NANO-01836; No of Pages 13

Nanomedicine: Nanotechnology, Biology, and Medicine

xx (2018) xxx – xxx

nanomedjournal.com

1Q1 Conformal coating by multilayer nano-encapsulation for the protection of

2 human pancreatic islets: In-vitro and in-vivo studies
Farooq Syed, PhD a,⁎, 1 , Marco Bugliani, PhD a , Michela Novelli, PhD b ,

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4 Francesco Olimpico, MSc a , Mara Suleiman, MSc a , Lorella Marselli, MD, PhD a ,

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5 Ugo Boggi, MD, PhD b , Franco Filipponi, MD, PhD b , Vittoria Raffa, PhD c , Silke Krol, PhD d, e ,
6 Daniela Campani, MD, PhD b , Pellegrino Masiello, MD, PhD b , Vincenzo De Tata, MD, PhD b, 2 ,
Piero Marchetti, MD, PhD a,⁎⁎, 2

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8 Department of Clinical and Experimental Medicine, University of Pisa, Italy

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9 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Italy
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10 Department of Biology, University of Pisa, Italy
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11 NanoMed lab, Fondazione IRCCS, Istituto Neurologico "Carlo Besta", IFOM-IEO-campus, Milan, Italy
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Laboratory for translational nanomedicine, IRCCS Istituto Tumori “Giovanni Paolo II”, Bari, Italy
13 Received 11 September 2017; accepted 28 June 2018
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14 Abstract
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15 To improve the efficiency of pancreatic islet transplantation, we performed in-vitro and in-vivo experiments with isolated human
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16 pancreatic islets coated by multi-layer nano-encapsulation using differently charged polymers [chitosan and poly(sodium styrene sulfonate)]
17 to obtain up to 9 layers. The islet coating (thickness: 104.2 ± 4.2 nm) was uniform, with ≥ 90% cell viability and well preserved beta- and
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18 alpha-cell ultrastructure. Nano-encapsulated islets maintained physiological glucose-stimulated insulin secretion by both static incubation
19 and perifusion studies. Notably, palmitate- or cytokine-induced toxicity was significantly reduced in nano-coated islets. Xenotransplantation
20 of nano-encapsulated islets under the kidney capsule of streptozotocin-induced C57Bl/6J diabetic mice allowed long term normal or near
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21 normal glycemia, associated with minimal infiltration of immune cell into the grafts, well preserved islet morphology and signs of re-
22 vascularization. In summary, the multi-layer nano-encapsulation approach described in the present study provides a promising tool to
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23 effectively protect human islets both in-vitro and in-vivo conditions.

24 © 2018 Elsevier Inc. All rights reserved.
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25 Key words: Human islets; Islets transplantation; Multilayer nanoencapsulation; Immune isolation; Diabetes
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26
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27 The islets of Langerhans represent the endocrine component cells) and the glucagon-producing alpha cells (approximately 30
28 of the pancreas and are mainly constituted by the insulin- 15%-30% of islet cells). Additional and less represented (around 31
29 producing beta cells (approximately 60%-70% of the total islet 5%) islet endocrine cells are the somatostatin-producing delta 32
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Abbreviations: T1D, Type 1 diabetes; MLBL, Multi-layer-by-layer; STZ, Streptozotocin; NA, Nicotinamide; IPGTT, Intraperitoneal glucose tolerance test;
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MPO, Myeloperoxidase; PSS, poly sodium styrene sulfonate; UC, Uncoated; NE, nano-encapsulated
Conflict of interest: The authors declare no conflict of interest.
Source of support: This work was supported by Caripisa Foundation (now Pisa Foundation), Pisa, Italy.
⁎Correspondence to: F. Syed, Center for Diabetes and Metabolic Disorders, Department of Pediatrics, Indiana University School of Medicine, 635 Barnhill
Drive, MS2031B, Indianapolis, IN.
⁎⁎Correspondence to: P. Marchetti, Department of Clinical and Experimental Medicine, Islet cell Laboratory, University of Pisa, AOUP-Via Paradisa, 2,
56124 Pisa, Italy. Tel: +39 050 995110.
E-mail addresses: fsyed@iupui.edu, (F. Syed), piero.marchetti@med.unipi.it. (P. Marchetti).
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Present address: Center for Diabetes and Metabolic Disorders, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, USA.
2
Equally contributed.

https://doi.org/10.1016/j.nano.2018.06.013
1549-9634/© 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Syed F., et al., Conformal coating by multilayer nano-encapsulation for the protection of human pancreatic islets: In-vitro and in-
vivo.... Nanomedicine: NBM 2018;xx:0-12, https://doi.org/10.1016/j.nano.2018.06.013
 2 F. Syed et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2018) xxx–xxx

