10 research outputs found
sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo
We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help with expressing the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at in the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.Yeshttp://www.plosone.org/static/editorial#pee
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Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells
Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity
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Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro
Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy
mvsFlt has longer residence time in the rat vitreous.
<p>A) Schematic depicting methods used to determine the half-life of fluorescently tagged sFlt and mvsFlt in the rat vitreous. The vitreous was injected with 5μl of Alexa Fluor 488-tagged sFlt or mvsFlt. After 0, 4, 12, 24, and 48 hours, the rats were sacrificed and their eyes were enucleated and frozen for analysis. The vitreous was then removed, immersed in RIPA buffer and homogenized for subsequent fluorescence measurements. B) Conjugation to HyA significantly improves residence time of sFlt in the vitreous after 48 hours in comparison to sFlt. Results are expressed as mean ±SD (*p<0.05, **p<0.01, ***p<0.001). * indicates a difference between the mvsFlt and sFlt at the given time point. Two-way ANOVA gives p-value ***<0.001.</p
sFlt and mvsFlt equally inhibit corneal angiogenesis.
<p>A) Schematic depicting methods utilized for carrying out corneal burn model. Mice were treated twice with 5μl of PBS, sFlt or mvsFlt at day 1 and 3 following the chemical burn. B) Representative images of eyes treated with PBS, sFlt and mvsFlt. CD31 positive (green) staining of corneal blood vessels. C) Quantification of corneal angiogenesis at day 10 following treatment. One-way ANOVA gives p value **<0.01 (n.s.- not significant; *p<0.05; **p<0.01). Scale bars correspond to 20 μm.</p
sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life <i>In Vivo</i>
<div><p>Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (<b>sFlt</b>) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These <i>in vivo</i> results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.</p></div
Synthesis of mvsFlt schematic.
<p>mvsFlt bioconjugates were synthesized using a 3-step reaction in which HyA was reacted with EDC and EMCH to create a thiol reactive HyA-EMCH intermediate. sFlt was then treated with 2-iminothiolane and reacted with the HyA-EMCH intermediate for the synthesis of the final mvsFlt bioconjugate.</p
Schematic demonstrating the proposed mechanism of mvsFlt action.
<p>A) sFlt (red, unconjugated) and mvsFlt (red conjugated blue chain of HyA) are injected into a diabetic retina where there is a high concentration of VEGF (green circles). B) After a given time, t, the majority of the sFlt has been cleared from the vitreous and VEGF is thus able to induce blood vessel growth. mvsFlt has a longer residence time in the vitreous and is able to bind and inhibit VEGF over much longer periods of time, leading to prolonged inhibition of retinal angiogenesis.</p