Impact of Four Common Hydrogels on Amyloid-ß (Aß) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer's Disease.

The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer's disease (AD) that could benefit from the in vivo-like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-ß (Aß), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aß cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aß is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aß structure and aggregation kinetics in solution and hydrogels composed of type I collagen, agarose, hyaluronic acid, and polyethylene glycol using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with enhanced Aß aggregation and Aß cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aß structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.

Protein folding and assembly in confined environments: Implications for protein aggregation in hydrogels and tissues.

In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein's structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more "in vivo-like" environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.

Collagen hydrogel confinement of Amyloid-ß (Aß) accelerates aggregation and reduces cytotoxic effects.

Alzheimer's disease (AD) is the most common form of dementia and is associated with the accumulation of amyloid-ß (Aß), a peptide whose aggregation has been associated with neurotoxicity. Drugs targeting Aß have shown great promise in 2D in vitro models and mouse models, yet preclinical and clinical trials for AD have been highly disappointing. We propose that current in vitro culture systems for discovering and developing AD drugs have significant limitations; specifically, that Aß aggregation is vastly different in these 2D cultures carried out on flat plastic or glass substrates vs. in a 3D environment, such as brain tissue, where Aß confinement alters aggregation kinetics and thermodynamics. In this work, we identified attenuation of Aß cytotoxicity in 3D hydrogel culture compared to 2D cell culture. We investigated Aß structure and aggregation in solution vs. hydrogel using Transmission Electron Microscopy (TEM), Fluorescence Correlation Spectroscopy (FCS), and Thioflavin T (ThT) assays. Our results reveal that the equilibrium is shifted to stable extended ß-sheet (ThT positive) aggregates in hydrogels and away from the relatively unstable/unstructured presumed toxic oligomeric Aß species in solution. Volume exclusion imparted by hydrogel confinement stabilizes unfolded, presumably toxic species, promoting stable extended ß-sheet fibrils. STATEMENT OF SIGNIFICANCE: Alzheimer's disease (AD) is a devastating disease and has been studied for over 100 years. Yet, no cure exists and only 5 prescription drugs are FDA-approved to temporarily treat the AD symptoms of declining brain functions related to thinking and memory. Why don't we have more effective treatments to cure AD or relieve AD symptoms? We propose that current culture methods based upon cells cultured on flat, stiff substrates have significant limitations for discovering and developing AD drugs. This study provides strong evidence that AD drugs should be tested in 3D culture systems as a step along the development pathway towards new, more effective drugs to treat AD.

The effect of glycation on arterial microstructure and mechanical response.

Like engineered materials, an artery's biomechanical behavior and function depend on its microstructure. Glycation is associated with both normal aging and diabetes and has been shown to increase arterial stiffness. In this study we examined the direct effect of glycation on the mechanical response of intact arteries and on the mechanical response and structure of elastin isolated from the arteries. Samples of intact arteries and isolated elastin were prepared from porcine aortas and glycated. The mechanical response of all samples was completed using a uniaxial material test system. Glycation levels were measured using ELISA. A confocal microscope was used to image differences in the structure of the glycated and untreated elastin fibers. We found that, under the conditions used in this study, glycation led to decreased stiffness of elastin isolated from arteries, which was associated with a thinning of elastin fibers as imaged by confocal microscopy. We observed no effect of glycation on collagen fibers under our treatment conditions. These results suggest that glycation leads to weakening of the elastin component of arteries that could contribute to vascular defects seen in diabetes and aging. Prevention of glycation reactions may be an important consideration for vascular health later in life.

The effect of oxidation on the mechanical response and microstructure of porcine aortas.

Reactive oxygen species (ROS), a product of many cellular functions, has been implicated in many age-related pathophysiological processes, including cardiovascular disease. The arterial proteins collagen and elastin may also undergo structural and functional changes due to damage caused by ROS. This study examined the effect of oxidation on the mechanical response of porcine aortas and aorta elastin and the associated changes in structural protein ultrastructure as a step in exploring the role of molecular changes in structural proteins with aging on elastic artery function. We examined the change in mechanical properties of aorta samples after various oxidation times as a first step in understanding how the oxidative environment associated with aging could impact mechanical properties of arterial structural proteins. We used confocal microscopy to visualize how the microstructure of isolated elastin changed with oxidation. We find that short term oxidation of elastin isolated from aortas leads to an increase in material stiffness, but also an increase in the fiber diameter, increase in void space in the matrix, and a decrease in the fiber orientation, possibly due to fiber cross-linking. The short term effects of oxidation on arterial collagen is more complex, with increase in material stiffness seen in the collagen region of the stress stretch curve at low extents of oxidation, but not at high levels of oxidation. These results may provide insight into the relationship between oxidative damage to tissue associated with aging and disease, structure of the arterial proteins elastin and collagen, and arterial mechanical properties and function.

