks, C. C. Benz, J. W. Park,Cancer Res. 2006, 66, 6732–6740.

[65] A. C. Antony, Blood 1992, 79, 2807 –2820.[66] J. J. Turek, C. P. Leamon, P. S. Low, J. Cell Sci. 1993, 106,

423�430.[67] P. Garin-Chesa, I. Campbell, P. E. Saigo, J. L. Lewis,

L. J. Jr. Old, W. J. Rettig, Am. J. Pathol. 1993, 142, 557�567.

[68] S. Wang, R. J. Lee, C. J. Mathias, M. A. Green, P. S. Low,Bioconjugate Chem. 1996, 7, 56�62.

[69] R. J. Lee, P. S. Low, J. Biol. Chem. 1994, 269, 3198�3204.[70] S. Gottschalk, R. J. Cristiano, L. C. Smith, S. L. Woo, Gene

Ther. 1994, 1, 185�191.[71] C. P. Leamon, P. S. Low, J. Drug Targeting 1994, 2, 101�

112.[72] A. Gabizon, A. T. Horowitz, D. Goren, T. Dinah, F. Man-

delbaun Shavit, M. M. Qazen, S. Zalipsky, BioconjugateChem. 1999, 10, 289–298.

[73] P. Caliceti, S. Salmaso, A. Semenzato, T. Carofiglio, R. For-nasier, M. Fermeglia, M. Ferrone, S. Pricl, BioconjugateChem. 2003, 14, 899–908.

[74] T. Higuchi, K. A. Connors. Adv. Anal. Chem. Instr. 1965,4, 117�212.

[75] Y. Ohtani, T. Irie, K. Uekama, K. Fukunaga, J. Pitha. Eur.J. Biochem. 1989, 186, 17�22.

[76] S. Salmaso, A. Semenzato, P. Caliceti, J. Hoebeke, F. Sonvi-co, C. Dubernet, P. Couvreur, Bioconjugate Chem. 2004,15, 997–1004.

[77] D. N. Salter, K. J. Scott, H. Slade, P. Andrews, Biochem. J.1981, 193, 469�476.

[78] F. Sonvico, C. Dubernet, V. Marsaud, M. Appel, H.Chacun, B. Stella, J. M. Renoir, P. Colombo, P. Couvreur,J. Drug Delivery Sci. Technol. 2005, 15, 407–410.

[79] S. Salmaso, S. Bersani, A. Semenzato, P. Caliceti, J. DrugTargeting 2007, 15, 379–390.




[80] J. Ishida, H. Ohtsu, Y. Tachibana, Y. Nakanishi, K. F.Bastow, M. Nagai, H. K. Wang, H. Itokawa, K. H. Lee,Bioorg. Med. Chem. 2002, 10, 3481 –3487.

[81] J. Kim, S. Park, J. E. Lee, S. M. Jin, J. H. Lee, I. S. Lee, I.Yang, J. S. Kim, S. K. Kim, M. H. Cho, T. Hyeon, Angew.Chem. 2006, 118, 7918–7922; Angew. Chem. Int. Ed. 2006,45, 7754 –7758.

[82] C. Wang, J. Chen, T. Talavage, J. Irudayaraj, Angew. Chem.2009, 121, 2797–2801; Angew. Chem. Int. Ed. 2009, 48,2759 –2763.

[83] P. K. Jain, I. H. El-Sayed, M. A. El-Sayed, Nano Today2007, 2, 18 –29.

[84] D. Pissuwan, S. M. Valenzuela, M. B. Cortie, Trends Nano-biotech. 2006, 24, 62–67.

[85] M. R. Choi, K. J. Stanton-Maxey, J. K. Stanley, C. S. Levin,R. Bardhan, D. Akin, S. Badve, J. Sturgis, J. P. Robinson,R. Bashir, N. J. Halas, S. E. Clare, Nano Lett. 2007, 7,3759 –3765.

[86] X. Yang, S. E. Skrabalak, Z. Y. Li, Y. Xia, L. V. Wang,Nano Lett. 2007, 7, 3798–3802.

[87] L. K. Chau, Y. F. Lin, S. F. Cheng, T. J. Lin, Sens. Actua-tors B 2006, 113, 100 –105.

[88] J. Perez-Juste, I. Pastoriza-Santos, L. M. Liz-Marzan, P.Mulvaney, G. Coord, Chem. Rev. 2005, 249, 1870 –1901.

[89] M. C. Daniel, D. Astruc, Chem. Rev. 2004, 104, 293–346.[90] M. Ferrari, Nat. Rev. Cancer 2005, 5, 161–171.[91] P. Alivisatos, Nat. Biotechnol. 2004, 22, 47–52.[92] J. M. de la Fuente, A. G. Barrientos, T. C. Rojas, J. Rojo, J.

