Biomaterials 126 (2017) 1e9

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Pathology-targeted cell delivery via injectable micro-scaffold capsule
mediated by endogenous TGase
Chunxiao Qi a, Yaqian Li a, b, 1, Patrick Badger c, 1, Hongsheng Yu a, 1, Zhifeng You a,
Xiaojun Yan a, Wei Liu a, Yan Shi d, Tie Xia d, Jiahong Dong e, Chenyu Huang f,
Yanan Du a, b, *
a
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, UT 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
Department of Plastic, Reconstructive and Aesthetic Surgery, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing 102218, China

a r t i c l e i n f o a b s t r a c t

Article history: Targeted cell delivery to lesion sites via minimally invasive approach remains an unmet need in
Received 13 December 2016 regenerative medicine to endow satisfactory therapeutic efficacy and minimized side-effects. Here, we
Received in revised form rationally designed a pathology-targeted cell delivery strategy leveraging injectable micro-scaffolds as
15 February 2017
cell-loading capsule and endogenous tissue transglutaminase (TGase) at lesion site as adhesive. Up-
Accepted 16 February 2017
Available online 17 February 2017
regulated TGase post-liver injury catalyzed chemical bonding between the glutamine and lysine resi-
dues on liver surface 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
Keywords:
Targeted cell delivery
cell protection from shear-induced damage and post-transplantation anoikis, adhered to the CCl4-treated
Micro-scaffold capsule liver with a hundred-fold improvement in targeting efficiency (70.72%) compared to free-cell injection,
Endogenous TGase which dramatically improved mice survival (33.3% vs. 0% for free-cell therapy) even with low-dosage
Pathological liver treatment. This unique and widely-applicable cell delivery mechanism and strategy hold great prom-
Cell therapy ise for transforming cell therapy for refractory diseases.
© 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Common delivery routes in cell therapy are based on either
systematic administration (e.g. intravenous or intraperitoneal in-
Along with pharmaceuticals and medical devices, cell therapy is jection) [2,3], or directed injection of cells into the damaged tissues
deemed to be the next therapeutic pillar of global healthcare which [4], whereas the therapeutic benefits of the transplanted cells are
has shown efficacy in alleviating symptoms of many end-stage and confined due to uncontrolled distribution, severe cell loss and
refractory diseases in animal models and clinical trials [1]. Just as death. Taking Mesenchymal Stem Cell (MSC)-based therapy for
drug delivery technologies (e.g. capsule and tablet-based formula- liver cirrhosis as an example, MSCs via intravenous transfusion are
tion) have transformed the entire pharmaceutical industry to ectopically distributed to almost all organs within 2 h in patients;
achieve improved drug efficacy and safety, a reliable cell delivery most cells experience anoikis within 2 days, hence leaving only
system is critical to achieve reproducible and satisfactory clinical approximately 0.02% surviving cells and 10% of them in liver,
outcomes, which is desired to transfer cells to targeted lesion site indicating poor targeting and engraftment efficiency [5].
and enhance cells' viability and therapeutic effects [2]. To overcome this hurdle, current strategies for improving cell
targeted-delivery include genetic modification, cytokine priming of
cells [6,7], and biomaterial-assisted implantation [8]. For instance,
genetically-modified or cytokines (e.g. IL-3, IL-6)-primed MSCs
* Corresponding author. Department of Biomedical Engineering, School of Med-
with upregulated expression of CXCR4, a receptor that specially
icine, Tsinghua University, Beijing 100084, China.
E-mail address: duyanan@tsinghua.edu.cn (Y. Du). interacts with SDF-1a in injured liver, exhibited elevated binding
1
These authors contributed equally to this work. and accumulation to fibrotic liver [6,7,9]. However, potential safety

http://dx.doi.org/10.1016/j.biomaterials.2017.02.021
0142-9612/© 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/
).