Structurally distinct toxicity inhibitors bind at common loci on ß-amyloid fibril.

The accumulation of aggregated ß-Amyloid (Aß) in the brain is a hallmark of Alzheimer's disease and is thought to play a role in the neurotoxicity associated with the disease. The mechanism by which Aß aggregates induce toxicity is uncertain. Nonetheless, several small molecules have been found to interact with Aß fibrils and to prevent their toxicity. In this paper we studied the binding of these known toxicity inhibitors to Aß fibrils, as a means to explore surfaces or loci on Aß aggregates that may be significant in the mechanism of action of these inhibitors. We believe knowledge of these binding loci will provide insight into surfaces on the Aß fibrils important in Aß biological activity. The program DOCK was used to computationally dock the inhibitors to an Aß fibril. The inhibitors docked at two shared binding loci, near Lys28 and at the C-termini near Asn27 and Val39. The docking predictions were experimentally verified using lysine specific chemical modifications and Aß fibrils mutated at Asn27. We found that both Congo red and Myricetin, despite being structurally different, bound at the same two sites. Additionally, our data suggests that three additional Aß toxicity inhibitors may also bind in one of the sites. Identification of these common binding loci provides targets on the Aß fibril surface that can be tested in the future for their role in Aß biological activity.

Can size alone explain some of the differences in toxicity between beta-amyloid oligomers and fibrils?

beta-Amyloid (Abeta) peptide is believed to play a key role in the mechanism of Alzheimer's disease (AD). Abeta tends to aggregate to form amyloid fibrils. A variety of evidence indicates that Abeta aggregates are toxic in vitro and in vivo. An early "Abeta hypothesis" postulated that AD was the consequence of neuron death induced by insoluble deposits of large Abeta fibrils. Newer findings indicate that small soluble Abeta oligomers are the neurotoxic species, yet their structure is still unknown. Many researchers have tried to probe the differences in molecular structure between Abeta oligomers, protofibrils, and fibrils that give rise to their unique toxicities, but with limited success. In this report, we examine the hypothesis that differences in the toxicity of different aggregated Abeta species are the result of differences in species concentration and diffusivity. Using a simple mathematical analysis based on the assumption of a diffusion-limited reaction, we demonstrate that near 10-fold differences in toxicity between spherical oligomers and fibrils can be explained from size and concentration arguments. While this work does not suggest that Abeta oligomers and fibrils have identical molecular structures, it highlights the possibility that simple physical phenomena may contribute to the biological processes induced by Abeta.

Two disaccharides and trimethylamine N-oxide affect Abeta aggregation differently, but all attenuate oligomer-induced membrane permeability.

Interaction between aggregates of amyloid beta protein (Abeta) and membranes has been hypothesized by many to be a key event in the mechanism of neurotoxicity associated with Alzheimer's disease (AD). Proposed membrane-related mechanisms of neurotoxicity include ion channel formation, membrane disruption, changes in membrane capacitance, and lipid membrane oxidation. Recently, osmolytes such as trehalose have been found to delay Abeta aggregation in vitro and reduce neurotoxicity. However, no direct measurements have separated the effects of osmolytes on Abeta aggregation versus membrane interactions. In this article, we tested the influence of trehalose, sucrose and trimethylamine-N-oxide (TMAO) on Abeta aggregation and fluorescent dye leakage induced by Abeta aggregates from liposomes. In the absence of lipid vesicles, trehalose and sucrose, but not TMAO, were found to delay Abeta aggregation. In contrast, all of the osmolytes significantly attenuated dye leakage. Dissolution of preformed Abeta aggregates was excluded as a possible mechanism of dye leakage attenuation by measurements of Congo red binding as well as hydrogen-deuterium exchange detected by mass spectrometry (HX-MS). However, the accelerated conversion of high order oligomers to fibril caused by vesicles did not take place if any of the three osmolytes presented. Instead, in the case of disaccharide, osmolytes were found to form adducts with Abeta, and change the dissociation dynamics of soluble oligomeric species. Both effects may have contributed to the observed osmolyte attenuation of dye leakage. These results suggest that disaccharides and TMAO may have very different effects on Abeta aggregation because of the different tendencies of the osmolytes to interact with the peptide backbone. However, the effects on Abeta membrane interaction may be due to much more general phenomena associated with osmolyte enhancement of Abeta oligomer stability and/or direct interaction of osmolyte with the membrane surface.

Kinetic study of beta-amyloid residue accessibility using reductive alkylation and mass spectrometry.

