In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.     

Abeta(1-40) peptide radiopharmaceuticals for brain amyloid imaging: (111)In chelation, conjugation to poly(ethylene glycol)-biotin linkers, and autoradiography with Alzheimer's disease brain sections

The amyloid plaques of Alzheimer's disease (AD) are formed by the neuropeptide Abeta(1)(-)(42/43), and carboxyl terminal truncated forms of this neuropeptide, designated Abeta(1)(-)(40), bind to amyloid plaques of AD autopsy tissue sections. Therefore, Abeta(1)(-)(40) is a potential peptide radiopharmaceutical that could be used for imaging brain amyloid in living subjects with AD, should this neuropeptide be made transportable through the blood-brain barrier (BBB). To accomplish this, the neuropeptide must be modified to enable (i) attachment to a BBB drug targeting system and (ii) labeling with a radionuclide, e.g., 111-indium, suitable for brain imaging by external detection modalities such as single photon emission computed tomography (SPECT). The present studies describe the synthesis of an Abeta(1)(-)(40) analogue that contains a biotin at the amino terminus and a diethylenetriaminepentaacetic acid (DTPA) moiety conjugated to one of the internal lysine residues. The DTPA-[N-biotin]-Abeta(1)(-)(40) was purified by gel filtration fast-protein liquid chromatography (FPLC) using two Superose 12HR columns in series, and the structure of the purified peptide was confirmed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The binding of the [(111)In]DTPA-[N-biotin]-Abeta(1)(-)(40) to amyloid plaques of AD autopsy tissue sections was demonstrated by film and emulsion autoradiography. A poly(ethylene glycol) (PEG) linker of 3400 Da molecular mass, designated PEG(3400), was inserted between the Abeta(1)(-)(40) and the biotin moiety, but this modification diminishes binding of Abeta(1)(-)(40) to the AD amyloid plaques. In summary, these studies describe a novel formulation of biotinylated Abeta(1)(-)(40) that allows radiolabeling with 111-indium. The peptide radiopharmaceutical may be conjugated to an avidin-based BBB drug targeting system to enable transport through the BBB and imaging of brain amyloid in vivo.     

Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent

The polymeric functionalization of superparamagnetic iron oxides nanoparticles is developed for cancer targeting capability and magnetic resonance imaging. Here the nanoparticles (NP) are decorated through the adsorption of a polymeric layer around the particle surface for the formation of core-shell. The synthesized magnetic nanoparticles (MNPs) are conjugated with fluorescent dye, targeting ligand, and drug molecules for improvement of target specific diagnostic and possible therapeutics applications. In this investigation doxorubicin was loaded into the shell of the MNPs and release study was carried out at different pH. The core-shell structure of magnetic NP coated chitosan matrix was visualized by TEM observation. The cytotoxicity of these magnetic NPs is investigated using MTT assay and receptor mediated internalization by HeLa and NIH3T3 cells are studied by fluorescence microscopy. Moreover, compared with T₂-weighted magnetic resonance imaging (MRI) in the above cells, the synthesized nanoparticles are showed stronger contrast enhancements towards cancer cells.     

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Effect of Pullulan Nanoparticle Surface Charges on HSA Complexation and Drug Release Behavior of HSA-Bound Nanoparticles

Nanoparticle (NP) compositions such as hydrophobicity and surface charge are vital to determine the presence and amount of human serum albumin (HSA) binding. The HSA binding influences drug release, biocompatibility, biodistribution, and intercellular trafficking of nanoparticles (NPs). Here, we prepared 2 kinds of nanomaterials to investigate HSA binding and evaluated drug release of HSA-bound NPs. Polysaccharides (pullulan) carboxyethylated to provide ionic derivatives were then conjugated to cholesterol groups to obtain cholesterol-modified carboxyethyl pullulan (CHCP). Cholesterol-modified pullulan (CHP) conjugate was synthesized with a similar degree of substitution of cholesterol moiety to CHCP. CHCP formed self-aggregated NPs in aqueous solution with a spherical structure and zeta potential of −19.9±0.23 mV, in contrast to −1.21±0.12 mV of CHP NPs. NPs could quench albumin fluorescence intensity with maximum emission intensity gradually decreasing up to a plateau at 9 to 12 h. Binding constants were 1.12×10⁵ M⁻¹ and 0.70×10⁵ M⁻¹ to CHP and CHCP, respectively, as determined by Stern-Volmer analysis. The complexation between HSA and NPs was a gradual process driven by hydrophobic force and inhibited by NP surface charge and shell-core structure. HSA conformation was altered by NPs with reduction of α-helical content, depending on interaction time and particle surface charges. These NPs could represent a sustained release carrier for mitoxantrone in vitro, and the bound HSA assisted in enhancing sustained drug release.     