or FITC-labeled FPP solution at 37  C for 2 h. has been widely used as a crosslinking agent for biomaterials containing glutamine (Q) and lysine (K) residues (e. and are thus not a viable option for patients with end. differentiation. tumori. Gelatin precursor solution (7% w/v gelatin and 0. via intraperitoneal injection (injured liver group). the livers were imaged using an IVIS fluorescence imaging system (Caliper Life Sciences).3% APS. and the excised livers were imaged one day after using an IVIS fluo- 2. was automatically absorbed thanks to GSs porous structure.g. injured. cell surface and ECM. and tissue fibrosis. FPSs. and 10% TEMED in cold could remodel ECM components at pathological sites [17.4. Therefore.g. liver parenchyma) [11e14].N0 -tetramethy- To achieve this goal. we rationally designed a micro-scaffold. and vacuum-packaged for later use in (e. Human adipose-derived mesenchymal stem cells (GA) solution was added with mild stirring.1.3. with the of tissue transglutaminase (TGase) from necrotic hepatocytes exception that they were cryogelated for 20 h at 20  C before [15.g. To inhibit CCl4- tion of TGase. surgical interventions which cause secondary damages to the tar- geted tissue. influence of the process of i. pathological states. contributing to wound healing. Anderson then chilled on ice for 5 min.18]. 1% w/v N-acryloxysuccinimide (NAS).p. excess zation. as functional PS (FPS) or control PS (CPS) respectively. CPSs. and vacuum-packaged for later use in characterization autoimmunity. upregulated TGase bonded to ECM by dissolving 6% w/v GelMA. Roland DG). The filled microstencil array chip subsequently pipetted onto 600 tightly packed GSs.5% w/v Odex in diH2O if Before cell loading. 2. 0. tional polypeptide or control polypeptide and incubated at 37  C for translational modifications of proteins involved in a variety of 2 h. reported protocol [27]. The quently lyophilized for 30 min (Boyikang). Ex vivo and in vivo scaffold adhesion experiments [19. cells could be loaded in hydrogel or scaffold vehicles oughly with diH2O. analogous to their pharmaceutical PEGDA (4000) precursor solution was prepared by dissolving counterparts. and cell culturing [1]. After lyophilizing. Cell loading and viability assessment cleaner (Mycro Technologies) to increase hydrophilicity. into which it then underwent cryogelation for 16 h at 20  C and was subse. GSs were harvested GSs were then maintained in a humidified chamber and incubated . After washing for 30 min. each containing 600 circular micro-wells reached the processus xiphoideus. after which 70 mL 0. These vehicles usually require characterization and cell loading [26]. GelMA (methacrylated gelatin) precursor solution was prepared within 24 h upon liver injury. TGase the same procedure used for PEG micro-cryogel scaffold. diH2O. and normal residues as cell delivery capsules to facilitate targeted cell delivery livers were excised after mice were anesthetized and incubated by preferentially adhering to TGase-overexpressed liver but not with GSs.N0 . collagen 2.g. lyophilized. then washed thor- Alternatively.2 C. there is an urgent need for suitable ‘cell capsules’. Materials and methods experiments. localized in multiple intracellular compartments.20]. harvested and dried GSs were sterilized with labeling is necessary) was dissolved at 50  C in a water bath and an ethylene oxide sterilization system (AN74j/Anprolene. A 60 mL MSCs suspension (8  106/mL) was face to ensure even distribution. was administered in a single dose of 7.15% w/v N. PEG- cross-link between side chains of lysine (ε-amine) and glutamine NAS scaffold was added to 100 mL 2% w/v solution of either func- (g-acyl). For in vivo 2. GSs were washed with clinical translation of these cells as therapeutic agents [4. lethylenediamine (TEMED) in cold diH2O maintained on ice. vascular remodeling. we hypothesized endogenous TGase bonded in induced TGase up-regulation. genesis/metastasis. anchor for the adhesion of materials containing Q and K residues. For We thereby rationally designed micro-scaffolds containing Q and K ex vivo adhesive experiments. mice were treated firstly with cyst- pathological tissues (e. 500 mM apoptosis. 0. migration.5% w/v the delivered cells or the targeted tissues/organs.5 mL per kg of body weight Considering the physiological function and industrial applica. and 2 days later injured with CCl4 (TGase-inhibited liver group). is 2. 