Beta-amyloid peptide (Abeta) is the major protein constituent found in senile plaques in Alzheimer's disease (AD). It is believed that Abeta plays a role in neurodegeneration associated with AD and that its toxicity is related to its structure or aggregation state. In this study, an approach based on chemical modification of primary amines and mass spectrometric (MS) detection was used to identify residues on Abeta peptide that were exposed or buried upon changes in peptide structure associated with aggregation. Results indicate that the N terminus was the most accessible primary amine in the fibril, followed by lysine 28, then lysine 16. A kinetic analysis of the data was then performed to quantify differences in accessibility between these modification sites. We estimated apparent equilibrium unfolding constants for each modified site of the peptide, and determined that the unfolding constant for the N terminus was approximately 100 times greater than that for K28, which was about six times greater than that for K16. Understanding Abeta peptide structure at the residue level is a first step in designing novel therapies for prevention of Abeta structural transitions and/or cell interactions associated with neurotoxicity in Alzheimer's disease.

Exploring the mechanism of beta-amyloid toxicity attenuation by multivalent sialic acid polymers through the use of mathematical models.

beta-Amyloid peptide (A beta), the primary protein component in senile plaques associated with Alzheimer's disease (AD), has been implicated in neurotoxicity associated with AD. Previous studies have shown that the A beta-neuronal membrane interaction plays a role in the mechanism of A beta toxicity. More specifically, it is thought that A beta interacts with ganglioside rich and sialic acid rich regions of cell surfaces. In light of such evidence, we have used a number of different sialic acid compounds of different valency or number of sialic acid moieties per molecule to attenuate A beta toxicity in a cell culture model. In this work, we proposed various mathematical models of A beta interaction with both the cell membrane and with the multivalent sialic acid compounds, designed to act as membrane mimics. These models allow us to explore the mechanism of action of this class of sialic acid membrane mimics in attenuating the toxicity of A beta. The mathematical models, when compared with experimental data, facilitate the discrimination between different modes of action of these materials. Understanding the mechanism of action of A beta toxicity inhibitors should provide insight into the design of the next generation of molecules that could be used to prevent A beta toxicity associated with AD.

Structural differences between Abeta(1-40) intermediate oligomers and fibrils elucidated by proteolytic fragmentation and hydrogen/deuterium exchange.

The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.

Development of photocrosslinked sialic acid containing polymers for use in Abeta toxicity attenuation.

beta-Amyloid peptide (Abeta), the primary protein component in senile plaques associated with Alzheimer's disease (AD), has been implicated in neurotoxicity associated with AD. Previous studies have shown that the Abeta-neuronal membrane interaction plays a crucial role in Abeta toxicity. More specifically, it is thought that Abeta interacts with ganglioside rich and sialic acid rich regions of cell surfaces. In light of such evidence, we have hypothesized that the Abeta-membrane sialic acid interaction could be inhibited through use of a biomimic multivalent sialic acid compound that would compete with the cell surface for Abeta binding. To explore this hypothesis, we synthesized a series of photocrosslinked sialic acid containing oligosaccharides and tested their ability to bind Abeta and attenuate Abeta toxicity in cell culture assays. We show that a polymer prepared via the photocrosslinking of disialyllacto-N-tetraose (DSLNT) was able to attenuate Abeta toxicity at low micromolar concentrations without adversely affecting the cell viability. Polymers prepared from mono-sialyl-oligosaccharides were less effective at Abeta toxicity attenuation. These results demonstrate the feasibility of using photocrosslinked sialyl-oligosaccharides for prevention of Abeta toxicity in vitro and may provide insight into the design of new materials for use in attenuation of Abeta toxicity associated with AD.

Nanofluidic biosensing for beta-amyloid detection using surface enhanced Raman spectroscopy.

Trace detection of the conformational transition of beta-amyloid peptide (Abeta) from a predominantly alpha-helical structure to beta-sheet could have a large impact in understanding and diagnosing Alzheimer's disease. We demonstrate how a novel nanofluidic biosensor using a controlled, reproducible surface enhanced Raman spectroscopy active site was developed to observe Abeta in different conformational states during the Abeta self-assembly process as well as to distinguish Abeta from confounder proteins commonly found in cerebral spinal fluid.

Simultaneous monitoring of peptide aggregate distributions, structure, and kinetics using amide hydrogen exchange: application to Abeta(1-40) fibrillogenesis.

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Abeta) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Abeta and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Abeta(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Abeta aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Abeta toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multi-component oligomer distributions of aggregating peptides and proteins.

Attenuation of beta-amyloid-induced toxicity by sialic-acid-conjugated dendrimers: role of sialic acid attachment.