33 cells and the pancreatic polypeptide-producing PP cells (these latter diabetic cadaveric organ donors (age: 62 ± 16 yrs.; body mass 88
34 are located mainly in islets in the head of the gland). In human index, BMI: 24.6 ± 3.3 kg/m 2; M/F: 17/15), in accordance with 89
35 islets, beta and alpha cells are intermingled to form the typical islet institutional guidelines and with approval of the local ethics 90
36 architecture 1 (Supplementary Figure 1). In type 1 diabetes, there is committee. Islets were then cultured in M199 medium 91
37 the selective destruction of beta cells, due to an autoimmune containing 5.5 mM glucose, 10% bovine serum (GIBCO, 92
38 process, leading to negligible or absent secretion of insulin and the Lifescience Technologies, UK), 100 UI/L penicillin, 100 mg/L 93
39 need of chronic exogenous insulin administration. 2 Pancreatic islet of streptomycin, 750 μg/L amphotericin and 50 mg/L 94
40 cell transplantation is a potential option for the treatment of type 1 gentamycin. 95
41 diabetes and significant advancements have been achieved,
42 particularly in the past few years. 3,4 However, the clinical success Multi-layer-by-layer nano-encapsulation 96
43 of this approach in humans is still hampered by several issues,
44 including the lifelong need of immunosuppressive drugs to prevent Multi-layer-by-layer nano-encapsulation was performed by 97

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45 islet cell destruction by the immune system of the recipient. 5,6 electrostatic binding of oppositely charged polymers. Chitosan 98

46 Unfortunately, the immunotherapies required to avoid immune [(Poly(D-glucosamine) deacetylated chitin)], a weak polycation 99

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47 rejection of transplanted islets can be toxic, impair islet function positively charged at a pH below 7, and PSS [(Poly(styrene- 100

48 and survival, and increase the susceptibility of recipients to serious sulfonic acid, sodium salt)], a strong polyanion permanently 101

49 infections and tumors. 7 A promising strategy to overcome chronic negatively charged (Sigma Aldrich Co, St. Louis, USA) were 102

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50 immunosuppression is the “immune isolation”, i.e., the dissolved (1 mg/ml) in medium M199 and stored at 37 °C for 103

51 encapsulation of islets with biocompatible materials which are approximately 48 h before the nano-encapsulation procedure. 104

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52 able to shield the cells from immune attacks, while allowing This step is necessary to form a random coil structure for the 105

53 nutrition supply to and insulin release from the beta cells. 8,9 More weak polycation. Then, the islets were suspended in the medium 106
containing chitosan (around 100 islets/ml) for 15 min, followed 107

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54 recently, nanomedical technologies have been employed in this
55 field, including conformal coating of isolated islets based on layer- by washing; next, islets were incubated with medium containing 108

56 by-layer nano-encapsulation. 10,11 Major advantages of this PSS for 15 min. This procedure was repeated to obtain nine 109
layers by alternate binding of the oppositely charged polymers. 110
57 approach are minimization of capsule size and graft volume
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58 (with possible reduction of islet core hypoxia) and better Finally, the nanocoated islets were collected and cultured in 111

59 preservation of the dynamics of insulin secretion. 12,13 medium M199. 112

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60 The concept of self-assembly of nano-particles around cells
61 was first introduced in the late 60’s, 14 and some years later this Electron microscopy 113
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62 approach was used to model multi-layer nano-films to be applied

To assess islet cell morphology, morphometry and ultrastruc- 114
63 in biological systems. 15 Since then, layer-by-layer nano-
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ture, uncoated (UC) and nano-encapsulated (NE) human islets 115

64 encapsulation methodologies have been implemented in various
were studied by electron microscopy, using methods previously 116
65 biomedical fields, 16 including islet transplantation. 17,18 The
described. 26,28 Briefly, samples were fixed with 2.5% glutaral-
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117
66 feasibility of multi-layer-by-layer (MLBL) approaches was
dehyde in 0.1 mol/l phosphate buffer with pH 7.4 for 2 h at 4 °C. 118
67 proved in rodent and pig islets, 17–22 with encouraging in-vitro
After rinsing with PBS, specimens were post-fixed in 1.0% 119
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68 and in-vivo results. Some in-vitro data are available with human
phosphate-buffered osmium tetroxide for 30 min at 4 °C, then 120
69 islets as well. Kozlovskaya et al 23 used hydrogen-bonded
dehydrated in a graded series of ethanol, transferred to propylene 121
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70 interactions of a natural polyphenol (tannic acid) with poly(N-

oxide, and embedded in Poly/bed 812 (Poliscience, USA). 122
71 vinylpyrrolidone) deposited on the human islet surface by non-
Ultrathin sections (60 to 80 nm thick) were obtained by using a 123
72 ionic layer-by-layer assembly, showing that in-vitro cell viability
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diamond knife and sections were placed on copper grids (200 124
73 and function were maintained for a few days.
mesh), and stained with uranyl acetate and lead citrate. 125
74 In another study, 10 poly-allylamine hydrochloride and poly-
Micrographs were obtained with varying degrees of magnifica- 126
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75 styrenesulfonate sodium salt were alternatively deposited on

tion, as appropriate. 127
76 human islets forming a nanometer-sized coating, which ensured
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77 in-vitro preservation of beta-cell ultrastructure and function and