250 mL of the resulting (MSCs) were isolated and cultured in mesenchymal stem cell precursor solution was pipetted onto the upper surface of each growth medium (BioWit Technologies) according to a previously microstencil array chip and manually scraped across the chip sur. 0. normal liver (Fig. GSs. neurodegeneration. namely the up-regulated secretion cedure used to fabricate gelatin micro-cryogel scaffolds. and 0. with diameters of 400 mm or 800 mm.10]. CPSs were injected intraperitoneally. facilitating the modification of PEG micro-cyogel scaffolds (PS) cellular processes. to achieve targeted cell delivery without interfering 10% w/v PEGDA. / Biomaterials 126 (2017) 1e9 issues associated with genetic modification and un-predictable cell using a PMMA ejector pin array fabricated with a desktop 3D functional alteration related with cytokine priming have hindered milling machine (MDX-40A.1 M NaBH4 to neutralize unreacted aldehyde. The protein.16]. 1A). Gelatin micro-cryogel scaffold fabrication rescence imaging system.5% glutaraldehyde Sterilization). Qi et al. ammonium persulfate (APS).5. and extracellular matrix (ECM) organi. FPSs. including adhesion. TGase is a Ca2þ-dependent enzyme which catalyzes the lyophilizing and washed only with diH2O. FPSs and involvement of this protein in various physiological responses and CPSs were then harvested into a dish in an even monolayer. Chips were fabricated us- ing a laser fabrication system (Rayjet) and treated using a plasma 2. The impact of TGase on these processes implicates the ethanolamine was removed by washing with PBS buffer. ethanolamine solution was used to block unreacted NAS. gelatin [21]) to fabricate scaffolds via ε(g-glutamyl)lysine bonds as surgical adhesives in medical industry and cling films in To induce liver injury in mice. GelMa micro-cryogel scaffolds fabrication known to exist ubiquitously at basal level in all types of mammalian tissue. To ensure fair comparison of targeting effect. lyophilized. PEG micro-cryogel scaffold fabrication stage symptoms (e. liver cirrhosis). but would be upregulated upon tissue injury. delivery operation was mini- Gelatin micro-cryogel scaffolds (GS) were prepared in PMMA mized by injecting the micro-scaffolds only when the needle tip microstencil array chips. growth. The established ε(g-glutamyl)lysine bonds result in post. TGase-inhibited. PEG based ‘capsule’ for targeted cell delivery to liver by exploiting the micro-cryogel scaffolds were then fabricated using the same pro- unique feature of injured liver. In particular. a 1:4 mixture of CCl4 in mineral oil food industry [22e25]. They were then harvested into a dish in an even to assist their implantation and retention at targeted tissue or organ monolayer.2. injured liver) would provide a molecular amine to inhibit TGase expression (at a dose of 112 mg/kg per day). inflammation. GelMA micro-croygel scaffolds were then fabricated using Mimicking the in vivo function as a cross-linker of proteins.N.

which were carried out with Animal experiments were performed in strict accordance with SYBR Premix Ex Taq (TaKaRa). 100 mg liver tissue along with otherwise identical conditions. Primers for all genes are listed in Supplementary Table 3. This initial step is accompanied by the release of one molecule of ammonia. In detail. The RNA was reversely transcribed using PrimeScript II 1st Strand cDNA mice were housed in microisolator cages. TGase forms a covalent thioester intermediate with the distal free amide group of protein-bound glutamine residues via their active side thiol group. (iv) GC group (treated with CCl4 and was added to each sample which is an aqueous solution of 4 M 5  105 cell suspension). TGase would be up-regulated on the liver surface. Accompanying with hepatocyte necrosis.7. After added for long-term culture. 