Accepted Manuscript Pathology-targeted cell delivery via injectable micro-scaffold capsule mediated by endogenous TGase Chunxiao Qi, Yaqian Li, Patrick Badger, Hongsheng Yu, Zhifeng You, Xiaojun Yan, Wei Liu, Yan Shi, Tie Xia, Jiahong Dong, Chenyu Huang, Yanan Du PII:
S0142-9612(17)30101-1
DOI:
10.1016/j.biomaterials.2017.02.021
Reference:
JBMT 17950
To appear in:
Biomaterials
Received Date: 13 December 2016 Revised Date:
15 February 2017
Accepted Date: 16 February 2017
Please cite this article as: Qi C, Li Y, Badger P, Yu H, You Z, Yan X, Liu W, Shi Y, Xia T, Dong J, Huang C, Du Y, Pathology-targeted cell delivery via injectable micro-scaffold capsule mediated by endogenous TGase, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.02.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Pathology-targeted Cell Delivery via Injectable Micro-scaffold Capsule Mediated by Endogenous TGase a
Chunxiao Qi , Yaqian Li a
a,b,
c,
a,
a
a
*, Patrick Badger *, Hongsheng Yu *, Zhifeng You , Xiaojun Yan ,
d
d
e
f
a,b, †
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Wei Liu , Yan Shi , Tie Xia , Jiahong Dong , Chenyu Huang , Yanan Du
Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China;
b
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China;
c
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA;
d
Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China;
e
Department of Hepatobiliary Surgery, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing 102218,
China;
f
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a
Department of Plastic, Reconstructive and Aesthetic Surgery, Beijing Tsinghua Changgung Hospital, Tsinghua
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University, Beijing 102218, China.
* These authors contributed equally to this work.
†
Corresponding author. Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing,
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Abstract:
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100084, PR China. Email:
[email protected]; Tel: +86 10 62781691
Targeted cell delivery to lesion sites via minimally invasive approach remains an unmet need in regenerative medicine to endow satisfactory therapeutic efficacy and minimized side-effects. Here, we rationally designed a pathology-targeted cell delivery strategy leveraging injectable micro-scaffolds as cell-loading capsule and endogenous tissue transglutaminase (TGase) at lesion site as adhesive. Up-regulated TGase post-liver injury catalyzed chemical bonding between the glutamine and lysine residues on liver surface 1
ACCEPTED MANUSCRIPT and micro-scaffolds both ex vivo and in vivo, facilitating sufficient adhesion on the pathological liver. Upon intraperitoneal injection, Mesenchymal Stem Cell-loaded capsules, exhibiting cell protection from shear-induced damage and post-transplantation
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anoikis, adhered to the CCl4-treated liver with a hundred-fold improvement in targeting efficiency (70.72%) compared to free-cell injection, which dramatically improved mice survival (33.3% vs. 0% for free-cell therapy) even with low-dosage treatment. This unique
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transforming cell therapy for refractory diseases.
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and widely-applicable cell delivery mechanism and strategy hold great promise for
Keywords: Targeted cell delivery; Micro-scaffold capsule; Endogenous TGase; pathological liver; Cell therapy
Introduction
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Along with pharmaceuticals and medical devices, cell therapy is deemed to be the next therapeutic pillar of global healthcare which has shown efficacy in alleviating symptoms of many end-stage and refractory diseases in animal models and clinical trials [1]. Just as
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drug delivery technologies (e.g. capsule and tablet-based formulation) have transformed
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the entire pharmaceutical industry to achieve improved drug efficacy and safety, a reliable cell delivery system is critical to achieve reproducible and satisfactory clinical outcomes, which is desired to transfer cells to targeted lesion site and enhance cells’ viability and therapeutic effects [2].
Common delivery routes in cell therapy are based on either systematic administration (e.g. intravenous or intraperitoneal injection) [2, 3], or directed injection of cells into the damaged tissues [4], whereas the therapeutic benefits of the transplanted cells are 2
ACCEPTED MANUSCRIPT confined due to uncontrolled distribution, severe cell loss and death. Taking Mesenchymal Stem Cell (MSC)-based therapy for liver cirrhosis as an example, MSCs via intravenous transfusion are ectopically distributed to almost all organs within 2 h in patients; most cells
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experience anoikis within 2 days, hence leaving only approximately 0.02% surviving cells and 10% of them in liver, indicating poor targeting and engraftment efficiency [5].