Experimental animals 128
78 protection against anti-GAD(+) antibody recognition.
79 Hereby we report the results of a comprehensive study aimed Male C57BL/6J mice (Harlan Laboratories, Italy), 8 weeks 129
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80 at evaluating the histological and ultrastructural features of old and weighing 20-25 g, were used as islet graft recipients. 130
81 human islets coated by an MLBL nano-encapsulation procedure, Mice were moderately diabetic due to the intraperitoneal 131
82 as well as their in-vitro and in-vivo function and survival injection of nicotinamide in saline (150 mg/kg b.w.) (Sigma, 132
83 following xenotransplantation into diabetic mice. Saint Louis, MO, USA), followed 15 min later by administration 133
of streptozotocin (Sigma, 190 mg/kg b.w., i.p) in buffer citrate 134
(pH 4.5) prepared immediately before use. 29,30 Control mice 135
84 Methods received vehicles of both substances. Ten days after diabetes 136

85 Isolation and culture of human islets induction, the mice showing in two separate determinations a 137
non-fasting blood glucose concentration of ≥200 mg/dl were 138
86 Human islets were isolated by enzymatic digestion and used as recipients. The experimental protocol followed the 139
87 density gradient purification 24–28 from the pancreas of 32 non- Principles of Laboratory Animal Care (US NH publication no. 140
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Figure 1. Uncoated (UC) and nanoencapsulated (NE) human islets. (a and b) Bright field images of UC and NE islets respectively, showing well maintained morphology. Scale bar, 50 μm; (c and d) Fluorescence
microscopy images of NE human islets coated with chitosan and polystyrene sulfonate sodium salt in alternate layers. The 7th and 9th layers were coated with FITC-tagged chitosan to reveal the conformal

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nanocoating, as visualized 2-3 h after the coating procedure. Scale bar, 50 and 10 μm; (e) 2D confocal microscopy images of a nanocoated islet in gray field. Scale bar,10 μm. (f) 3D confocal image of an NE islet.
Scale bar,10 μm; (g and h) Cross-sections of nanocoated islets, showing that the nanolayers cover the islet cell surface completely; (i and j) Electron micrographs of an NE islet showing a beta cell and a portion of
the nanocoating layer at higher magnification. Scale bar, 0.5 and 0.15 μm. (N: nucleus; M: mitochondrion; IG: insulin granules; yellow arrow heads in panel j indicate the nanocoating over the plasma membrane).

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Figure 2. In-vitro function of UC and NE human islets. (A) Insulin secretion of batches of 15 UC and NE islets in response to 3.3 and 16.7 mM glucose during

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45 min static incubation; values are mean ± SEM of 8 separate incubation experiments; * P b 0.001 vs 3.3 mM glucose. (B) Dynamics of insulin release from
batches of 50 UC and NE human islets during perifusion experiments in response to varying glucose concentrations. Periods of glucose stimulation and glucose
concentrations were as indicated. Values are mean ± SEM of four separate perifusion experiments.

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141 83-85, revised 1985), including the humane care principle, and Endocrine cells were identified by the anti-Chromogranin A
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142 was approved by our institution's Ethical Committee. antibody (mouse monoclonal antibody, clone LK2H10, 172
prediluted; Ventana Medical Systems). Infiltrating mononuclear 173
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143 Transplantation procedure cells were detected by an anti-CD45 antibody (mouse monoclo- 174
nal antibody, clone RP2/18, prediluted; Ventana Medical 175
144 Mice were anesthetized with an intraperitoneal injection of
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Systems), an anti-CD68 antibody (mouse monoclonal antibody, 176

145 50 mg/kg b.w. penthobarbital sodium and transplanted with 500 clone KP-1, prediluted; Ventana Medical Systems) and an anti- 177
146 islets under the kidney capsule, according to the procedures
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myeloperoxidase (rabbit polyclonal antibody prediluted, 178

147 described by Zmuda et al 31 and Zhi et al. 17 Briefly, a lumbar Ventana Medical Systems). Citrate buffer with pH 7.3 was 179
148 incision was made and the kidney exposed. NE and UC human used for antigen retrieval and to ensure antibody specificity, 180
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149 islets were pelleted by centrifugation and placed underneath the consecutive sections were incubated with isotype matched 181
150 kidney capsule using a Hamilton syringe. All islets were control immunoglobulins and in the absence of primary 182
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151 transplanted with a delay of no more 3 h after nano- antibody. In these cases, no specific immunostaining was 183
152 encapsulation. detected. The images were acquired using the Leica DM5500 184
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B microscope (Leica, Germany) and analyzed using the Leica 185

153 In vivo function of islet graft
MMAF software. 186
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154 Blood glucose concentrations of recipient mice were

Statistical analysis 187
155 monitored every 2-3 days in non-fasting animals. Fifteen days
156 after transplant, we assessed the in-vivo acute function of the
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All results are expressed as mean ± SEM. Statistical 188