50 nM sodium citrate and 1 M Tris. 2. and relative gene expression was BALB/c nude mice were randomized to one of seven groups: (i) analyzed. (F) FTIR spectrum of functional polypeptide (FPP). at 37  C for 2 h to allow for cell adhesion. / Biomaterials 126 (2017) 1e9 3 Fig.6. GSs were cultured for varying times under samples surrounding GCs adhesion. a standard curve was made to precipitated with 400 mL of isopropanol. The free thiol of the enzyme is regenerated by reaction of the thioester intermediate with the ε-amine in the side chain of lysine to form the ε(g-glutamyl)lysine isopeptide bond. PS-NAS. In vivo treatment of mice with acute liver failure Cell or tissue RNA was isolated using TRIzol reagent (Invitrogen) BALB/c nude mice were maintained in a specific pathogen-free according to the manufacturer's instructions. Culture medium was then guanididium thiocyanate. The resulting amplification was standards approved by the Animal Ethics Committee of Tsinghua monitored with the CFX96 Real-Time System (Bio-Rad). CellTiter-Blue® cell viability assay was vortexing. the samples were centrifuged at 16. (A) Mechanism by which endogenous TGase mediated micro-scaffolds adhesion to pathological liver. Live/dead assay was also performed for adhesion.000g for 15 min. (iii) Low FC group (treated with CCl4 and removed for genomic DNA extraction. Care Committees at Tsinghua University. Samples for FCs group were collected at same sites as in GCs group. (ii) FC group (treated with CCl4 and Following RNA extraction. was used for DNA extraction. and cell viability was assessed. For and cell number. Cell the adhered GCs (always located at sites surrounding gall bladder) number per micro-scaffold was calculated according to the estab. Experimental protocols were approved by the Animal expression of genes of interest was normalized against the refer. Qi et al. All reverse primers. As to samples distant from GCs lished standard curve. control (no treatment of CCl4). catalyzing the ε(g-glutamyl)lysine bonds between liver and micro-scaffolds containing Q and K residues. C. and PS-NAS-FPP (FPP was modified on PEG micro- scaffold via N-acryloxysuccinimide (NAS)) to confirm the conjugation of FPP on FPS. as well as forward and Animal Center) were used for experiments at 7e8 weeks of age. SEM images of porous FPS (C) and GS (E).5 ng determine the linear relationship between fluorescence intensity of genomic DNA was used as template for real time PCR [28]. 1. (BeE) Photograph of clusters of polypeptide-functionalized PEG micro-scaffolds (FPS) (B) and gelatin microscaffolds (GS) (D). (v) Low GC group (treated with CCl4 and . was added to PCRs. ence gene GAPDH in all samples. applied to determinate cell loading-capacity and proliferation The aqueous phase was transferred to a new tube and DNA within each microcryogel: first. 2. 100 mg liver tissue from right lobe (without GCs adhe- visualization of cell viability. Then. sion) was used for DNA extraction. The University. One microgram of animal facility at the Animal Center of Tsinghua University. 2.5 ng of cDNA. (G) Quantification of lysine residues in GS gelated by different concentrations of glutaraldehyde (GA) via a modified TNBS assay. As in RNA analysis. the remaining aqueous layer was 106 cell suspension). Mice (Purchased by the Synthesis Kit (TaKaRa). 400 mL back extraction buffer 105 cell suspension). Targeting efficiency quantification and gene expression analysis 2. Micro-scaffolds preferentially adhere to pathological liver.

which is well amount of FPSs was retained on injured liver as showed by higher known for its anti-fouling features in minimizing non-specific ab. (Mindray ®. containing N and R residues in replacement of Q and K injured liver were examined by image analysis showing 3. and in vivo experiments were not randomized. S3G and H).74-fold increase in fluorescent area and total mometer to test the adhesive force between two TGase-bound fluorescence intensity respectively) and no signal was detected on scaffolds (both with surface areas of 2  4 cm2) (Fig. incubated with FITC-labeled FPP revealed specific bonding of Q- No statistical methods were used to predetermine sample sizes for and K-containing polypeptide to the apical surface of the liver in vivo experiments. Mice received one dose and soluble lysine (K) inhibited macro-scale adhesive force as it did of 7.and 13. 2B. S1C). TGase-catalyzed crosslink of Q and K residues was first verified at the molecular level by employing atomic force microscopy 3. (Fig. indicating the specific catalytic function xiphoideus (Fig.and 28. 4C and D. Similarly.54-fold in total tional polypeptide (GQLKHLEQQEG.29] (Fig. 2C and D). 