Design,synthesis, and biological evaluation of biotinylated colchicine derivatives as potential antitumor agents

Chemical drug design based on the biochemical characteristics of cancer cells has become an important strategy for discovering new anti-tumour drugs to improve tumour targeting effects and reduce off-target toxicities. Colchicine is one of the most prominent and historically microtubule-targeting drugs, but its clinical applications are hindered by notorious adverse effects. In this study, we presented a novel tumour-specific conjugate 9 that consists of deacetylcolchicine (Deac), biotin, and a cleavable disulphide linker. 9 was found to exhibit potent anti-tumour activity and exerted higher selectivity between tumour and nontarget cells than Deac. The targeting moiety biotin might enhance the transport capability and selectivity of 9 to tumour cells via biotin receptor-mediated endocytosis. The tubulin polymerisation activity of 9 (with DTT) was close to the parent drug Deac. These preliminary results suggested that 9 is a high potency and reduced toxicity antitumor agent and worthy of further investigation.     

Temporal control over cellular targeting through hybridization of folate-targeted dendrimers and PEG-PLA nanoparticles

Polymeric nanoparticles (NPs) and dendrimers are two major classes of nanomaterials that have demonstrated great potential for targeted drug delivery. However, their targeting efficacy has not yet met clinical needs, largely because of a lack of control over their targeting kinetics, which often results in rapid clearance and off-target drug delivery. To address this issue, we have designed a novel hybrid NP (nanohybrid) platform that allows targeting kinetics to be effectively controlled through hybridization of targeted dendrimers with polymeric NPs. Folate (FA)-targeted generation 4 poly(amidoamine) dendrimers were encapsulated into poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) NPs using a double emulsion method, forming nanohybrids with a uniform size (~100 nm in diameter) at high encapsulation efficiencies (69-85%). Targeted dendrimers encapsulated within the NPs selectively interacted with FA receptor (FR)-overexpressing KB cells upon release in a temporally controlled manner. The targeting kinetics of the nanohybrids were modulated using three different molecular weights (MW) of the PLA block (23, 30, and 45 kDa). The release rates of the dendrimers from the nanohybrids were inversely proportional to the MW of the PLA block, which dictated their binding and internalization kinetics with KB cells. Our results provide evidence that selective cellular interactions can be kinetically controlled by the nanohybrid design, which can potentially enhance targeting efficacy of nanocarriers.     

Biotinylated granulocyte/macrophage colony-stimulating factor analogues: effect of linkage chemistry on activity and binding.

Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF) and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF) were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein (EC50 = 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.     

Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer (M(r) 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1-4 positive charges in the headgroup (referred to as CPL(1)-CPL(4), respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios < or =0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60 degrees C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca(2+). The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL(2) displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL(3), while for CPL(4)-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.     