157 transplanted islets by an intraperitoneal glucose tolerance test. significance was evaluated by using the Student's t test, or the 189
158 Glucose (1.5 g/kg b.w., administered intraperitoneally as 16.5%
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analysis of variance (ANOVA) followed by post-test analysis, as 190

159 solution) was given to 3 h fasted, conscious mice. Drops of appropriate. 191
160 blood samples were sequentially collected from the tail vein at 0,
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161 15, 60, 120 and 180 min after the glucose injection, and
162 immediately used for glucose determination by a glucometer Results 192
163 (LifeScan Inc., CA, USA). After 4-5 weeks the islet bearing
164 kidneys were harvested and assessed for various immunohisto- Multi-layer-by-layer (MLBL) nano-encapsulation 193
165 chemistry and EM analysis.
Multi-layer-by-layer nano-encapsulation was achieved by 194
deposition of oppositely charged polymers (chitosan/PSS) up to 195
166 Immunohistochemistry
9 layers, using electrostatic binding. As shown in Figure 1, A and 196
167 Consecutive paraffin embedded tissue sections (2-4 μm B, NE and UC islets showed similar morphology as the uncoated 197
168 thick) were dewaxed and subjected to immunostaining by the islets, indicating that the nanoencapsulation procedure did not 198
169 streptavidin-peroxidase technique, using the automated Bench- alter overall islet architecture. Conformal coating was assessed 199
170 Mark XT staining system (Ventana Medical Systems, USA). by using chitosan tagged with FITC in the 7th and 9th (external) 200
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Figure 3. In-vitro protection studies. (A and B) Glucose-induced insulin stimulation index of UC or NE islets exposed for 72 h to a cocktail of pro-inflammatory
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cytokines (IL-1β 50, U/mL + IF-γ, 1000 U/mL) or 0.5 mM palmitate, respectively; values are mean ± SEM of four separate experiments; *P b 0.05 vs UC
islets; § P b 0.05 vs UC + cytokines. (C and D) Percentage of apoptotic beta cells in UC or NE islets exposed for 72 h to a cocktail of pro-inflammatory
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cytokines or palmitate, respectively; beta cells without or with signs of apoptosis were identified by electron microscopy ultrastructure analysis as shown in E
and F (yellow arrows indicate the nucleus of a normal beta cell in E and that of a beta cell with signs of apoptosis in F). Scale bar, 1 μm; values are mean ± SEM
of four separate experiments; *P b 0.05 vs UC islets; § P b 0.05 vs UC + cytokines.
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201 layers, followed by fluorescence microscopy analysis. A strong cultured for 7 days, and then hand-picked and stained with 222
202 fluorescence was observed on the surface of the islets 2-3 h after propidium iodide (PI), a dye binding to DNA in cells with 223
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203 nano-encapsulation (Figure 1, C and D), revealing complete and reduced cell membrane integrity (late apoptotic or dead cells), 224
204 uniform coverage. The NE islets also showed well maintained and the vital dye Hoechst 33342, to assess cell viability. As 225
205 plasma membrane (Figure 1, E) and the presence of bright shown in Supplementary Figure 2, no apparent difference was 226
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206 fluorescent spots on the islet surface, as observed by confocal detected in terms of cell survival between UC (84 ± 11%) and 227
207 microscopy (Figure 1, F), has also been noticed by other authors NE (87 ± 10%) islets, further confirming that nano- 228
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208 using mouse islets, and this might be due to the higher abundance of encapsulation did not affect cell viability. 229
209 extracellular surface charges. 17 In addition, cross-sections of NE
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210 islets (Figure 1, G and H), further confirmed that the nano layers In-vitro insulin secretion 230
211 covered the islet cell surface completely. Electron microscopy
212 analysis (Figure 1, I and J), performed on day 7 after the MLBL After 7 days of culture, in-vitro function of islets was 231
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213 procedure, confirmed the presence of a continuous coating of nano assessed by measuring glucose-stimulated insulin secretion 232
214 layers, whose thickness was 100.4 ± 4.2 nm, as measured in 12 (GSIS). Static incubation experiments showed that insulin 233
215 different preparations (in each the coating of at least 30 islets was release at both 3.3 and 16.7 mM glucose was similar between 234
216 determined). In addition, EM showed that islet cells (including beta UC and NE islets, achieving in both cases a four-fold increase at 235
217 cells as seen in Figure 1, I and J) maintained an apparently intact the higher glucose concentration (Figure 2, A). Moreover, 236
218 ultrastructural appearance. perifusion experiments confirmed that NE human islets main- 237
tained a quite normal glucose-induced insulin secretory response 238