To overcome this hurdle, current strategies for improving cell targeted-delivery include
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genetic modification, cytokine priming of cells [6, 7], and biomaterial-assisted implantation
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[8]. For instance, genetically-modified or cytokines (e.g. IL-3, IL-6)-primed MSCs with upregulated expression of CXCR4, a receptor that specially interacts with SDF-1α in injured liver, exhibited elevated binding and accumulation to fibrotic liver [6, 7, 9]. However, potential safety issues associated with genetic modification and un-predictable cell
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functional alteration related with cytokine priming have hindered clinical translation of these cells as therapeutic agents [4, 10]. Alternatively, cells could be loaded in hydrogel or scaffold vehicles to assist their implantation and retention at targeted tissue or organ (e.g.
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liver parenchyma) [11-14]. These vehicles usually require surgical interventions which
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cause secondary damages to the targeted tissue, and are thus not a viable option for patients with end-stage symptoms (e.g. liver cirrhosis). Therefore, there is an urgent need for suitable ‘cell capsules’, analogous to their pharmaceutical counterparts, to achieve targeted cell delivery without interfering the delivered cells or the targeted tissues/organs. To achieve this goal, we rationally designed a micro-scaffold-based ‘capsule’ for targeted cell delivery to liver by exploiting the unique feature of injured liver, namely the up-regulated secretion of tissue transglutaminase (TGase) from necrotic hepatocytes [15, 3
ACCEPTED MANUSCRIPT 16]. TGase is a Ca2+-dependent enzyme which catalyzes the cross-link between side chains of lysine (ε-amine) and glutamine (γ-acyl). The established ε(γ-glutamyl)lysine bonds result in post-translational modifications of proteins involved in a variety of cellular
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processes, including adhesion, migration, growth, apoptosis, differentiation, and extracellular matrix (ECM) organization. The impact of TGase on these processes implicates the involvement of this protein in various physiological responses and
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pathological states, contributing to wound healing, inflammation, autoimmunity,
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neurodegeneration, vascular remodeling, tumorigenesis/metastasis, and tissue fibrosis. The protein, localized in multiple intracellular compartments, cell surface and ECM, is known to exist ubiquitously at basal level in all types of mammalian tissue, but would be upregulated upon tissue injury. In particular, within 24 h upon liver injury, upregulated
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TGase bonded to ECM could remodel ECM components at pathological sites [17, 18]. Mimicking the in vivo function as a cross-linker of proteins, TGase has been widely used as a crosslinking agent for biomaterials containing glutamine (Q) and lysine (K) residues
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(e.g. collagen [19, 20], gelatin [21]) to fabricate scaffolds via ε(γ-glutamyl)lysine bonds as
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surgical adhesives in medical industry and cling films in food industry [22-25]. Considering the physiological function and industrial application of TGase, we hypothesized endogenous TGase bonded in pathological tissues (e.g. injured liver) would provide a molecular anchor for the adhesion of materials containing Q and K residues. We thereby rationally designed micro-scaffolds containing Q and K residues as cell delivery capsules
to
facilitate
targeted
cell
delivery
by
preferentially
adhering
to
TGase-overexpressed liver but not normal liver (Fig. 1A). 4
ACCEPTED MANUSCRIPT Materials and Methods Gelatin micro-cryogel scaffold fabrication. Gelatin micro-cryogel scaffolds (GS) were prepared in PMMA microstencil array chips, each containing 600 circular micro-wells with
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diameters of 400 µm or 800 µm. Chips were fabricated using a laser fabrication system (Rayjet) and treated using a plasma cleaner (Mycro Technologies) to increase hydrophilicity. Gelatin precursor solution (7% w/v gelatin and 0.5% w/v Odex in diH2O if
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labeling is necessary) was dissolved at 50 °C in a water bath and then chilled on ice for 5
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min, after which 70 µL 0.5% glutaraldehyde (GA) solution was added with mild stirring. 250 µL of the resulting precursor solution was pipetted onto the upper surface of each microstencil array chip and manually scraped across the chip surface to ensure even distribution. The filled microstencil array chip then underwent cryogelation for 16 h at
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−20 °C and was subsequently lyophilized for 30 min (Boyikang). GSs were harvested using a PMMA ejector pin array fabricated with a desktop 3D milling machine (MDX-40A; Roland DG). GSs were washed with 0.1 M NaBH4 to neutralize unreacted aldehyde, then
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washed thoroughly with diH2O. They were then harvested into a dish in an even
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monolayer, lyophilized, and vacuum-packaged for later use in characterization and cell loading [26].
