Letter pubs.acs.org/journal/abseba
Controlling Angiogenic Response of Endothelial Cells via Intracellular Microparticle Depot containing Growth Factors Jing Luo,† Yuan Yuan,† and Debanjan Sarkar*,†,‡ †
Department of Biomedical Engineering and ‡Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States S Supporting Information *
ABSTRACT: Controlling of endothelial cell and its microenvironment is crucial to promote angiogenic response during tissue regeneration. However, current strategies are limited to genetic cellular engineering or matrix-based methods which are complex and highly variable. To overcome this, we engineer endothelial cells by intracellular loading of growth factor containing microparticles. These microparticle depots control the host cells and its microenvironment through intracellular and extracellular release of growth factors. Specifically, human endothelial cells engineered with vascular endothelial growth factor loaded poly lactic-co-glycolic acid (PLGA) microparticle show enhanced angiogenic response through extended endothelial network and sprouting. Enhancement of angiogenic response is attributed to autocrine and paracrine-endocrine like signaling from the growth factors. KEYWORDS: endothelial cells, microparticle, angiogenesis, cell-engineering
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with genetic changes and eliminate the need for an external source of growth factors. Biocompatible and biodegradable poly lactic-co-glycolic acid (PLGA) particles provide a feasible platform to engineer ECs because PLGA particles are widely used to deliver therapeutics in or via ECs, mostly using nanoparticles. However, for in situ functionalization of ECs with growth factor containing particles, microparticles are preferable compared to nanoparticles (which are more appropriate for systemic delivery) because nanoparticles are rapidly exocytosed because of their smaller size, leading to lower retention efficiency, and microparticles can deliver more payloads.10,12 In fact, ECs tend to internalize large number of microparticles mostly due to the phagocytotic nature of cells,11,13 and thus by using this approach, we can regulate the functional behavior of ECs for angiogenic response. In this study, we aimed to demonstrate that EC response can be engineered by stably functionalizing the cells using a microparticle-based approach and achieve a functional angiogenic response. To achieve this, we functionalized human umbilical vein endothelial cells (HUVECs) with PLGA microparticles which contain vascular endothelial growth factor (VEGF). PLGA particles were coated with poly-L-lysine to induce positive surface charge which facilitates cell internalization.1,10 Following internalization, intracellular delivery of VEGF through these microparticles depot is expected to promote the angiogenic response via autocrine and paracrine/
article-based cell engineering, where the cells are doped with nano- or microparticles containing therapeutic molecules,1−4 is a viable approach to control cell fate and their microenvironment as an alternate to genetic engineering and matrix-based tissue engineering strategies. Controlled release of therapeutic molecules from the particle engineered cells has demonstrated their efficacy to regulate cellular functions and their surrounding microenvironment.1,2 Endothelial cells (EC) are therapeutically relevant for vascular application during angiogenesis, and graft remodeling, and therefore regulating the fate of these cells is critical to define the therapeutic outcomes in these applications. While genetic engineering5,6 and various biomaterial strategies7,8 are predominantly used for these purposes, the limitations of these strategies arise from cellular toxicity related to genetic manipulation and from the complex molecular design of artificial matrices. This necessitates alternative and viable approach to regulate EC functions. ECs are widely characterized for their interactions with nano- and microparticles where it is demonstrated that ECs can internalize particles of different sizes and shapes using a wide range of mechanisms and the particles are capable of delivering therapeutic molecules including macromolecular proteins.9−11 Thus, by harnessing the potential of ECs to internalize particles and release the encapsulated payload, ECs can be functionalized with microparticles which can deliver growth factors to regulate their angiogenic response. This response is triggered by the intracellular release of angiogenic growth factor instead of genetic engineering to overexpress these factors or by the presence of extracellular soluble factors or matrices. Particleengineering can reduce the effect of cellular toxicity associated © XXXX American Chemical Society
Received: July 28, 2016 Accepted: August 10, 2016
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DOI: 10.1021/acsbiomaterials.6b00434 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