219 Islet cell viability (Figure 2, B). Indeed, the increase of glucose concentration from 239
3.3 to 16.7 mM caused a relevant and sustained stimulation of 240
220 To further ascertain the biocompatibility of polymers used in insulin secretion from NE islets, reversible to basal values upon 241
221 the nano-encapsulation, free and encapsulated islets were discontinuation of high glucose, which was similar to the 242
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Figure 4. Palmitic acid uptake by UC and NE human islet cells. UC and NE human islets were metabolically labeled with azido-palmitic acid for 8-12 h and
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allowed to react with Alexa Fluor 594-alkyne. (A) Fluorescence microscopy images of UC and NE islets; the bright points, clearly visible in UC islets (a), but not
in NE islets (b) denote the presence of palmitic acid within the islet cells; in a1 and b1, cross sections of metabolically labeled UC and NE islets showing nuclei
stained with DAPI are reported; in a2 and b2, the increased presence of azido-palmitic acid counter-stained with alkylne-Alexa Fluor 594 in UC islets is shown;
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in a3 and b3, merged panels are reported, confirming the higher presence of palmitic acid inside the UC islets. Scale bar, 10 μm. (B) Bar graph showing
quantification of results described in A, assessed by fluorescence spectrophotometry and expressed as relative fluorescence units; values are mean ± SEM of 4
separate experiments.

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243 secretory pattern observed with UC islets. These results show Protective action of nano-encapsulation in-vitro
244 that NE human islets maintained appropriate secretory response In order to determine whether the MLBL coating has 248
245 in-vitro, thus indicating that the multilayers stratified on the cell protective effects in-vitro, we investigated islet insulin secretion 249
246 surface does not compromise physiological performances. and beta cell survival after 72-h exposure to cytotoxic levels of 250
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251 palmitate or cytokines. As shown in Figure 3, A and B, exposure

252 to either pro-inflammatory cytokines or palmitate was associated
253 with a significant decrease of glucose-stimulated insulin release
254 in UC islets, which was prevented in NE islets. In agreement
255 with these findings, when the frequency of apoptotic beta cells
256 was assessed by morphometric analysis of electron microscopy
257 images (Figure 3, E and F), pro-inflammatory cytokine or
258 palmitate treatment was unable to exert significant pro-apoptotic
259 effects in NE islets (Figure 3, C and D). Finally, to determine
260 whether the protective action of nano-encapsulation against
261 palmitate effects could be due to reduced palmitate entry into the
262 islets, we visualized and quantified palmitate uptake from both

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263 UC and NE islets by a fluorescent based click-iT approach. The
264 results (Figure 4, A and B). demonstrated that NE islets displayed

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265 a significantly reduced palmitate uptake when compared to UC
266 islets. Together, these data are consistent with the concept that in
267 our experimental conditions the MLBL coating could protect

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268 human islets from cytokines and palmitate toxicity.

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269 Nano-encapsulated islet transplantation studies

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270 We next asked if nano-encapsulated islets can induce
271 normoglycemia in-vivo in diabetic mice as well as immunopro-
272 tection. For this purpose, approximately 500 UC or NE human D
273 islets were xeno-transplanted under the kidney capsule of
274 C57BL/6J mice made moderately diabetic by combined
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275 administration of streptozotocin and nicotinamide. The results are
276 shown in Figure 5. As expected, diabetic mice showed elevated
277 blood glucose levels when compared to their age matched non-
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Figure 5. In-vivo assessment of transplanted islet grafts. (A) Non-fasting

278 diabetic controls throughout the experimental period. A slight blood glucose values in control mice (C), untreated diabetic mice (DM),
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279 reduction of hyperglycemia was observed when diabetic mice were diabetic mice transplanted with uncoated islets (TU) and diabetic mice
280 transplanted with UC human islets, with glycemic levels remaining transplanted with nanoencapsulated islets (TN). Data are expressed as
281 below those of untransplanted diabetic mice, yet not achieving a mean ± SEM of 3-6 mice for each group. (B) Glucose tolerance test results:
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282 statistically significant difference (Figure 5, A). On the other hand, Blood glucose levels were determined during an intraperitoneal glucose
tolerance test (glucose: 1.5 g/kg b.w.) performed after 15 days from
283 the reduction of glycemia was faster and quantitatively stronger in
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transplant. Data are expressed as means ± SEM of 3-6 mice for each
284 the diabetic mice transplanted with NE islets, in which hypergly- group. The inset in panel B shows the area under the curve (AUC) for the
285 cemic values decreased to levels slightly but not significantly higher glycemic values; symbols and colors as in panel A; * P b 0.05 or less vs
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286 than in non-diabetic controls and remained stable until the end of the diabetic mice (post-hoc Fisher test).
287 experimental time period (31 days) (Figure 5, I).
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To better understand the state of metabolic control in transplanted