PEG micro-cryogel scaffold fabrication. PEGDA (4000) precursor solution was prepared by dissolving 10% w/v PEGDA, 1% w/v N-acryloxysuccinimide (NAS), 0.5% w/v ammonium persulfate (APS), and 0.15% w/v N,N,N',N'-tetramethylethylenediamine (TEMED) in cold diH2O maintained on ice. PEG micro-cryogel scaffolds were then fabricated using the same procedure used to fabricate gelatin micro-cryogel scaffolds, 5
ACCEPTED MANUSCRIPT with the exception that they were cryogelated for 20 h at -20 °C before lyophilizing and washed only with diH2O. After lyophilizing, PEG-NAS scaffold was added to 100 µL 2% w/v solution of either functional polypeptide or control polypeptide and incubated at 37 °C
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for 2 h, facilitating the modification of PEG micro-cyogel scaffolds (PS) as functional PS (FPS) or control PS (CPS) respectively. 500 mM ethanolamine solution was used to block unreacted NAS; excess ethanolamine was removed by washing with PBS buffer. FPSs
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and CPSs were then harvested into a dish in an even monolayer, lyophilized, and
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vacuum-packaged for later use in characterization and cell culturing [1].
GelMa micro-cryogel scaffolds fabrication. GelMA (methacrylated gelatin) precursor solution was prepared by dissolving 6% w/v GelMA, 0.3% APS, and 10% TEMED in cold diH2O. GelMA micro-croygel scaffolds were then fabricated using the same procedure
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used for PEG micro-cryogel scaffold.
Ex vivo and in vivo scaffold adhesion experiments. To induce liver injury in mice, a 1:4 mixture of CCl4 in mineral oil was administered in a
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single dose of 7.5 mL per kg of body weight via intraperitoneal injection (injured liver
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group). To inhibit CCl4-induced TGase up-regulation, mice were treated firstly with cystamine to inhibit TGase expression (at a dose of 112 mg/kg per day), and 2 days later injured with CCl4 (TGase-inhibited liver group). For ex vivo adhesive experiments, injured, TGase-inhibited, and normal livers were excised after mice were anesthetized and incubated with GSs, FPSs, CPSs, or FITC-labeled FPP solution at 37 °C for 2 h. After washing for 30 min, the livers were imaged using an IVIS fluorescence imaging system (Caliper Life Sciences). For in vivo experiments, GSs, FPSs, CPSs were injected 6
ACCEPTED MANUSCRIPT intraperitoneally, and the excised livers were imaged one day after using an IVIS fluorescence imaging system. To ensure fair comparison of targeting effect, influence of the process of i.p. delivery operation was minimized by injecting the micro-scaffolds only
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when the needle tip reached the processus xiphoideus. Cell loading and viability assessment.
Before cell loading, harvested and dried GSs were sterilized with an ethylene oxide
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sterilization system (AN74j/Anprolene; Anderson Sterilization). Human adipose-derived
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mesenchymal stem cells (MSCs) were isolated and cultured in mesenchymal stem cell growth medium (BioWit Technologies) according to a previously reported protocol [27]. A 60 µL MSCs suspension (8×106/mL) was subsequently pipetted onto 600 tightly packed GSs, into which it was automatically absorbed thanks to GSs porous structure. The GSs
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were then maintained in a humidified chamber and incubated at 37 °C for 2 h to allow for cell adhesion. Culture medium was then added for long-term culture. CellTiter-Blue® cell viability assay was applied to determinate cell loading-capacity and proliferation within
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each microcryogel: first, a standard curve was made to determine the linear relationship
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between fluorescence intensity and cell number. Then, GSs were cultured for varying times under otherwise identical conditions, and cell viability was assessed. Cell number per micro-scaffold was calculated according to the established standard curve. Live/dead assay was also performed for visualization of cell viability. Targeting efficiency quantification and gene expression analysis. Cell or tissue RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. One microgram of RNA was reversely transcribed using 7
ACCEPTED MANUSCRIPT PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa). 2.5 ng of cDNA, as well as forward and reverse primers, was added to PCRs, which were carried out with SYBR Premix Ex Taq (TaKaRa). The resulting amplification was monitored with the CFX96
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Real-Time System (Bio-Rad). The expression of genes of interest was normalized against the reference gene GAPDH in all samples, and relative gene expression was analyzed. Primers for all genes are listed in Supplementary Table 3.