CaÇada, A. Fernandez, S. Penades, Angew. Chem. 2001,113, 2317 –2321; Angew. Chem. Int. Ed. 2001, 40, 2257 –2261.

[93] Y. W. Cao, R. Jin, C. A. Mirkin, J. Am. Chem. Soc. 2001,123, 7961 –7962.

[94] G. Han, P. Ghosh, V. M. Rotello, Nanomedicine 2007, 2,113–123.

[95] Z. Zhong, A. S. Subramanian, J. Highfield, K. Carpenter,A. Gedanken, Chem. Eur. J. 2005, 11, 1473–1478.

[96] N. Singh, L. A. Lyon, Chem. Mater. 2007, 19, 719 –726.[97] M. Q. Zhu, L. Q. Wang, G. J. Exarhos, A. D. Q. Li, J. Am.

Chem. Soc. 2004, 126, 2656–2657.[98] J. Raula, J. Shan, M. Nuopponen, A. Niskanen, H. Jiang,

E. I. Kauppinen, H. Tenhu, Langmuir 2003, 19, 3499–3504.[99] P. Zheng, X. Jiang, X. Zhang, W. Zhang, L. Shi, Langmuir

2006, 22, 9393–9396.[100] J. H. Kim, T. R. Lee, Chem. Mater. 2004, 16, 3647–3651.[101] J. Ye, Y. Hou, G. Zhang, C. Wu, Langmuir 2008, 24, 2727 –

2731.

[102] Y. Hou, J. Ye, Z. Guiand, G. Zhang, Langmuir 2008, 24,9682 –9685.

[103] J. He, J. Y. Chen, P. Wang, P. N. Wang, J. Guo, W.-L. Yang,C.-C. Wang, Q. Peng, Nanotechnology 2007, 18, 415101.

[104] H. M. Joshi, D. R. Bhumkar, K. Joshi, V. Pokharkar, M.Sastry, Langmuir 2006, 22, 300 –305.

[105] P. Sharrna, S. Brown, G. Walter, S. Santra, B. Moudgil,Adv. Colloid Interface Sci. 2006, 123, 471 –485.

[106] S. G. Penn, L. He, M. J. Natan, Curr. Opin. Chem. Biol.2003, 7, 609 –615.

[107] K. K. Sandhu, C. M. McIntosh, J. M. Simard, S. W. Smith,V. M. Rotello, Bioconjugate Chem. 2002, 13, 3–6.

[108] J. M. Nam, C. S. Thaxton, C. A. Mirkin, Science 2003, 301,1884 –1886.

[109] D. O. Lapotko, E. Lukianova, A. A. Oraevsky, LasersSurg. Med. 2006, 38, 631 –642.

[110] L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Sershen,B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas, J. L. West,Proc. Natl. Acad. Sci. USA 2003, 100, 113549–113554.

[111] X. H. Huang, I. H. El-Sayed, W. Qian, M. A. El-Sayed, J.Am. Chem. Soc. 2006, 128, 2115 –2120.

[112] B. D. Chithrani, A. A. Ghazani, W. C. W. Chan, Nano Lett.2006, 6, 662 –668.

[113] B. D. Chithrani, W. C. W. Chan, Nano Lett. 2007, 7, 1542 –1550.

[114] T. B. Huff, M. N. Hansen, Y. Zhao, J. X. Cheng, A. Wei,Langmuir 2007, 23, 1596–1599.

[115] D. A. Giljohann, D. S. Seferos, P. C. Patel, J. E. Millstone,N. L. Rosi, C. A. Mirkin, Nano Lett. 2007, 7, 3818 –3821.

[116] G. Sonavane, K. Tomoda, K. Makino. Colloids Surf., B:Biointerfaces 2008, 66, 274–280.

[117] S. Salmaso, P. Caliceti, V. Amendola, M. Meneghetti, G.Pasparakis, C. Alexander, J. Mater. Chem. 2009, 19, 1608 –1615.

[118] V. Amendola, S. Polizzi, M. Meneghetti, J. Phys. Chem. B2006, 110, 7232–7237.

[119] C. Stefanadis, C. Chrisochoou, D. Markou, K. Petraki,D. B. Panagiotakos, C. Fasoulakis, A. Kyriakidis, C. Papadi-mitriou, K. Toutouzas, J. Clin. Oncol. 2001, 19, 676–681.

[120] N. Shamim, L. Hong, K. Hidajat, M. S. Uddin, J. ColloidInterface Sci. 2006, 304, 1–8.

Received: April 14, 2010Accepted: June 3, 2010

Published online: July 27, 2010




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Supramolecular Bioconjugates for Protein and Small Drug Delivery