1.82. Livers were isolated. S1B).4 C. Q and K residues) did not (Fig. / Biomaterials 126 (2017) 1e9 105 cell suspension). TGase mediated pathology-preferential adhesion of Q. To investigate the specific interactions be. was used in fabricating control PEG micro-scaffolds (CPS) fluorescence intensity compared to TGase-inhibited and normal (Fig. 4B. and GSs. 3G and H. we investigated the TGase-mediated adhesiveness be. S2A).2.23- Graphpad Prism software was used to perform all statistical fold increase in fluorescent area. (vii) PT Similar observations were made for adhesions between two GSs. Fig. fluorescent area and total fluorescence intensity. upon incubation with soluble K amino acid due to competitive influence of manual operation was minimized by injecting the binding. 1BeE). S3D). capsules in vitro suggesting pathology-preferential adhesion mediated by endoge- nous TGase in injured livers (Fig. Such adhesion was blocked by soluble K. FPP) (Fig.05 was considered statistically significant. Meanwhile gelatin-based micro-scaffolds (GS) livers respectively (Fig. 1B and C. prior treatment). Fig. S1G and these results confirmed the necessity of sufficient endogenous H).) injections of FPSs. 2EeG). Standard H-E stained sections and Sirius Red stained sections were examined and scored. Data are presented as mean ± SD. TGase to induce pathology-preferential adhesion of micro-scaffolds mediated adhesion of biomaterials containing Q and K residues. physiochemical properties proved as beneficial 3D cellular niches Injured and normal livers were excised and then incubated with [26. was selected as the polymer for analysis of TGase-inhibited livers showed that signal decreased micro-scaffold fabrication. a threshold concentration of endogenous TGase to generate suffi- Fig. (vi) sham (treated with CCl4 and PBS). liver. CPSs. Such force was diminished ensure reproducible and fair comparison of the targeting effect. Fig. Qi et al.5- .8. neally injected free cell suspension or cell capsule suspension. 1G. A value of P < 0.and 11-fold increase in fluorescent area and 2. aspartate aminotransferase (AST) and albumin glutaminase activity of the TGase in injured livers. Significant tween TGase and capsules. 3E troscopy [31] (Fig. A Q. 5 and 14 days later. While none of FPSs and GSs adhered to normal of TGase on bond formation between Q and K residues (Fig. and normal livers were 2. 2A. TGase catalyzes bonding between micro-scaffold-based CPP-modified beads. S1D and E). livers (4. S4A).11. S3AeC) than those on TGase- to determine the significance of observed differences between test inhibited and normal livers. Likewise. as confirmed by Fourier Transform Infrared (FTIR) spec. fixed in 4% paraformaldehyde. injured increased reaction time and FPP concentration (Fig.and 2.and K- ficacy. cient adhesiveness to retain micro-scaffolds on liver surface (Fig.51-fold increase in average intensity) for times.2. S3E and F). 3. and imaged.and 85-fold in total bility). bonding of (ALB) levels were measured using a Mindray BS-200 analyzer rhodamine-labeled FPP to excised injured livers (induced by CCl4). Results molecular level. S1A) was successfully fluorescence intensity less than injured liver) suggesting there was grafted to form functional PEG micro-scaffolds (FPS) (Fig. Rhodamine signal was significantly higher (2. S3I and J). Plasma alanine amino To verify the presence and indicate Ca2þ-dependent trans- transferase (ALT). 1F). 3D. S4B and C). significantly (2. As schematized in Fig. was examined at macro-scale using large gelatin scaffolds which sules. All experiments were performed a minimum of three tal intensity. which can measure the force to break TGase-mediated capsule in vivo chemical bonding. China).5 mL/kg 1:4 mixture of CCl4 in mineral oil were intraperito. Injured liver signifi- Next. Confocal microscopy of injured livers groups. Pathology-preferential adhesion of micro-scaffold-based cell (AFM). 4. Shenzhen. TGase-inhibited livers (TGase synthesis was inhibited by cystamine treatment prior to CCl4 induction [32]). A control polypeptide (GNLRHLENNEG.5. modification of silica beads with CPP yielded micro-scaffolds only when the needle tip reached the processus negligible retractile force. polyethylene glycol (PEG). Fig. Fig. in the AFM tests (Fig. (Fig. normal livers (Fig. But fluorescence sorptions and interactions [30]. rhodamine-labeled FPSs ex vivo for 2 h. mice were sacrificed to analysis therapeutic ef.5. S2B and C). Fig. group (treated with CCl4. Larger retractile force was required to detach the FPP or gelatin-modified beads from injured liver slices compared to 3. and containing micro-scaffolds to injured liver ex vivo embedded in paraffin.p.1.63-fold in fluorescent area and 3. 