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Following RNA extraction, the remaining aqueous layer was removed for genomic DNA
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extraction. 400 µL back extraction buffer was added to each sample which is an aqueous solution of 4 M guanididium thiocyanate, 50 nM sodium citrate and 1 M Tris. After vortexing, the samples were centrifuged at 16,000×g for 15 min. The aqueous phase was transferred to a new tube and DNA precipitated with 400 µL of isopropanol. As in RNA
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analysis, 2.5 ng of genomic DNA was used as template for real time PCR [28]. For samples surrounding GCs adhesion, 100 mg liver tissue along with the adhered GCs (always located at sites surrounding gall bladder) was used for DNA extraction. As to
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samples distant from GCs adhesion, 100 mg liver tissue from right lobe (without GCs
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adhesion) was used for DNA extraction. Samples for FCs group were collected at same sites as in GCs group.
In vivo treatment of mice with acute liver failure. BALB/c nude mice were maintained in a specific pathogen-free animal facility at the Animal Center of Tsinghua University. The mice were housed in microisolator cages. Mice (Purchased by the Animal Center) were used for experiments at 7 to 8 weeks of age. All Animal experiments were performed in strict accordance with standards approved by the 8
ACCEPTED MANUSCRIPT Animal Ethics Committee of Tsinghua University. Experimental protocols were approved by the Animal Care Committees at Tsinghua University. BALB/c nude mice were randomized to one of seven groups: (i) control (no treatment of
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CCl4), (ii) FC group (treated with CCl4 and 106 cell suspension), (iii) Low FC group (treated with CCl4 and 105 cell suspension), (iv) GC group (treated with CCl4 and 5×105 cell suspension), (v) Low GC group (treated with CCl4 and 105 cell suspension), (vi) sham
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(treated with CCl4 and PBS), (vii) PT group (treated with CCl4, prior treatment). Mice
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received one dose of 7.5 mL/Kg 1:4 mixture of CCl4 in mineral oil were intraperitoneally injected free cell suspension or cell capsule suspension. 5 and 14 days later, mice were sacrificed to analysis therapeutic efficacy. Livers were isolated, fixed in 4% paraformaldehyde, and embedded in paraffin. Standard H-E stained sections and Sirius
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Red stained sections were examined and scored. Plasma alanine amino transferase (ALT), aspartate aminotransferase (AST) and albumin (ALB) levels were measured using a Mindray BS-200 analyzer (Mindray ®, Shenzhen, China).
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Statistical Analysis.
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Graphpad Prism software was used to perform all statistical analyses. All experiments were performed a minimum of three times. Data are presented as mean ± SD. One-way ANOVA was used to determine the significance of observed differences between test groups. A value of P < 0.05 was considered statistically significant. No statistical methods were used to predetermine sample sizes for in vivo experiments, and in vivo experiments were not randomized.
Results 9
ACCEPTED MANUSCRIPT TGase catalyzes bonding between micro-scaffold-based capsules in vitro. Cryogelated micro-scaffolds were applied as cell delivery capsules, owing to their superior elasticity and injectability to protect cells from shear damage during injection as well as
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tenability of physiochemical properties proved as beneficial 3D cellular niches [26, 27, 29] (Fig. 1B-E). To investigate the specific interactions between TGase and capsules, polyethylene glycol (PEG), which is well known for its anti-fouling features in minimizing
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non-specific absorptions and interactions [30], was selected as the polymer for
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micro-scaffold fabrication. A Q- and K-residue containing functional polypeptide (GQLKHLEQQEG, FPP) (Fig. S1A) was successfully grafted to form functional PEG micro-scaffolds (FPS) (Fig. 1B,C; Fig. S1C), as confirmed by Fourier Transform Infrared (FTIR) spectroscopy [31] (Fig. 1F). A control polypeptide (GNLRHLENNEG, CPP) (Fig.
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S1B), containing N and R residues in replacement of Q and K residues respectively (with no change in overall charge and solubility), was used in fabricating control PEG micro-scaffolds (CPS) (Fig. S1D,E). Meanwhile gelatin-based micro-scaffolds (GS) (Fig.
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1D,E; Fig. S1F), containing K residues (Fig. 1G; Fig. S1G,H), were also chosen to confirm
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the general applicability of TGase-mediated adhesion of biomaterials containing Q and K residues.