low concentration of particles (described in the Supporting Information), cells were engineered to achieve high and low particle content inside the cells, i.e., HP-Cells and LP-Cells, respectively. Twenty-four hours following the treatment with the particles, fluorescently imaged HP-Cells showed higher number of particles compared to LP-Cells (Figure 1D). Quantitative analysis (Figure 1E) shows almost all the cells, in both conditions, were functionalized with particles. However, the distribution of particles per cell in two conditions was different; majority of HP-cells contained more than 10 particles per cells (with a maximum of ∼25 particles per cell observed in some cells) whereas majority of LP-Cells contained 1−4 particles per cell. Because endothelial cells can engulf large number of microparticles,11,13 our results confirm that HUVECs can be loaded with a significantly high number of particles that can be easily engineered by altering particle concentration. Effective and stable internalization of these particles over a 4-day period was observed from 3D confocal images showing internalized PLGA particles in HUVECs (Figure 1F). Qualitatively, internalized PLGA particles evade the endosome to localize in cytoplasm, likely due to positive surface charges,10 but large aggregate of particles in HP-Cells tends to localize in endosomal/lysosomal compartment, observed from colocalization of fluorescence (Figure S1). PLGA microparticles can enter ECs involving both endocytic and phagocytic pathways, as these are known mechanisms for particle internalization.10,11 However, detailed analysis is required to identify the exact internalization mechanism and the time-dependent subcellular localization of particles. Overall, this process represents a facile approach to load microparticles inside HUVECs with controlled efficacy. To assess if particle engineering of HUVECs induces any adverse effect on cellular response or impairs the normal angiogenic response of endothelial cells, we characterized cell viability, proliferation, and endothelial tubulogenesis and sprouting of particle engineered HUVECs. For this purpose, we used fluorescent PLGA particles containing rhodamine, which can be easily detected with the cells. Particle loaded HUVECs showed similar viability (Figure 2A) relative to unmodified cells, however, statistically nonsignificant ∼10% reduction was observed in HP-Cells, likely because of membrane rupture from excessive interaction between positively charged particles and cell membrane.16 Both HPCells and LP-Cells proliferated at a similar rate compared to unmodified cells (Figure 2B). Significantly, during proliferation, the number of particle containing cells did not increase in either HP-Cell or LP-Cells implying that the particles were not transferred to daughter cells unlike some nanoparticles which are transferred to daughter during division.17 This is likely due to larger size of the particles, and essentially showing that microparticle engineering is not permanent. Next we examined if microparticle engineering of HUVEC impairs the inherent angiogenic character of endothelial cells. To assess this, we analyzed endothelial network formation (Figure 2C) and sprouting (Figure 2D) of particle modified cells embedded within Matrigel. Both HP-Cells and LP-Cells formed networks at 24 h (Figure S2A) and 48 h, where length of networks and their interconnection to form branched structure was not affected by particle modification and was comparable to that of unmodified HUVECs. Endothelial sprouting from cellular aggregates of HP-Cell and LP-Cell in Matrigel, examined at 24 h (Figure S2B) and 48 h showed similar response in terms of sprout length and sprouting area, compared to unmodified
endocrine route (Figure 1A). PLGA particles used to engineer HUVECs were fabricated with an encapsulated fluorescent dye
Figure 1. Particle engineering of endothelial cells. (A) Schematic representation of endothelial cell functionalization with positively charged poly lactic-co-glycolic (PLGA) microparticles to regulate host cell and its microenvironment. (B) Spherical PLGA particles from fluorescent images of rhodamine containing PLGA particles and SEM image. (C) Average size and zeta potential of PLGA microparticle with and without poly-L-lysine coating. (D) Bright-field and fluorescent microscopic images of human umbilical vein endothelial cells (HUVEC) functionalized with low particle (LP-Cell) and high particle (HP-Cell) content. PLGA particles are fluorescent from rhodamine and cell nuclei are stained with DAPI. (E) Quantification of particle functionalization of HUVEC from number of cells with particle and from number of particles (particle distribution) per cell for HP-Cell and LP-Cell. (F) Confocal microscopic images showing internalized PLGA microparticles in LP-Cell and HP-Cells which remains stably internalized over 4 day (scale bar: ∼2 μm).