3C). both of them adhered to injured liver and CPSs (containing no Fig. To dependent on TGase concentration. 1D and E. Statistical analysis assessed ex vivo respectively via IVIS fluorescence imaging system [33]. owing to their superior elasticity and injectability to protect showed higher adhesive force to injured liver than normal liver cells from shear damage during injection as well as tenability of (could be also blocked by soluble K) (Fig.09-fold increase in to- analyses. 4A). were also chosen to confirm the general applicability of TGase.and 3. Fig. the retractile force required to detach silica bead (modified with soluble FPP or gelatin) The pathology-preferential adhesion was further validated glued on AFM cantilever from soluble gelatin coated coverslips was in vivo via intraperitoneal (i.and K-residue containing func. cantly adhered with more FPSs compared with TGase-inhibited tween Q and K residues on the macro-scale using a friction dyna. S1F). One-way ANOVA was used injured livers (Fig.3. containing Q and K residues. In terms of GSs (as Adhesive force between FPSs and GSs was shown to correlate with preferred cell capsules due to degradability of gelatin). Similarly. containing K residues (Fig. Taken together. CPP) and F.27. Fig. livers exhibited 3. Adhesive force between ex vivo liver slices and bio- materials containing Q and K residues was measured by AFM at the 3. adhesion of dextran-labeled GSs to (Fig. adhesion Cryogelated micro-scaffolds were applied as cell delivery cap.and 17- residues respectively (with no change in overall charge and solu. fold increase in fluorescent area as well as 3. 3A and B.

. 4E and F. FPP. free lysine was used as an inhibitor to TGase (n ¼ 25e30). (G) The adhesive force was blocked by soluble lysine. Table S1). 2. CPP (control polypeptide) was used as a negative control. (A. S4D and E).F) and FITC- dextran-labeled GSs (G. (C) Confocal micrographs of injured (up). by which the force to break chemical bonds can be accurately measured.B) TGase accumulation on liver surface via IVIS fluorescence imaging system (n ¼ 6). injection (Fig. days culture) through a 23G needle minimally impacted micro- Fig. All above results as the transplantation point (Fig. methacrylated gelatin was crosslinked with APS/TEMED cell delivery in mice. FI (a.): Fluorescence Intensity (arbitrary units). and gelatin. (A) Schematic of atomic force microscopy (AFM) testing. S5A and B.4. To rule out the possible contribution of remaining glutaraldehyde (crosslinker to fabricate gelatin micro-scaffolds) in mscMSCs-loaded GSs (GC) were tested for pathology-targeted adhesion. 4B. TGase-inhibited (middle) and normal (down) liver incubated with FITC-FPP to confirm fluorescent signal coming from liver surface.H) ex vivo respectively and corresponding data analysis (n ¼ 6)(normalized to TGase-inhibited liver). injection of GCs (after 2 mediate Q. TGase mediated pathology-preferential adhesion of micro-scaffolds ex vivo. as measured via AFM.and K-containing cell capsules adhesion to injured liver. Data were normalized to normal liver. as measured via AFM with lysine as TGase inhibitor (n ¼ 25e30). confirmed that sufficient endogenous TGase after liver injury could Attributing to the high elasticity of GSs. Fig. S4A).u. C. 5AeC. / Biomaterials 126 (2017) 1e9 5 Fig. (B) Retractile force required to separate FPP or gelatin modified silica beads and gelatin coated coverslips in the presence of different concentrations of TGase. Cumulative targeting efficiency of MSCs to injured liver TGase-inhibited and normal livers respectively (Fig. Qi et al. FPS (Function) TGase concentration (E) and reaction time (F) influence the adhesive force between GSs. Sequences (C) and concentration (D) of polypeptide used to modify PSs influence adhesive force between PSs and GSs. and 13-fold increase in total fluorescence intensity compared to 3. Fig. MSCs were evenly distributed and showed to fabricate GelMA micro-scaffolds which also adhered to injured highest proliferation ability after 2 days culture which was chosen liver upon i. (EeH) Quantification of fluorescent signal on excised livers incubated with rhodamin-labeled FPSs (E. 3. delivered by injectable cell capsules Fig.p. TGase catalyzes Q and K residues bonding in vitro. (D) Retractile force required to separate liver slices and silica beads coated with CPP.