289 diabetic mice, we performed an intraperitoneal glucose tolerance test enzyme richly expressed in neutrophil granulocytes), cluster of 307
290 (IPGTT) at 15 days after islet transplantation. The results (Figure 5, differentiation 68 (CD68) (that identifies circulating and tissue 308
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291 B) showed that the increased post-loading blood glucose profile resident macrophages) and cluster of differentiation 45 (CD45) 309
292 observed in untreated diabetic animals, clearly indicative of a severe (expressed in differentiated hematopoietic cells, including B and 310
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293 glucose intolerance, was significantly ameliorated (and almost T lymphocytes), with respect to UC islet grafts (Figure 6). 311
294 normalized) in the diabetic group transplanted with NE human islets, Furthermore, immunofluorescence analysis of islet graft showed 312
295 whereas the improvement was much less apparent in mice better preserved insulin- and glucagon-containing cells in NE 313
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296 transplanted with UC islets. The inset of Figure 5, B refers to than UC islets (Figure 7). Semi-thin sections from explanted NE 314
297 calculation of the areas under the blood glucose curve (AUC) and islet grafts confirmed the presence of numerous islets with well- 315
298 confirms the efficiency of the transplanted nanocoated islets in preserved morphology (Figure 8, A). Notably, in NE encapsu- 316
299 improving glucose tolerance. lated islet grafts, signs of graft revascularization around the islets 317
300 Finally, the islet grafts explanted 4-5 weeks after transplan- could be observed (Figure 8, B and C), indicating that the nano- 318
301 tation were analyzed by light and electron microscopy. coating allowed vascular formation, which in turn might 319
302 Hematoxylin/eosin staining and Chromogranin A immune- contribute to improve long-term survival and function of the 320
303 staining showed that islet cell morphology appeared to be well transplanted islets. Ultrastructural analysis also established the 321
304 preserved in the nano-coated grafts. These latter also exhibited presence of typical endocrine cells in explanted NE islet grafts 322
305 decreased immune cell infiltration, as determined by immune- (Figure 8, D and E) as well as the persistence of a well-defined 323
306 staining of multiple markers like myeloperoxidase (MPO) (an nanocoating system around the islets (Figure 8, E and F). 324
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Figure 6. Immunohistochemical analysis of transplanted islets grafts. Cross sections of islet grafts containing UC (upper panels) or NE (lower panels) human islets one month after transplantation. Better

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expression of Chromogranin-A (endocrine cells marker) containing cells and reduced expression of immune cells (MPO: myeloperoxidase, marker of leukocyte presence; CD68, marker of macrophages; CD45,
marker of T cells) were observed with nanoencapsulated (NE) islets. Scale bar, 20 μm.
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Figure 7. Expression of insulin and glucagon in transplanted islet grafts. Cross sections of islet grafts containing UC (upper panels) or NE (lower panels)
human islets one month after transplantation. Immunofluorescence staining for insulin (red) and glucagon (green) showed more insulin or glucagon containing
cells with NE islet grafts. Scale bar, 50 and 10 μm.

325 Discussion beyond the scarcity of donors, by the need for immunosuppres- 330
sive intervention following transplantation. 33 In the last few 331
326 Islet cell transplantation is generally viewed as one of the years, immune protection of transplanted islets by means of 332
327 most dependable clinical approaches to achieve insulin inde- suitable preventive micro-encapsulation has been introduced as a 333
328 pendence in patients with type 1 diabetes. 4, 32 However, new and ambitious approach to avoid life-long pharmacological 334
329 widespread utilization of this procedure is still discouraged, immune suppression. 10 Materials that are employed to 335
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Figure 8. Ultrastructural analysis of transplanted islet grafts. Electron microscopy analysis was performed with grafts removed one month after transplantation.
(a-c) Semi-thin sections of kidney tissue showing the presence of several NE islets, indicated by black arrows in a and b; in c, a human islet (HI) with preserved
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morphology is shown, together with endothelial cells (long red arrow), collagen fibers (CF) and a newly formed blood capillary (BC) with red blood cells inside
(red arrow heads). (d-f) Electron micrographs of NE islet graft showing: (d) the presence of different islet endocrine cells (β, α and δ) with normal ultrastructure;
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(e) insulin and glucagon granules with normal ultrastructure; (f) enlargement of e, also better showing the presence of the nanocapsule (black arrow heads).
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336 encapsulate pancreatic islets to be transplanted must fulfill two main Our results show that our method led to a continuous and 359
337 criteria: they should provide islets with an efficient shield against uniform coverage of the islets by a multilayer coating whose 360
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338 immune attack, and at the same time they should not interfere with thickness was approximately 100 nm, without any evidence of 361
339 the diffusion of oxygen, nutrients, glucose in and insulin out. 8–10 ultrastructural alterations of islet cells induced by the coating 362
340 In the present study, we have developed a novel multi-layer- procedure. The first step to assess the biocompatibility of this 363
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341 by-layer (MLBL) electrostatic binding method for the conformal multi-layer coating was to verify whether islet cells might be 364
342 coating of islet cells, using two differently charged polymers stressed by the encapsulation process itself in terms of both 365
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343 (positively charged chitosan and negatively charged polystyrene survival and preservation of functional properties, with particular 366
344 sulfonate sodium salts-PSS) and we have evaluated several reference to insulin secretory capabilities. Our targeted in-vitro 367
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345 features of such nano-encapsulated (NE) human islets both in- experiments actually showed that, after 7 days of culture, nano- 368
346 vitro and following xenotransplantation in diabetic mice. The use encapsulation had no deleterious effects on both cell survival and 369
347 of multilayer systems in order to modify the surface properties of glucose-stimulated insulin secretion (assayed by both static 370
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348 cells is well established and in this case it has been achieved by incubation and perifusion experiments). In this regard, it is worth 371
349 the layer-by-layer technique, based on the attachment of to mention that some conventional micro-encapsulation methods 372
350 oppositely charged polyions onto charged islet surfaces in a are actually associated with delayed and impaired insulin 373
351 self-assembly process. 34, 35 This technique enables the complete secretion due to the presence of a high diffusion barrier around 374
352 coverage of the islets because the perinsular capsule will serve as the islets. 36, 37 Overall, our in-vitro data on islet cell viability and 375
353 a template for polyion adhesion. 10 Moreover, we can take insulin secretory function provide evidence that the polymers 376
354 advantage of other favorable circumstances: the nanometer size used in the present study for conformal coating neither 377
355 of the capsule, the possibility to modulate islet functionality and compromise cell survival nor perturb cellular physiology. 378
356 immune protection by selecting different polyelectrolyte pairs, Moreover, ultrastructural analysis of NE islets further confirms 379
357 and the possibility to bind selected factors or molecules to the that our nano-encapsulation methodology NE does not alter sub- 380
358 polyelectrolyte layers. cellular organelles (e.g., mitochondria and endoplasmic 381
 F. Syed et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2018) xxx–xxx 11