rhodamine for visualization. Fluorescent images of rhodamine containing PLGA particles and scanning electron microscopy image of PLGA particles (Figure 1B) show nearly uniform size and shape of the particles. Particle size and surface charge measured from dynamic light scattering (DLS) (Figure 1C) show, prior to functionalization with poly lysine, particle size is around ∼2 μm and zeta potential is −42 mV because of carboxylic acid functionalities. Following surface adsorption of poly lysine, particle size increased to 2.9 μm with a zeta potential of +23 mV, indicating effective coating with poly lysine and positively charged surface. Poly lysine on PLGA particles can induce effective cellular internalization due to the electrostatic interaction with cell membrane and/or its influence on membrane lipid bilayer.1,14,15 HUVECs were engineered with these PLGA particles under adherent condition to maximize the interaction of particles with cell membrane through extended cell surface area. Using high and B
DOI: 10.1021/acsbiomaterials.6b00434 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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where free VEGF was added in the media. Quantitative analysis of network (Figure 3B) from 3-point junctions (a characteristics signature for branching network) and closed loop structure (indicative of endothelial lumen-like organization) shows HP-Cell formed robust networks with increased branching which formed closed lumens. Networks from HPCells were enhanced and sustained over 48 h compared to LPCells. Cellular aggregates of HP-Cells and LP-Cells showed increased sprouting in Matrigel compared to unmodified cells at 48 h (Figure 3C). Effect of particle engineering on sprouting was more pronounced at 48 h compared to 24 h response (Figure S3B). Compared to LP-Cells, HP-Cells showed more sprouting with increased sprout length and areas, which were significantly greater than unmodified cells. Similar to network behavior, sprouting of HP-Cells were comparable to free VEGF group. This finding suggests that enhanced angiogenic network and sprouts in particle engineered cells is due to the intracellular VEGF loaded particles which stimulated these responses in endothelial cells. Engineered enhancement of angiogenic response was comparable to free VEGF, and increased particle content enhanced and sustained these behaviors. In contrast to genetic viral based cellular engineering, direct intracellular loading of angiogenic growth factor through biodegradable microparticle induced autocrine and paracrine as well as endocrine like signaling to yield a robust angiogenic response. Enhancement of angiogenic performance of endothelial cells is due to controlled intracellular signaling of growth factors and its ability to influence the extracellular microenvironment through controlled regulation of endothelial secretome. To investigate the mode of action through which particle engineered HUVECs enhanced angiogenic response, we performed three separate analyses. When conditioned media from VEGF loaded PLGA particles (without cells) were transferred to HUVECs, cell proliferation was significantly higher compared to regular media (Figure 4A). The enhanced proliferation was comparable to free VEGF control group which indicates VEGF released from PLGA particle in the media (Figure 4A′) enhanced the proliferation in a concentration-dependent manner, i.e., higher particle content (HP-Cell) releases more VEGF to induce more proliferation than lower particle content (LP-Cell). Next we analyzed the proliferation of VEGF-loaded PLGA particle engineered HUVECs. Particle engineered cells proliferated more than unmodified cells and proliferation was higher in HP-Cell compared to LP-Cell but was comparable to free VEGF group (Figure 4B). This indicates intracellular delivery of VEGF through loaded particle enhanced HUVEC proliferation compared to control. Previous studies have also shown cytoplasmic delivery of proteins in endothelial cells through PLGA particles.10 Finally we examined the potential of extracellular release of VEGF from particle engineered HUVECs and its ability to induce proliferation of unmodified cells. To analyze this, we transferred conditioned media from particle engineered HUVECs (from HP-Cells and LP-Cells) to unmodified cells and characterized the proliferation. Media transferred from particle engineered HUVEC induced more proliferation than the media transferred from unmodified HUVECs, and particularly, media from HP-Cell induced proliferation to the same extent as free VEGF (Figure 4C). In fact, the conditioned media from particle engineered HUVECs contained excess VEGF in a concentration dependent manner (Figure 4C′), which shows extracellular release of
Figure 2. Characterization of particle engineered HUVEC. (A) Viability of particle engineered HUVEC for HP-Cell and LP-Cell and unmodified cells measured after 24 h of cell modification. (B) Proliferation of particle engineered HUVEC for HP-Cell and LP-Cell and unmodified cells measured over 5 days. For HP-Cell and LP-Cell, cells with and cell without particles were quantified during proliferation. (C) Endothelial network formation by PLGA particle engineered HUVEC with HP-Cell and LP-Cell and unmodified cells in Matrigel. Brightfield images of endothelial network at 48 h. Quantification of endothelial network from tube length and 3-point junctions measurement (* p < 0.05). (D) Endothelial sprouts from PLGA particle engineered HUVEC aggregates with HP-Cell and LPCell and unmodified cells in Matrigel. Bright-field images of endothelial sprouts at 48 h. Quantification of endothelial sprouts from sprout length and area measurement (* p < 0.05).