5H and I. but adhered GCs) during the 14 days. GCs with 105 cells (low GCs). MSCs in GC group were transplantation mainly restricted in the GSs adhered to the liver surface immedi- ately after transplantation. planted MSCs was most critical during the first several days. 4C. suggesting a small number of cells slightly enhance the targeting effect (70. MSCs were in an in vitro cell migration model (Sup. liver tissue) were detected at liver tissues surrounding GCs. Notably.7% of the mice (n ¼ 6) survived Hundred-folds more human Alu tandems (~105 MSCs per gram in groups treated with FCs or GCs during a 2-week observation. TGase mediated pathology-preferential adhesion of micro-scaffolds in vivo. CPSs.4%)] and 800 mm GSs [54. respectively. injection and corresponding data analysis (n ¼ 6) (normalized to TGase-inhibited liver). 4.p. These enriched signals in GCs-adhered livers vival curve revealed that all deaths occurred within the first 4 days (~2  103 MSCs per g liver tissue) remained to be at least ten folds of treatment.3% mice while adhered sites 2 days after transplantation compared to FCs low FCs failed to rescue any (n ¼ 6) (Fig.1%e78. Size of longed cell survival via GCs delivery (Fig.8%e87. 63. Fig. PBS (sham group). S5D.D) and GSs (E.1%)]. One day after CCl4 induction. In contrast. The loaded MSCs were found to MSCs per gram liver tissue). or compared with FCs with dosage of 106 (normally-used dosage [35]). Improved therapeutic efficacy via injectable GCs different sites of the liver.9% (57. 5J). mice were subjected to a delivered by GCs in comparison with FC injection [28. GCs with 5  105 cells (GCs). providing direct evidence for efficient targeting of MSCs and pro- providing protection to loaded cells during transplantation. Qi et al.7% for 400 mm GSs and migrated into liver parenchyma from the adhesion sites (Fig. (A) Schematic demonstrating the processus xiphoideus at which the needle should reach before injection of the micro-scaffolds. (CeF) Quantification of fluorescent signal on livers followed by FPSs (C.5. GCs with single injection of FCs with 106 cells (FCs). uniformly dispersed in abdominal cavity and have similar chance to contact with liver leading to relatively uniform distribution in 3.G.p. / Biomaterials 126 (2017) 1e9 Fig. 6A. 5E-K). Around 102 MSCs per gram liver tissue was detected statistical difference in targeting ability between 400 mm GSs distant from GCs-adhered sites (at the right superior lobe without [65.) injection. with no statistical difference in GSs with smaller size showed relatively higher average targeting signal to FC treatment groups (which remained at a level of 102 ratio and more stable adhesion.6 C. 5F. and GelMA micro-scaffold after intraperitoneal (i. (B) In vivo adhesion of FPSs. 6B). scaffold morphology and cell viability (Fig. S5C). GCs and cell loading effects were next evaluated. migration of MSCs from GCs. A brief schematic outlining the targeted treatment is shown in fication was used to quantify targeting and survival of human MSCs Fig. FCs with 105 cells (low 5  105 cells were injected into mice with acute liver injury as FCs). Fig. 66.0% (37. . Table S2). low GCs were able to maintain survival of 33. There was no Table S4). hence suggesting that therapeutic support of trans- higher than FCs treated livers 5 and 9 days after treatment. Examination of sur- treated livers. Fig. qPCR-based human Alu tandem ampli. we chose 400 mm GSs as cell capsules for the following (PDGF) post liver injury may play a chemotactic role in inducing applications. GSs.F) i. which was preliminarily investigated In conventional free cell (FC) injection method.34]. Given these We assumed that up-regulation of platelet derived growth factor results. 5D and E.7% for 800 mm GSs) (Fig.