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease and is believed to be associated with neurotoxicity in the disease. We and others have shown that Abeta binds with relatively high affinity to clustered sialic acid residues on cell surfaces and that removal of cell surface sialic acids attenuates Abeta toxicity. We have also shown that sialic acid functionalized dendrimeric polymers can act as mimics of cell surface sialic acid clusters and attenuate Abeta-induced neurotoxicity. In the current study, we prepared sialic-acid-conjugated dendrimers using a physiologically relevant attachment of the sialic acid to the dendrimeric termini, and evaluated the Abeta toxicity attenuation properties of the dendrimers. We compared performance of sialic-acid-conjugated dendrimeric polymers in which the sialic acid moieties were attached to dendrimeric termini via the anomeric hydroxyl group of the sialic acid, a physiological attachment, to polymers in which the attachment was made via the carboxylic acid group on the sialic acid, a non-physiological attachment. This work enhances our understanding of Abeta-cell surface binding and is a step towards the development of new classes of sequestering agents as therapeutics for the prevention of Abeta toxicity in AD.

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway.

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.

The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation.

Calcitonin, a peptide hormone associated with medullary carcinoma of the thyroid, has the potential to form amyloid fibrils and may be a valuable model for investigating the role of peptide-membrane interactions in beta-sheet and amyloid formation. Via a new model peptide system, bovine calcitonin, we found that the exposure of peptide to phospholipid membranes altered its structure relative to the structures formed in aqueous solutions. Of particular relevance to the amyloidoses, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant enrichment in the beta-sheet and amyloid content of the peptide. The formation of amyloid was also accelerated in these systems. A correlation between the phospholipid-induced structural alterations and calcitonin binding affinities to phospholipid membranes was evident. Bovine calcitonin has considerably higher binding affinity for the phospholipid systems that enhanced its beta-sheet and amyloid structure. Electrostatic forces were not the governing forces behind the observed behavior, as supported by the fact that the ionic strength did not affect the peptide structures or binding affinities. A Van't Hoff analysis of the temperature-dependent peptide binding affinities indicated that binding led to an increase in enthalpy and possibly an increase in entropy of the peptide-membrane systems. Experiments with other amyloid-forming peptides such as beta-amyloid of Alzheimer's disease have also shown similar results and may indicate the need to manipulate peptide-membrane interactions in order to control amyloid formation and its associated disease.

The influence of phospholipid membranes on bovine calcitonin peptide's secondary structure and induced neurotoxic effects.

The peptide hormone, calcitonin, which is associated with medullary carcinoma of the thyroid, has a marked tendency to form amyloid fibrils and may be a useful model in probing the role of peptide-membrane interactions in beta-sheet and amyloid formation and amyloid neurotoxicity. Using bovine calcitonin, we found that, like other amyloids, the peptide was toxic only when in a beta-sheet-rich, amyloid form, but was non-toxic, when it lacked an amyloid structure. We found that the peptide bound with significant affinity to membranes that contained either cholesterol and gangliosides. In addition, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant changes in peptide structure yielding a peptide enriched in beta-sheet and amyloid content. Because the cholesterol- and ganglioside-rich phospholipid systems enhanced the calcitonin beta-sheet and amyloid contents, and peptide amyloid content was associated with neurotoxicity, we then investigated whether depleting cellular cholesterol and gangliosides affected calcitonin neurotoxicity. We found that cholesterol and ganglioside removal significantly reduced the calcitonin-induced PC12 cell neurotoxicity. Similar results have been observed with other amyloid-forming peptides such as beta-amyloid (A beta) of Alzheimer's disease and suggest that modulation of membrane composition and peptide-membrane interactions may prove useful in the control of amyloid formation and amyloid neurotoxicity.

Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli.

With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.

Efficacy of different targeting agents in the photolysis of interleukin-2 receptor bearing cells.

The multichain interleukin-2 receptor (IL-2R) has been proposed as a target for immunotherapy in the treatment of certain cancers including adult T-cell leukemia and cutaneous T-cell lymphoma as well as certain autoimmune diseases. The IL-2R is abnormally expressed on cells associated with each of these diseases; while normal, non-activated T-cells do not express the receptor. This report describes the selective photolysis of activated and non-activated IL-2R expressing cells using several immunoconjugates synthesized with one of two photosensitizers, hematoporphyrin (HP) or chlorin-e(6) (Ce(6)), covalently linked to IL-2 or an anti-IL-2R antibody. Destruction of IL-2R bearing cells was achieved after photosensitizer internalization and irradiation using all tested photosensitizer conjugates. Chlorin containing conjugates were more effective, by a factor of 4 or more, than HP containing conjugates. Conjugates made with IL-2 were up to 30 times more effective than conjugates that used a monoclonal antibody against the IL-2R for targeting. Activation of the cells to increase IL-2R expression decreased the internalization time required for optimal therapeutic efficacy; however, stimulation of the cell to increase IL-2 secretion greatly reduced conjugate effectiveness. This work could lead to the development of more effective strategies to treat T-cell diseases.