382 reticulum) and preserves islet cell architecture, implying a high thus prevent rejection. Actually, both UC and NE transplanted 440
383 degree of biocompatibility. Furthermore, we also found that the human islets determined a reduction of glycemia in diabetic 441
384 nano-coating was able to reduce the damage caused by exposure mice, but only mice transplanted with NE islets lowered 442
385 of the islets in culture to harmful metabolic (palmitate) or glycemic levels nearby those of healthy control mice. This 443
386 inflammatory (cytokines) stimuli. Palmitate is known to induce blood glucose-lowering effect was in agreement with previously 444
387 beta cell dysfunction and death by multiple mechanisms, which reported results 17,48 and lasted (with the exception of a single 445
388 include stress of the endoplasmic reticulum, mitochondrial case, as mentioned) for the entire follow-up period (1 month). 446
389 alterations, increased oxidative stress and dysregulated autoph- Moreover, when glucose tolerance was evaluated 15 days after 447
390 agy. Similarly, beta cell exposure to cytokine causes endoplas- transplant by an IPGTT, it was observed that mice transplanted 448
391 mic reticulum stress, inflammation and apoptosis. 38–41 Whereas with NE human islets showed a better response to the glucose 449
392 the full understanding of the mechanisms involved in such an load with respect to mice transplanted with UC islets, as 450
393 effect is beyond the scope of this study, it is worth mentioning indicated by the reduction of the area under the blood glucose 451

F
394 that chitosan has been found to trap lipid molecules and curve to values not significantly different from non-diabetic 452
395 cytokines by electrostatic and/or hydrophobic interaction 42,43 controls. 453

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396 which could prevent their entry through the MLBL system After one month, the graft material was retrieved from the 454
397 described in the present study. kidneys of all surviving transplanted mice for histological 455
398 Based on these encouraging in-vitro results, we then analysis. It is known that early graft loss is significantly higher in 456

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399 investigated whether nano-encapsulation was also effective in islet than in pancreas transplantation and that this adverse 457
400 the in-vivo setting. To be profitably utilized for islet transplan- outcome is mainly due to the innate inflammatory response 458

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401 tation, a biocompatible nanocapsule must ensure: i) immune triggered by islet-derived pro-coagulant and pro-inflammatory 459
402 shielding of transplanted islets; ii) protection against inflamma- mediators. 49 Indeed, in animal models of islet transplantation, a 460