TGase-catalyzed crosslink of Q and K residues was first verified at the molecular level by employing atomic force microscopy (AFM), which can measure the force to break TGase-mediated chemical bonding. As schematized in Fig. 2A, the retractile force required to detach silica bead (modified with soluble FPP or gelatin) glued on AFM cantilever from soluble gelatin coated coverslips was dependent on TGase concentration. 10
ACCEPTED MANUSCRIPT Such force was diminished upon incubation with soluble K amino acid due to competitive binding. Likewise, modification of silica beads with CPP yielded negligible retractile force, indicating the specific catalytic function of TGase on bond formation between Q and K
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residues (Fig. 2B, Fig. S2A). Next, we investigated the TGase-mediated adhesiveness between Q and K residues on the macro-scale using a friction dynamometer to test the adhesive force between two
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TGase-bound scaffolds (both with surface areas of 2×4 cm2) (Fig. S2B,C). Adhesive force
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between FPSs and GSs was shown to correlate with increased reaction time and FPP concentration (Fig. 2C,D). Similar observations were made for adhesions between two GSs, and soluble lysine (K) inhibited macro-scale adhesive force as it did in the AFM tests (Fig. 2E-G) mediated
pathology-preferential
adhesion
of Q-
and
K-containing
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TGase
micro-scaffolds to injured liver ex vivo.
To verify the presence and indicate Ca2+-dependent transglutaminase activity of the
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TGase in injured livers, bonding of rhodamine-labeled FPP to excised injured livers
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(induced by CCl4), TGase-inhibited livers (TGase synthesis was inhibited by cystamine treatment prior to CCl4 induction [32]), and normal livers were assessed ex vivo respectively via IVIS fluorescence imaging system [33]. Rhodamine signal was significantly higher (2.82- and 3.23-fold increase in fluorescent area, 1.5- and 2.09-fold increase in total intensity, 4.11- and 13.51-fold increase in average intensity) for injured livers (Fig. 3A,B; Fig. S3A-C) than those on TGase-inhibited and normal livers. Confocal microscopy of injured livers incubated with FITC-labeled FPP revealed specific bonding of 11
ACCEPTED MANUSCRIPT Q- and K-containing polypeptide to the apical surface of the liver (Fig. 3C). Adhesive force between ex vivo liver slices and biomaterials containing Q and K residues was measured by AFM at the molecular level. Larger retractile force was required to detach the FPP or
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gelatin-modified beads from injured liver slices compared to CPP-modified beads. Such adhesion was blocked by soluble K, suggesting pathology-preferential adhesion mediated by endogenous TGase in injured livers (Fig. 3D; Fig. S3D). Similarly, adhesion was
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examined at macro-scale using large gelatin scaffolds which showed higher adhesive
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force to injured liver than normal liver (could be also blocked by soluble K) (Fig. S3E,F). Injured and normal livers were excised and then incubated with rhodamine-labeled FPSs ex vivo for 2 h, and imaged. Significant amount of FPSs was retained on injured liver as showed by higher fluorescent area and total fluorescence intensity. But fluorescence
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analysis of TGase-inhibited livers showed that signal decreased significantly (2.63-fold in fluorescent area and 3.54-fold in total fluorescence intensity less than injured liver) suggesting there was a threshold concentration of endogenous TGase to generate
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sufficient adhesiveness to retain micro-scaffolds on liver surface (Fig. 3E,F; Fig. S3G,H).
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Similarly, adhesion of dextran-labeled GSs to injured liver were examined by image analysis showing 3- and 17-fold increase in fluorescent area as well as 3.5- and 85-fold in total fluorescence intensity compared to TGase-inhibited and normal livers respectively (Fig. 3G,H; Fig. S3I,J). Taken together, these results confirmed the necessity of sufficient endogenous TGase to induce pathology-preferential adhesion of micro-scaffolds containing Q and K residues. Pathology-preferential adhesion of micro-scaffold-based cell capsule in vivo. 12
ACCEPTED MANUSCRIPT The pathology-preferential adhesion was further validated in vivo via intraperitoneal (i.p.) injections of FPSs, CPSs, and GSs. To ensure reproducible and fair comparison of the targeting effect, influence of manual operation was minimized by injecting the
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micro-scaffolds only when the needle tip reached the processus xiphoideus (Fig. 4A). While none of FPSs and GSs adhered to normal liver, both of them adhered to injured liver and CPSs (containing no Q and K residues) did not (Fig. 4B; Fig. S4A). Injured liver
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significantly adhered with more FPSs compared with TGase-inhibited livers (4.2- and
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28.74-fold increase in fluorescent area and total fluorescence intensity respectively) and no signal was detected on normal livers (Fig. 4C,D; Fig. S4B,C). In terms of GSs (as preferred cell capsules due to degradability of gelatin), injured livers exhibited 3- and 11-fold increase in fluorescent area and 2.5- and 13-fold increase in total fluorescence
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intensity compared to TGase-inhibited and normal livers respectively (Fig. 4E,F; Fig. S4D,E). To rule out the possible contribution of remaining glutaraldehyde (crosslinker to fabricate gelatin micro-scaffolds) in adhesion, methacrylated gelatin was crosslinked with
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APS/TEMED to fabricate GelMA micro-scaffolds which also adhered to injured liver upon
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i.p. injection (Fig. 4B; Fig. S4A). All above results confirmed that sufficient endogenous TGase after liver injury could mediate Q- and K-containing cell capsules adhesion to injured liver.
