Tatiana Segura

A novel intracellular protein delivery platform based on single-protein nanocapsules

An average cell contains thousands of proteins that participate in normal cellular functions, and most diseases are somehow related to the malfunctioning of one or more of these proteins. Protein therapy, which delivers proteins into the cell to replace the dysfunctional protein, is considered the most direct and safe approach for treating disease. However, the effectiveness of this method has been limited by its low delivery efficiency and poor stability against proteases in the cell, which digest the protein. Here, we show a novel delivery platform based on nanocapsules consisting of a protein core and a thin permeable polymeric shell that can be engineered to either degrade or remain stable at different pHs. Non-degradable capsules show long-term stability, whereas the degradable ones break down their shells, enabling the core protein to be active once inside the cells. Multiple proteins can be delivered to cells with high efficiency while maintaining low toxicity, suggesting potential applications in imaging, therapy and cosmetics fields.

Anchorage of VEGF to the extracellular matrix conveys differential signaling responses to endothelial cells

Matrix-bound VEGF elicits more distinct vascular effects than soluble VEGF, including prolonged VEGFR2 activation with altered patterns of tyrosine activation and downstream enhancement of the p38/MAPK pathway.

The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels

Synthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites were present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000 μm) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100 μm).

Biocompatible Hydrogels by Oxime Click Chemistry

Oxime Click chemistry was used to form hydrogels that support cell adhesion. Eight-armed aminooxy poly(ethylene glycol) (PEG) was mixed with glutaraldehyde to form oxime-linked hydrogels. The mechanical properties, gelation kinetics, and water swelling ratios were studied and found to be tunable. It was also shown that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenchymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells demonstrated biocompatibility of the material.

siRNA applications in nanomedicine

The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease or the augmentation of tissue formation. RNAi is the sequence-specific gene silencing mediated by a 21-25 nucleotide double-stranded small interfering RNA (siRNA) molecule. siRNAs are incorporated into the RNA-induced silencing complex (RISC), which mediates mRNA sequence-specific binding and cleavage. Although RNAi has the potential to be a powerful therapeutic drug, its delivery remains a major limitation. The generation of nanosized particles is being investigated to enhance the delivery of siRNA-based drugs. These nanoparticles are generally designed to overcome one or more of the barriers encountered by the siRNA when trafficked to the cytosol. In this review, we will discuss recent advances in the design of delivery strategies for siRNA, focusing our attention to those strategies that have had in vivo success or have introduced novel functionality that allowed enhanced intracellular trafficking and/or cellular targeting. First, we will discuss the different barriers that must be overcome for efficient siRNA delivery. Second, we will discuss the approaches for siRNA delivery by size including direct modification of siRNAs (less than 10 nm), self-assembled particles based on cationic polymers and cationic lipids (100-300 nm), neutral liposomes (<200 nm), and macroscale matrices that contain naked siRNA or siRNA loaded nanoparticles (>100 microm). Finally, we will briefly discuss recent in vivo therapeutic success.

The chicken chorioallantoic membrane model in biology, medicine and bioengineering

Abstract The chicken chorioallantoic membrane (CAM) is a simple, highly vascularized extraembryonic membrane, which performs multiple functions during embryonic development, including but not restricted to gas exchange. Over the last two decades, interest in the CAM as a robust experimental platform to study blood vessels has been shared by specialists working in bioengineering, development, morphology, biochemistry, transplant biology, cancer research and drug development. The tissue composition and accessibility of the CAM for experimental manipulation, makes it an attractive preclinical in vivo model for drug screening and/or for studies of vascular growth. In this article we provide a detailed review of the use of the CAM to study vascular biology and response of blood vessels to a variety of agonists. We also present distinct cultivation protocols discussing their advantages and limitations and provide a summarized update on the use of the CAM in vascular imaging, drug delivery, pharmacokinetics and toxicology.

Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds.

The design of hyaluronic acid (HA)-based hydrogel scaffolds to elicit highly controlled and tunable cell response and behavior is a major field of interest in developing tissue engineering and regenerative medicine applications. This review will begin with an overview of the biological context of HA, which is needed to better understand how to engineer cell-matrix interactions in the scaffolds via the incorporation of different types of signals in order to direct and control cell behavior. Specifically, recent methods of incorporating various bioactive, mechanical and spatial signals are reviewed, as well as novel HA modifications and crosslinking schemes with a focus on specificity.

DNA Delivery from Matrix Metalloproteinase Degradable Poly(ethylene glycol) Hydrogels to Mouse Cloned Mesenchymal Stem Cells

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP-degradable peptides via Michael addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches: clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7 days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.

Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery

Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.

The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell-surface receptors. Although a myriad of possible cell fates stems from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor--soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.