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Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery

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Abstract

Introduction

Biomaterials can provide localized reservoirs for controlled release of therapeutic biomolecules and drugs for applications in tissue engineering and regenerative medicine. As carriers of gene-based therapies, biomaterial scaffolds can improve efficiency and delivery-site localization of transgene expression. Controlled delivery of gene therapy vectors from scaffolds requires cell-scale macropores to facilitate rapid host cell infiltration. Recently, advanced methods have been developed to form injectable scaffolds containing cell-scale macropores. However, relative efficacy of in vivo gene delivery from scaffolds formulated using these general approaches has not been previously investigated. Using two of these methods, we fabricated scaffolds based on hyaluronic acid (HA) and compared how their unique, macroporous architectures affected their respective abilities to deliver transgenes via lentiviral vectors in vivo.

Methods

Three types of scaffolds—nanoporous HA hydrogels (NP-HA), annealed HA microparticles (HA-MP) and nanoporous HA hydrogels containing protease-degradable poly(ethylene glycol) (PEG) microparticles as sacrificial porogens (PEG-MP)—were loaded with lentiviral particles encoding reporter transgenes and injected into mouse mammary fat. Scaffolds were evaluated for their ability to induce rapid infiltration of host cells and subsequent transgene expression.

Results

Cell densities in scaffolds, distances into which cells penetrated scaffolds, and transgene expression levels significantly increased with delivery from HA-MP, compared to NP-HA and PEG-MP, scaffolds. Nearly 8-fold greater cell densities and up to 16-fold greater transgene expression levels were found in HA-MP, over NP-HA, scaffolds. Cell profiling revealed that within HA-MP scaffolds, macrophages (F4/80+), fibroblasts (ERTR7+) and endothelial cells (CD31+) were each present and expressed delivered transgene.

Conclusions

Results demonstrate that injectable scaffolds containing cell-scale macropores in an open, interconnected architecture support rapid host cell infiltration to improve efficiency of biomaterial-mediated gene delivery.

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Abbreviations

HA:

Hyaluronic acid

HA-SH:

Thiolated hyaluronic acid

PEG:

Poly(ethylene glycol)

FLuc:

Firefly luciferase

FLuc-LV:

Firefly luciferase encoding lentivirus

PEG-VS:

Vinyl sulfone-terminated poly(ethylene glycol)

PEG-mal:

Maleimide-terminated poly(ethylene glycol)

DTT:

Dithiothreitol

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Acknowledgments

The authors would like to acknowledge funding for this work from a National Science Foundation CAREER Award 1653730 (SKS), a UCLA Henry Samueli School of Engineering and Applied Sciences (HSSEAS) Faculty Research Grant (SKS) and a UCLA Faculty Career Development Award (SKS). We thank the UCLA Tissue Pathology Core Laboratory (TPCL) for cryosectioning and hematoxylin and eosin staining, the UCLA Crump Institute for Molecular Imaging for use of the IVIS imaging system, and the UCLA Molecular Instrumentation Center for use of proton NMR facilities. Confocal laser scanning microscopy was performed at the California NanoSystems Institute Advanced Light Microscopy/Spectroscopy Shared Resource Facility at UCLA, supported with funding from NIH-NCRR shared resources grant (CJX1-443835-WS-29646) and NSF Major Research Instrumentation Grant (CHE-0722519).

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical Standards

All animal studies were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the UCLA Institutional Animal Care and Use Committee. No human studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Bioengineering, University of California Los Angeles, Los Angeles, CA, 90095, USA

    Arshia Ehsanipour, Tommy Nguyen, Tasha Aboufadel, Mayilone Sathialingam, Phillip Cox, Weikun Xiao, Christopher M. Walthers & Stephanie K. Seidlits