Cumulative targeting efficiency of MSCs to injured liver delivered by injectable cell capsules. MSCs-loaded GSs (GC) were tested for pathology-targeted cell delivery in mice. MSCs were evenly distributed and showed highest proliferation ability after 2 days culture which 13
ACCEPTED MANUSCRIPT was chosen as the transplantation point (Fig. 5A-C; Fig. S5A,B; Table S1). Attributing to the high elasticity of GSs, injection of GCs (after 2 days culture) through a 23G needle minimally impacted micro-scaffold morphology and cell viability (Fig. 5D,E; Fig. S5C),
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providing protection to loaded cells during transplantation. Size of GCs and cell loading effects were next evaluated. There was no statistical difference in targeting ability between 400 µm GSs [65.9% (57.1% - 78.4%)] and 800 µm GSs [54.0% (37.8% - 87.1%)],
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but GSs with smaller size showed relatively higher average targeting ratio and more
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stable adhesion. The loaded MSCs were found to slightly enhance the targeting effect (70.7% for 400 µm GSs and 63.7% for 800 µm GSs) (Fig. 4C; Fig. 5F,G; Table S2). Given these results, we chose 400 µm GSs as cell capsules for the following applications. In conventional free cell (FC) injection method, MSCs were uniformly dispersed in
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abdominal cavity and have similar chance to contact with liver leading to relatively uniform distribution in different sites of the liver. In contrast, MSCs in GC group were mainly restricted in the GSs adhered to the liver surface immediately after
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transplantation. qPCR-based human Alu tandem amplification was used to quantify
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targeting and survival of human MSCs delivered by GCs in comparison with FC injection [28, 34]. GCs with 5×105 cells were injected into mice with acute liver injury as compared with FCs with dosage of 106 (normally-used dosage [35]). Hundred-folds more human Alu tandems (~105 MSCs per gram liver tissue) were detected at liver tissues surrounding GCs-adhered sites 2 days after transplantation compared to FCs treated livers. These enriched signals in GCs-adhered livers (~2x103 MSCs per g liver tissue) remained to be at least ten folds higher than FCs treated livers 5 and 9 days after treatment, providing direct 14
ACCEPTED MANUSCRIPT evidence for efficient targeting of MSCs and prolonged cell survival via GCs delivery (Fig. 5H,I; Fig. S5D; Table S4). Around 102 MSCs per gram liver tissue was detected distant from GCs-adhered sites (at the right superior lobe without adhered GCs) during the 14
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days, with no statistical difference in signal to FC treatment groups (which remained at a level of 102 MSCs per gram liver tissue), suggesting a small number of cells migrated into liver parenchyma from the adhesion sites (Fig. 5J). We assumed that up-regulation of
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platelet derived growth factor (PDGF) post liver injury may play a chemotactic role in
cell migration model (Sup. 5E-K).
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inducing migration of MSCs from GCs, which was preliminarily investigated in an in vitro
Improved therapeutic efficacy via injectable GCs transplantation. A brief schematic outlining the targeted treatment is shown in Fig. 6A. One day after CCl4
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induction, mice were subjected to a single injection of FCs with 106 cells (FCs), FCs with 105 cells (low FCs), GCs with 5X105 cells (GCs), GCs with 105 cells (low GCs), or PBS (sham group), respectively. 66.7% of the mice (n=6) survived in groups treated with FCs
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or GCs during a 2-week observation. Notably, low GCs were able to maintain survival of
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33.3% mice while low FCs failed to rescue any (n=6) (Fig. 6B). Examination of survival curve revealed that all deaths occurred within the first 4 days of treatment, hence suggesting that therapeutic support of transplanted MSCs was most critical during the first several days. Therefore, mice with CCl4-induced liver injury were sacrificed prior treatment (denoted as PT) or 5 days after treatment for liver function evaluation to assess the therapeutic efficiency. Albumin (ALB) recovered to almost normal levels in all surviving mice
upon
treatment
(Fig.
6C).