cells. These analyses showed intracellular localization of PLGA microparticles does not impair angiogenic character of HUVECs, and therefore can serve as a platform for delivery of growth factors. To deliver angiogenic growth factors, we loaded VEGF within PLGA microparticle using standard double-emulsion approach with a loading efficiency of ∼15%. HUVECs were engineered with VEGF containing PLGA particles as described earlier with high (HP-Cells) and low (LP-Cells) concentration of particles. Particle treated cells were examined for their angiogenic responses through network formation and sprouting response in Matrigel (Figure 3). Matrigel provides a supportive matrix for endothelial cells to form angiogenic network and sprouts, due to the presence of natural extracellular matrix components. HP-Cells and LP-Cells formed extensive endothelial networks with interconnected morphologies compared to unmodified cells at 48 h (Figure 3A) and at an earlier time point (24 h, Figure S3A). Significantly, endothelial networks of HP-Cell and LP-Cell were similar to VEGF control C
DOI: 10.1021/acsbiomaterials.6b00434 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 3. Enhancement of angiogenic response of HUVECs functionalized with vascular endothelial growth factor (VEGF) containing PLGA microparticles. VEGF-PLGA particle engineered HUVEC with high particle (HP-Cell) and low particle (LP-Cell) content and control groups of unmodified cells in free VEGF (VEGF) and in ECM (cell). (A) Endothelial network at 48 h from brightfield images in Matrigel. (B) Quantification of endothelial network from number of 3-point junctions and closed loops at 24 and 48 h (* p < 0.05). (C) Endothelial sprouts from cellular aggregates at 48 h from brightfield images in Matrigel. (D) Quantification of endothelial sprouts from sprout length and area measurement at 24 and 48 h (* p < 0.05).