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403 tory reaction and fibrotic overgrowth, which in turn would severe inflammation at the graft site has been repeatedly 461
404 permit long-term islet cell survival; iii) adequate insulin secretion documented with intense leukocytes infiltration into and around 462
405 in response to changes in blood glucose levels and, even better, islets that could negatively affect beta-cell viability and
D 463
406 ability to normalize hyperglycemia in diabetic animals. To function. 50–52 In our experiments, HE staining of most explanted 464
407 ascertain the fulfillment of these objectives, we transplanted UC grafts revealed a local infiltration of inflammatory cells, whose 465
408 and NE human islets in mice made diabetic by the combined nature was displayed by specific markers. Interestingly, and 466
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409 administration of streptozotocin (STZ) and nicotinamide (NA). 44 consistently with their longer survival, in the explanted grafts of 467
410 This non-obese diabetes mouse model was developed on the NE islets, we observed a lower infiltration of inflammatory cells 468
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411 basis of a previous similar rat model 45 and it is characterized by associated with a better preserved islet cell morphology with 469
412 moderate hyperglycemia, hypoinsulinemia, growth impairment respect to those of UC islets. This result was further confirmed 470
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413 and approximately 75% reduction of pancreatic insulin stores.- by immunofluorescence analysis (showing the persistence of 471
44,46,47
414 These features make this model particularly suitable for insulin- and glucagon-containing cells in the explanted grafts), 472
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415 pharmacological research in diabetes. 29,30,46 The immune and by electron microscopy observation of intact endocrine cells. 473
416 protection was tested by using a xenotransplantation of human We have also to take into account that factors others than 474
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417 islets in mice without immune suppression. biocompatibility could play a relevant role in long-term survival 475
418 We choose the kidney subcapsular space as the site for islet of islet grafts. As isolated islets are completely de-vascularized 476
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419 transplantation because it offers several benefits over transplan- because of the permanent damage of the vascular supply caused 477
420 tation in the liver. In particular, after liver transplantation, islets by the enzymatic degradation during the isolation procedure, 53 478
421 spread out largely and are hardly detectable, whereas in the an important factor in graft failure could be represented by 479
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422 kidney they are located in a definite place and can be easily insufficient oxygenation and nutrition due to the post-transplant 480
423 explanted later for further studies. After transplantation, we lack or scarcity of blood vessels. 54 Indeed, as previously 481
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424 periodically monitored basal blood glucose levels of control, reported, vascularization of encapsulated islet grafts is critical 482
425 diabetic and transplanted mice for one month. A significant for islet survival and function, given the high oxygen demand of 483
islet cells. 55 In our experiments, signs of revascularization
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426 decrease of glycaemic levels few days after transplantation was 484
427 regarded as an index of successful engraftment, while the around the islets were seen in encapsulated islet grafts, indicating 485
428 subsequent failure to maintain lowered glycaemic levels was that the nano-coating we used allowed vascular formation, which 486
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429 considered a sign of rejection. In our study, we performed in turn might contribute to improve long-term survival and 487
430 separate transplantation experiments for each type of human function of the transplanted islets. Thus, the detected occurrence 488
431 islets (uncoated or nano-encapsulated) and, on the basis of the of some re-vascularization in the explanted grafts of NE islets 489
432 above mentioned indicators, we observed that during the follow- (and not in those of UC islets) might prompt further studies to 490
433 up time (1 month), 50% of UC islet grafts (i.e., 3 out of 6) were confirm this observation and address the underlying 491
434 rejected (and the corresponding mice were sacrificed), whereas mechanisms. 492
435 only 17% (i.e., 1 out of 6) of NE islet grafts failed. Although the In conclusion, islet nano-encapsulation with multi-layered 493
436 number of cases is still small to draw definite conclusions from coating could represent a profitable approach for immune 494
437 these results, nevertheless a clear and promising indication protection of transplanted islets. Our results demonstrate the 495
438 emerges that nano-encapsulation could be able to effectively feasibility of islet encapsulation with chitosan/PSS nano-thin 496
439 protect islet grafts from the host immune recognition system and coating, whose biocompatibility was testified by the in-vitro 497
 12 F. Syed et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2018) xxx–xxx

498 preserved islet cell survival and secretory function. Finally, 18. Dong H, Fahmy TM, Metcalfe SM, Morton SL, Dong X, Inverardi L, et 556
499 xenotranspantation of human islets in STZ/NA diabetic mice al. Immuno-isolation of pancreatic islet allografts using pegylated 557
nanotherapy leads to long-term normoglycemia in full MHC mismatch 558
500 provided evidence of a better glucose tolerance and an improved
recipient mice. PLoS One 2012;7e50265. 559
501 prolonged survival of NE with respect to UC islets, confirming 19. Wilson JT, Cui W, Chaikof EL. Layer-by-layer assembly of a conformal 560
502 that MLBL nano-encapsulation could represent a very helpful nanothin PEG coating for intraportal islet transplantation. Nano Lett 561
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506 Supplementary data to this article can be found online at 22. Blasi P, Luca G, Mancuso F, Schoubben A, Calvitti M, Giovagnoli S, et 569

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507 https://doi.org/10.1016/j.nano.2018.06.013. al. Conformal polymer coatings for pancreatic islets transplantation. 570
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 Supplementary Figure 1 Schematic representation of a human pancreatic islet showing the presence of different cell types and the
corresponding hormones.

Supplementary Figure 2 Viability of UC and NE human islets.(A) UC and NE human islets were cultured for 7 days, and then randomly
handpicked; bright fields (a, b) and immune-fluorescence images are shown; staining with propidium iodide (PI) identifies dead cells (a1, b1),
whereas staining with the vital dye Hoechst 3342 identifies live cells (a2, b2) and the overlay showing PI and Hoechst in a3 and b3. (B)
Quantification as summarized in the bar graph was performed using the Image J software (NIH) and is expressed as percentage of total cells.

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