Aspartate
transaminase
(AST)
and
alanine 15
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deposition and resulting fibrosis might have been attenuated after treatment (Fig. 6F,G). Increment of regeneration-associated gene (e.g. HGF) and decrease of multifunctional cytokine gene (e.g. TGFβ) in the treated mice further indicated partial recovery of the
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injured livers (Fig. 6H,I). H-E and Sirius Red staining demonstrated significantly
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decreased necrotic areas of liver tissues, as well as remarkably reduced collagen deposition (Fig. 6J,K). For long-term observation, the therapeutic benefits of GCs and FCs treatment could both last for 14 days (Fig. S6), demonstrating sustained therapeutic effect on injured livers.
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Above data support the claim that improved cell accumulation to injured liver via GCs delivery can successfully reverse liver damage at lower cell dosage. As humans can survive with only 30% liver function, our approach of localized cell accumulation may
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provide a promising therapeutic approach to reverse local liver injury with high efficiency
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and assist in the overall delay of further hepatocyte necrosis. The superior therapeutic effects may be attributed to enhanced paracrine secretion of growth factors (e.g. HGF, bFGF and EGF) by MSCs in GCs, as proved in our previous studies (Fig. S7A,B) [26, 27, 29], which would promote hepatocyte proliferation and modulate inflammatory conditions.
Discussion Taken together, we have, for the first time, developed a new formulation for cell therapy using injectable cell capsules as targeted localization of cell carriers by leveraging the 16
ACCEPTED MANUSCRIPT specific feature of injured liver to achieve pathological-preferential adhesion. To a limited extent, the adhered capsules allowed sustained release of cells into liver parenchyma (secondary targeting) for prolonged treatment effect, in analogy with controlled release of
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pharmaceuticals after targeted delivery. Just as drug delivery technologies have transformed the entire pharmaceutical industry to achieve improved drug efficacy and safety, targeted cell delivery capsules developed in this work controlled risks of cell
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therapy by realizing targeted and high-efficient cell therapy with lower dosage, hence
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reducing non-specific cell incorporation and unwanted side-effects to healthy tissues. Our approach could potentially be applied to many other cell types since the pathology-preferential property belongs to the cell capsules rather than the loaded cells. Furthermore, the 3D micro-scaffold-based cell capsule also provides an optimal niche for
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enhanced cell survival, functionalities and therapeutic effects at the harsh lesion site (Fig. 5I,J; Fig. S7C,D) [26]. As we have yet to achieve the goal of complete resolution of liver failure with just a single dose of cell transplantation, further experiments with more
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stringent end-point evaluations and analysis of whole body distribution of transplanted
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cells, much like the concept of pharmacokinetics in drug development, need to be carried out. Meanwhile, we do not exclude the possibility of additional factors in play to promote cell capsule adhesion in vivo. Also, i.p. injection method chosen in this work facilitated liver targeting but would have no access to other organs lying beneath the liver and intestines in the abdominal cavity. In the future, we expect to generalize this approach by incorporating endoscopic instruments (e.g. gastroscope, cystoscope, hysteroscope, colonscopy) to aid cell capsule delivery, which guides cell capsule targeted adhesion to 17
ACCEPTED MANUSCRIPT organs with natural orifice (e.g. stomach lining, endometrium of uterus and bladder, intestinal lining). Author contributions: C.Q. engineered micro-scaffold capsules, carried out most
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experiments, analyzed data and wrote the manuscript; Y.L., P.B., Y.S. and T.X. processed experiments and analyzed corresponding data; H.Y. carried out Figures processing; Z.Y., X.Y. and W.L. fabricated scaffolds; J.D. and C.H. provided clinical samples and
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consultation. Y.D. conceived of micro-scaffold capsule construct, provided overall
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intellectual guidance, edited the manuscript and is the principal investigator of the supporting grants. Acknowledgements
The authors would like to acknowledge Prof. Yonghui Zhang from School of
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Pharmaceutical Science in Tsinghua University for providing experimental mice. This work is financially supported by the National Natural Science Foundation of China (Grant Nos:
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Figure legend
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Fig. 1. Micro-scaffolds preferentially adhere to pathological liver. (A) Mechanism by
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which endogenous TGase mediated micro-scaffolds adhesion to pathological liver. Accompanying with hepatocyte necrosis, TGase would be up-regulated on the liver surface, catalyzing the ε(γ-glutamyl)lysine bonds between liver and micro-scaffolds containing Q and K residues. In detail, TGase forms a covalent thioester intermediate with the distal free amide group of protein-bound glutamine residues via their active side thiol group. This initial step is accompanied by the release of one molecule of ammonia. The free thiol of the enzyme is regenerated by reaction of the thioester intermediate with the 20
ACCEPTED MANUSCRIPT ε-amine in the side chain of lysine to form the ε(γ-glutamyl)lysine isopeptide bond. (B-E) Photograph of clusters of polypeptide-functionalized PEG micro-scaffolds (FPS) (B) and gelatin microscaffolds (GS) (D); SEM images of porous FPS (C) and GS (E). (F) FTIR
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spectrum of functional polypeptide (FPP), PS-NAS, and PS-NAS-FPP (FPP was modified on PEG micro-scaffold via N-acryloxysuccinimide (NAS)) to confirm the conjugation of FPP on FPS. (G) Quantification of lysine residues in GS gelated by different
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concentrations of glutaraldehyde (GA) via a modified TNBS assay.