feasibility of delivering protein based therapeutic molecules into the external environment using particle based approach.22 Collectively, these mechanisms contributed to enhanced angiogenic response and provide a facile approach to modulate endothelial angiogenesis with growth factor containing microparticle depot. The goal of this work was to demonstrate the feasibility of loading growth factor containing microparticle as depot in endothelial cells and to control endothelial angiogenesis using these particle-containing cells. This strategy enhanced angiogenesis of HUVECs by inducing intracellular and extracellular release of VEGF. Designing a nongenetic and non-matrix-based cellular engineering approach to regulate angiogenic response of endothelial cells provides a platform to regulate endothelial cell behavior through delivery of repertoire of therapeutic molecules. From a therapeutic standpoint, these particle engineered HUVECs can control cellular functions and the microenvironment during tissue regeneration and repair e.g. promoting capillary angiogenesis or endothelialization of grafts. In particular, these engineered cells can have greater reparative potency through enhanced survival and improved functioning under inflammatory “growth factor deprived” conditions because of their in-built cargo, which can provide the therapeutic growth factors. Particle-in-cell strategy provides a local control of endothelial phenotype and their angiogenic behavior without requiring soluble growth factors, which in vivo, has shown limited efficacy due to lower retention and
VEGF from VEGF-PLGA particle engineered HUVECs. In endothelial cells, VEGF can act through intracellular autocrine stimulation or extracellular paracrine and endocrine mechanism through the receptors on cell surface.18,19 Intracellular release of VEGF from microparticles in particle engineered HUVECs enhanced proliferation and angiogenic response through an autocrine manner. In addition, to influence the external environment, extracellular release of VEGF from these particle engineered HUVECs acted in a paracrine and endocrine like mode. Since, the proliferation of particle containing HUVECs do not transfer the particles to daughter cells, it is likely that intracellularly released VEGF acts in a autocrine manner to stimulate the host cell and extracellularly released VEGF stimulated the neighboring cells in a paracrine and endocrine like manner. Extracellular release of VEGF showed enhanced time-dependent increase for LP-and HP-Cells compared to free VEGF-PLGA particles (Figure 4). Particularly for low concentration particles, i.e., LP-Cells, this response was obvious indicating the involvement of intracellular mechanisms to release the VEGF from particle modified cells. Although the mechanism of extracellular release of VEGF from particleengineered HUVECs is not obvious, it is likely that combination of different cellular machineries, e.g., permeability glycoprotein, exosomes, can promote the transport of growth factor from intracellular to extracellular environment.20,21 In fact, extracellular release of macromolecular peptides from particle-engineered mesenchymal stem cells has shown the D
DOI: 10.1021/acsbiomaterials.6b00434 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Functioning of HUVECs functionalized with vascular endothelial growth factor (VEGF) containing PLGA microparticles. (A) Cell proliferation measured after transferring of particle conditioned media from high (HP-Cell) and low (LP-Cell) particle content to unmodified HUVECs. Particle-free media with free VEGF (VEGF) and without VEGF (cell) are transferred as control. (A′) Amount of VEGF released from VEGF- PLGA particles normalized with respect to blank particle at day 2 and day 4. (B) Cell proliferation of VEGF-PLGA particle engineered HUVEC with high particle (HP-Cell) and low particle (LP-Cell) content and control groups of unmodified cells in free VEGF (VEGF) and in ECM (cell). (C) Cell proliferation measured after transferring of conditioned media from VEGF-PLGA particle engineered HUVEC with high (HP-Cell) and low (LP-Cell) particle content to unmodified HUVECs. Media transferred from unmodified HUVECs (cells) and media with free VEGF (VEGF) were used as controls. (C′) Amount of VEGF released from VEGF- PLGA particle functionalized HUVECs normalized with respect to unmodified HUVECs at day 2 and day 4. All cell proliferations were measured at day 2 and 4 (* p < 0.05).
Notes
smaller half-life. The critical factors to maximize the utility of this system depend on increased particle uptake and retention as well as efficient escape of the endosomal compartment to avoid denaturing of the growth factors, as large aggregates of microparticles can localize in lysosome. Using positively charged PLGA particle with poly-L-lysine ensures effective cellular internalization and also can avoid endosomal routes to deliver the growth factors both intracellularly and extracellularly,10 but an optimization of this approach is essential for maximum efficacy. Long-term effectiveness of this approach should also be analyzed in terms of final fate of these particles and the particle engineered cells. Additionally, their therapeutic efficacy should be established with relevant in vivo models to expand the utility of this approach. Therapeutically, this approach can provide a clinically feasible system for cellbased therapies where direct or systemic delivery of selfsupporting particle-engineered cells can control their functions and the microenvironment.
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The authors declare no competing financial interest.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00434. Materials and methods and supplementary data (PDF)
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ACKNOWLEDGMENTS
D.S. acknowledges UBF for financial support.
AUTHOR INFORMATION
Corresponding Author
*E-mail: debanjan@buffalo.edu. Ph: 716-645-8497. Fax: 716645-2207. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. E
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