The cumulative targeting efficiency of MSCs to injured liver. 5. HGF) and decrease of multi- treatment (denoted as PT) or 5 days after treatment for liver functional cytokine gene (e. RT-PCR revealed down-regulated expression of days (Fig. / Biomaterials 126 (2017) 1e9 7 Fig. H-E (ALB) recovered to almost normal levels in all surviving mice upon and Sirius Red staining demonstrated significantly decreased treatment (Fig. 5.g. demonstrating sustained therapeutic effect on ECM-related genes (e. LML-left median lobe. 6H and I). ICL-inferior caudate lobe. Albumin indicated partial recovery of the injured livers (Fig. and 14 days after treatment. the all mice upon treatment as compared to the pre-treatment levels therapeutic benefits of GCs and FCs treatment could both last for 14 (Fig. as well as remarkably reduced aminotransferase (ALT) concentrations decreased significantly in collagen deposition (Fig.g. For long-term observation. RML-right median lobe. TGFb) in the treated mice further function evaluation to assess the therapeutic efficiency. Aspartate transaminase (AST) and alanine necrotic areas of liver tissues. LLL-left lateral lobe. Therefore. 6J and K). Col 1A1 and TIMP) upon treatment. (H) Coronal plane map of liver showing sites surrounding or distant from GCs adhesion for cell number quantification after cell transplantation. C. (D) Morphology of GCs before (top) and after (bottom) injection. implying ECM deposition and resulting fibrosis might have been Above data support the claim that improved cell accumulation attenuated after treatment (Fig. Increment of to injured liver via GCs delivery can successfully reverse liver . (I. RSL-right superior lobe. 6C). (F) Influence of size and MSCs loaded in GSs on pathology-adhesion efficiency. S6). SCL-superior caudate lobe. 6D and E). (C) MSCs proliferation in GSs with diameter of 400 mm during 5 days.g. 9. 2. mice with CCl4-induced liver injury were sacrificed prior regeneration-associated gene (e.J) Cells detected in liver tissues surrounding (I) or distant from (J) the GCs adhesion in GCs and FCs treatment groups respectively. 6F and G). (A) Live and dead staining of MSCs in GSs. (G) Quantification of GSs and GCs adhered to injured livers. (B) SEM image of MSC adhered to GSs. Qi et al. (E) Injection influence on MSCs proliferation. RIL-right inferior lobe. injured livers.

in analogy with controlled release of pharma- injury with high efficiency and assist in the overall delay of further ceuticals after targeted delivery. and GCs treatment with dosage of 5  105 and 105 during a 14 days observation (n ¼ 6). GCs treated. we have. Col 1A1 (F).27. 5I and J. Qi et al. H-E (J) and Sirius Red (K) staining of normal. (B) Survival rate of mice receiving FCs treatment with dosage of 106 and 105. developed a new more. Fig. and ALT (E). Further- Taken together. TIMP (G). and multifunctional cytokine gene. (A) Schematic of GCs treatment for mice with acute liver injury. hence cyte proliferation and modulate inflammatory conditions. TGFb (I). bFGF and EGF) by MSCs in GCs. Serum levels of main hepatic function markers: ALB (C). which would promote hepato.g. / Biomaterials 126 (2017) 1e9 Fig. optimal niche for enhanced cell survival. improved drug efficacy and safety. The superior therapeutic effects may be have transformed the entire pharmaceutical industry to achieve attributed to enhanced paracrine secretion of growth factors (e. As humans can survive with only 30% limited extent. reducing non-specific cell incorporation and unwanted side-effects to healthy tissues. Our approach could potentially be applied to 4. our approach of localized cell accumulation may cells into liver parenchyma (secondary targeting) for prolonged provide a promising therapeutic approach to reverse local liver treatment effect. FCs treated. As we have yet to achieve the goal of complete resolution of . low GCs treated livers 5 days after cell transplantation. AST (D). HGF (H). S7C and D) of injured liver to achieve pathological-preferential adhesion. S7A and B) [26. targeted and high-efficient cell therapy with lower dosage. (FeI) Relative gene expression of ECM related genes. damage at lower cell dosage.29]. targeted cell delivery capsules HGF. Just as drug delivery technologies hepatocyte necrosis. 6. and prior treatment liver (PT). the adhered capsules allowed sustained release of liver function. as proved in our previous developed in this work controlled risks of cell therapy by realizing studies (Fig.8 C. hepatic regeneration-related genes. To a [26]. Treatment assessment of GCs and FCs transplantation. Discussion many other cell types since the pathology-preferential property belongs to the cell capsules rather than the loaded cells. for the first time. functionalities and ther- geted localization of cell carriers by leveraging the specific feature apeutic effects at the harsh lesion site (Fig. the 3D micro-scaffold-based cell capsule also provides an formulation for cell therapy using injectable cell capsules as tar.

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