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Fig. 2. TGase catalyzes Q and K residues bonding in vitro. (A) Schematic of atomic force microscopy (AFM) testing, by which the force to break chemical bonds can be accurately measured. (B) Retractile force required to separate FPP or gelatin modified silica beads and gelatin coated coverslips in the presence of different concentrations of
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TGase, as measured via AFM, CPP (control polypeptide) was used as a negative control, free lysine was used as an inhibitor to TGase (n=25~30). Sequences (C) and concentration (D) of polypeptide used to modify PSs influence adhesive force between
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PSs and GSs. FPS (Function) TGase concentration (E) and reaction time (F) influence the
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adhesive force between GSs. (G) The adhesive force was blocked by soluble lysine. Fig. 3. TGase mediated pathology-preferential adhesion of micro-scaffolds ex vivo. (A,B) TGase accumulation on liver surface via IVIS fluorescence imaging system (n=6). Data were normalized to normal liver. (C) Confocal micrographs of injured (up), TGase-inhibited (middle) and normal (down) liver incubated with FITC-FPP to confirm fluorescent signal coming from liver surface. FI (a.u.): Fluorescence Intensity (arbitrary units). (D) Retractile force required to separate liver slices and silica beads coated with 21
ACCEPTED MANUSCRIPT CPP, FPP, and gelatin, as measured via AFM with lysine as TGase inhibitor (n=25~30). (E-H)
Quantification
of
fluorescent
signal
on
excised
livers
incubated
with
rhodamin-labeled FPSs (E,F) and FITC-dextran-labeled GSs (G,H) ex vivo respectively
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and corresponding data analysis (n=6)(normalized to TGase-inhibited liver). Fig. 4. TGase mediated pathology-preferential adhesion of micro-scaffolds in vivo. (A) Schematic demonstrating the processus xiphoideus at which the needle should reach
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before injection of the micro-scaffolds. (B) In vivo adhesion of FPSs, CPSs, GSs, and
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GelMA micro-scaffold after intraperitoneal (i.p.) injection. (C-F) Quantification of fluorescent signal on livers followed by FPSs (C,D) and GSs (E,F) i.p. injection and corresponding data analysis (n=6) (normalized to TGase-inhibited liver). Fig. 5. The cumulative targeting efficiency of MSCs to injured liver. (A) Live and dead
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staining of MSCs in GSs. (B) SEM image of MSC adhered to GSs. (C) MSCs proliferation in GSs with diameter of 400 µm during 5 days. (D) Morphology of GCs before (top) and after (bottom) injection. (E) Injection influence on MSCs proliferation. (F) Influence of size
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and MSCs loaded in GSs on pathology-adhesion efficiency. (G) Quantification of GSs and
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GCs adhered to injured livers. (H) Coronal plane map of liver showing sites surrounding or distant from GCs adhesion for cell number quantification after cell transplantation. ICL-inferior caudate lobe, LML-left median lobe, LLL-left lateral lobe, RIL-right inferior lobe, RML-right median lobe, RSL-right superior lobe, SCL-superior caudate lobe. (I,J) Cells detected in liver tissues surrounding (I) or distant from (J) the GCs adhesion in GCs and FCs treatment groups respectively, 2, 5, 9, and 14 days after treatment. Fig. 6. Treatment assessment of GCs and FCs transplantation. (A) Schematic of GCs 22
ACCEPTED MANUSCRIPT treatment for mice with acute liver injury. (B) Survival rate of mice receiving FCs treatment with dosage of 106 and 105, and GCs treatment with dosage of 5×105 and 105 during a 14 days observation (n=6). Serum levels of main hepatic function markers: ALB (C), AST (D),
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and ALT (E). (F-I) Relative gene expression of ECM related genes, Col 1A1 (F), TIMP (G), hepatic regeneration-related genes, HGF (H), and multifunctional cytokine gene, TGFβ (I). H-E (J) and Sirius Red (K) staining of normal, FCs treated, GCs treated, low GCs treated
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