  2. Broad Stem Cell Research Center, University of California Los Angeles, Los Angeles, CA, 90095, USA

    Stephanie K. Seidlits

  3. Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA, 90095, USA

    Stephanie K. Seidlits

  4. Brain Research Institute, University of California Los Angeles, Los Angeles, CA, 90095, USA

    Stephanie K. Seidlits

  5. Center for Minimally Invasive Therapeutics, University of California Los Angeles, Los Angeles, CA, 90095, USA

    Stephanie K. Seidlits

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  1. Arshia Ehsanipour
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Stephanie K. Seidlits is an Assistant Professor in the Department of Bioengineering at the University of California Los Angeles (UCLA). Dr. Seidlits received her Ph.D. in Biomedical Engineering from the University of Texas at Austin in 2010. Under the mentorship of Dr. Christine Schmidt and Dr. Jason Shear, her dissertation research focused on developing biomaterial-based strategies to promote nerve regeneration. Dr. Seidlits received a National Science Foundation (NSF) Integrated Graduate Education and Research Trainee (IGERT) Fellowship and a Scholar Award from the Philanthropic Education Organization (PEO). Dr. Seidlits then completed a post-doctoral fellowship at Northwestern University under the mentorship of Dr. Lonnie Shea, where she worked on several projects including development of biomaterial scaffolds with gene delivery capabilities for spinal cord injury repair and high-throughput arrays for monitoring dynamic activities transcription factors. During this time, she received the Rice University Outstanding Bioengineering Undergraduate Alumna Award, Northwestern University Institute for BioNanotechnology in Medicine-Baxter Early Career Award and a National Institutes of Health (NIH) F32 Ruth L. Kirchstein National Research Service Award (NRSA) for Post-Doctoral Training under the co-mentorship of Dr. Lonnie Shea and Dr. Aileen Anderson. Dr. Seidlits started her independent lab at UCLA in 2014, where her research uses biomaterial platforms to better understand the mechanisms underlying dysfunction and disease in central nervous system tissues and ultimately to develop new therapies. She has received an NSF CAREER Award, a UCLA Hellman Fellow Award, an American Brain Tumor Association Discovery Award and the 2019 Society for Biomaterials Young Investigator Award.

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This article is part of the CMBE 2019 Young Innovators special issue.

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12195_2019_593_MOESM1_ESM.tif

Figure S1 Schematics of FITC-dextran incubation and confocal reconstruction of scaffolds (A) and hydraulic conductivity (B) experiments. Scaffolds were incubated in PBS containing high molecular weight FITC-dextran before imaging by confocal microscopy. Confocal stacks were taken near the surface of the scaffolds and imaged to a depth of 200 µm (A). Hydraulic conductivity was measured by forming scaffolds on top of a permeable membrane within a custom 3D printed device. PBS was placed on top of the scaffolds and the change in height of PBS was measured before and after at least 3 hours of incubation (B). (TIFF 4181 kb)

12195_2019_593_MOESM2_ESM.tif

Figure S2 Size distributions for microparticles produced by water-in-oil emulsion. PEG-MPs (A) are notably smaller and have a tighter distribution than HA-MPs (B) with a mean diameter of 20 ± 9 µm compared to that of 42 µm ± 23 µm for HA-MPs (C). Microparticle size distributions were assessed across three different batches and at least 500 microparticles of each type were cumulatively measured. Scale bars = 50 µm (TIFF 72170 kb)

12195_2019_593_MOESM3_ESM.tif

Figure S3. Rheological testing of NP-HA, PEG-MP, and HA-MP scaffolds. Rheological testing of NP-HA, PEG-MP, and HA-MP scaffolds show non-significant differences in storage moduli of NP-HA and HA-MP scaffolds. PEG-MP scaffolds had significantly greater moduli than either NP-HA or HA-MP scaffolds. Error bars represent standard deviation (*p<0.05, n=5, Kruskal Wallis test). (TIFF 21524 kb)

12195_2019_593_MOESM4_ESM.tif

Figure S4. Nuclei density at scaffold centers were measured at the radial center of scaffolds, and at least three sections per scaffolds were used for analysis. Immunostained tissue sections were imaged for nuclei (A, B), and cell density was calculated using thresholding and object analysis in CellProfiler to obtain cell counts over the section (C, D). Objects outlined in green are considered positive nuclei, whereas objects outlined in purple are excluded for not meeting size criteria. Scale bars = 500 µm (A, C) or 100 µm (B, D). (TIFF 137176 kb)

12195_2019_593_MOESM5_ESM.tif

Figure S5. Densities of nuclei were measured through the scaffold boundary in radial sections. Measurements were performed in radial sections and analysis were performed based on nuclei stained area on three separate regions per image and three images per condition. Immunostained tissue sections were imaged for nuclei (A), thresholded, and nuclei area per scaffold area were quantified (B). Scaffolds are indicated within the dashed lines. Scale bars = 200 µm. (TIFF 63386 kb)

12195_2019_593_MOESM6_ESM.tif

Figure S6. Differences in viral load did not affect cell infiltration or types of infiltrating cells within scaffolds. Quantification of cell density within all scaffolds showed no significant difference depending on viral load (A). Similarly, immunostaining did not exhibit any notable differences (B). Scaffolds are indicated on the left-hand side of the dashed lines. (N = 4, Unpaired t-test with Welch's correction). Scale bars = 200 µm. (TIFF 90442 kb)

12195_2019_593_MOESM7_ESM.tif

Figure S7. Quantification of immunostaining expression. Expression was quantified for immunostained sections (A) by applying a mask (B) around the scaffold area, applying it to the F4/80, CD31, or ERTR7 immunostained image (C), and quantifying staining density using CellProfiler software. Scaffolds are indicated within the dashed lines. Scale bars = 200 µm. (TIFF 116557 kb)

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Ehsanipour, A., Nguyen, T., Aboufadel, T. et al. Injectable, Hyaluronic Acid-Based Scaffolds with Macroporous Architecture for Gene Delivery. Cel. Mol. Bioeng. 12, 399–413 (2019). https://doi.org/10.1007/s12195-019-00593-0

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