Two-step fluorescence screening of CD21-binding peptides with one-bead one-compound library and investigation of binding properties of N-(2-hydroxypropyl)methacrylamide copolymer-peptide conjugates

Using the one-bead one-compound (OBOC) combinatorial method, four heptapeptide ligands of CD21 receptor, a cell surface marker of malignant B cell lymphoma, were identified with an innovative two-step fluorescence screening method to overcome the limitation caused by autofluorescence of TentaGel resin. The binding affinities of selected peptides, YILIHRN (B1), PTLDPLP (B2), and LVLLTRE (B3), were in the micromolar region as determined by a fluorescence quenching assay. Peptide B1 was conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via spacers of different lengths, composed of one to four repeats of the 8-amino-3,6-dioxaoctanoic acid (A) group. The evaluation of the biorecognizability of HPMA copolymer-B1 conjugates by the CD21 receptor revealed that increasing the number of repeats of A in the spacer from one to three resulted in continuous improvements in the biorecognition by the CD21 receptor; the increase from three to four repeats showed no significant effect. This work showed the potential of the OBOC combinatorial approach to select peptide ligands as targeting moieties for CD21 specific polymeric drug carriers.     

UV-patterned poly(ethylene glycol) matrix for microarray applications

A versatile method to fabricate polymeric matrixes for microarray applications is demonstrated. Several different design strategies are presented where a variety of organic films, such as plastic polymers and self-assembled monolayers (SAMs) on planar silica and gold substrates, act as supports for the graft polymerization procedure. An ensemble of poly(ethylene glycol) methacrylate monomers are combined to obtain a matrix with desired properties: low nonspecific binding and easily accessible groups for postimmobilization of ligands. The free radical graft polymerization process occurs under irradiation with UV light in the 254-266 nm range, which offers the possibility to introduce patterns by means of a photomask. The arrays are created on inert and homogeneous coatings prepared either by graft polymerization of a methoxy-terminated PEG-methacrylate or self-assembly of a methoxy-terminated oligo(ethylene glycol) thiol. Carboxylic acid groups, introduced in the array spots either during graft polymerization or upon wet chemical conversion of hydroxyls, grant the capability to immobilize proteins and other molecules via free amine groups. Immobilization of fluorescent species as well as biotin followed by exposure to a fluorescently labeled antibody directed toward biotin display both excellent integrity of the spots and low nonspecific binding to the surrounding framework. Beside patterns of uniform height and size, an array of spots with varying thickness (a sort of gradient) is demonstrated. Such gradient samples enable us to address critical issues regarding the mechanism(s) behind spatially resolved free radical polymerization of methacrylates. It also offers a convenient route to optimize the matrix properties with respect to thickness, loading capacity, protein diffusion/penetration, and nonspecific binding.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors

Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein / ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1) / inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann / surface area (MM / PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects.     

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.     

Fluorogen-Activating scFv Biosensors Target Surface Markers on Live Cells Via Streptavidin or Single-Chain Avidin

Fluorescence biosensors are indispensable tools for understanding protein behavior and function in cells. Recent advancements utilize fluorogen-activating-proteins (FAPs) that form complexes with small organic molecules (fluorogens) and result in their fluorescence activation. The technology has found multiple uses in protein discovery applications; however, the current method of detection requires the expression of FAPs as gene fusion tags in cells—a process that is time- and labor-intensive. In this report, we present an alternate method that utilizes FAPs as affinity reagents. Accordingly, we isolated soluble reagents based on FAP fusions with streptavidin (Strep) or avidin proteins, both highly selective for biotin. When tested in vitro, the reagents displayed bi-functional activity, fluorogen activation, and biotin affinity. For live-cell protein discovery, surface targets were biotinylated via biotin-tagged immunoglobulins or a genetically encoded biotin acceptor peptide. As a result, when the cells were labeled with FAP–Strep or FAP–avidin reagent, the in vivo fluorescence measurements indicated high target specificity, minimal background, and bright signal detection. In summary, we present a novel FAP reagent platform that offers a rapid and efficient approach for cell surface protein detection.     

Flow injection analysis of binding reaction between fluorescent lectin and cells

A fluorometric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported (Y. Oda, M. Kinoshita, and K. Kakehi, Anal. Biochem. 254, 41-48, 1997). However, the true amount of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amount. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1. 3-1.6 x 10(8) binding sites per cell for Concanavalin A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10(6) M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.     

Fluorescence imaging with multifunctional polyglycerol sulfates: novel polymeric near-IR probes targeting inflammation

We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ~70 sulfate groups) targets factors of inflammation (IC(50) of 3-6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1-3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.