Effects of Transplanted Islets Nano-Encapsulated with Hyperbranched

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Effects of Transplanted Islets Nano-Encapsulated with Hyperbranched Polyethylene Glycol and Heparin on Microenvironment Reconstruction and Glucose Control Muhammad R. Haque, Jee-Heon Jeong, Kyo Won Lee, Du Yeon Shin, Geun-Soo Kim, Sung Joo Kim, and Youngro Byun Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00364 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Bioconjugate Chemistry

Effects of Transplanted Islets Nano-Encapsulated with Hyperbranched Polyethylene Glycol and Heparin on Microenvironment Reconstruction and Glucose Control

Muhammad R. Haque†, Jee-Heon Jeong ‡, Kyo Won Lee §, ⊥, Du Yeon Shin §, ⊥, ǁ, ∇, Geun-Soo Kim §, ⊥, ǁ, Sung Joo Kim §, ⊥, and Youngro Byun*, †, O

†

Research Institute of Pharmaceutical Science, College of Pharmacy, Seoul National University,

1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea ‡

College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Republic of

Korea §

Transplantation Research Center, Samsung Biomedical Research Institute, 81 Ilwon-ro,

Gangnam-gu, Seoul 06351, Republic of Korea ⊥Department

of Surgery, Samsung Medical Center, Sungkyunkwan University School of

Medicine, Seoul 06351, Republic of Korea ǁ

Stem Cell & Regenerative Medicine Institute, Samsung Medical Center, Seoul 06351, Republic

of Korea. ∇Department

of Health Sciences & Technology, Samsung Advanced Institute for Health Sciences

& Technology, Graduate School, Sungkyunkwan University, Seoul 06351, Republic of Korea O

Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of

Convergence Science and Technology, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea 1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

*Address for Correspondence: Youngro Byun, Ph.D., Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea; Tel: +82-2-880-7866, Fax: +82-2-872-7864, E-mail: [email protected]

# Supporting Information

2 ACS Paragon Plus Environment

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ABSTRACT: The microenvironment of pancreatic islets gets disrupted during enzyme digestion and causes islets to remain in vulnerable state, leading to poor outcome in the initial days of transplantation. To avoid immune invasion while allowing the reconstruction of microenvironment of the transplanted site, we propose immunoisolation polymers, which can nano-encapsulate islets quickly without cytotoxicity. Here, non-human primate (NHP) islets were nano-encapsulated with hyperbranched polyethylene glycol (hb-PEG) and heparin by layerby-layer technology and transplanted into the kidney subcapsular space of diabetic C57BL/6 mice. An immunosuppressive drug protocol was applied to increase the survival time until the animals were sacrificed. The recipients of NHP islets exhibited high non-fasting blood glucose level (BGL) for 2-3 weeks, which was normalized afterwards. Immunohistochemical (IHC) analysis revealed an immature vascular basement membrane and cell surface integrins directly associated with poor initial insulin production. The transplanted grafts regained their own microenvironment within a month without any outside stimuli. No lymphocyte infiltration was observed in the grafts at any time. Humoral and cell-mediated immune responses were prominently diminished by the hb-PEG/Heparin nano-encapsulated islets. Immunoisolation accompanied by an immunosuppressive drug protocol protects islets by helping them avoid immunogenesis while at the same time allowing them to reconstruct their microenvironment.

KEYWORDS: hyperbranched polyethylene glycol, heparin, nano-encapsulation, non-human primate pancreatic islets, xenotransplantation.



■ INTRODUCTION Islet transplantation is a preferred treatment option in terms of invasiveness and immunogenicity over whole pancreas transplantation for treating type-1 diabetes mellitus (T1DM).1 However, it employs a suboptimal isolation technique and uses enzymes to digest extracellular matrix components of pancreas, all of which disrupts the islet microenvironment and results in inferior engraftment efficiency.2-3 Enzyme digestion includes several critical steps that involve factors such as digestion time, concentration of the enzyme, and temperature, and a small variation of these steps may result in poor isolation yield.1-2 For instance, a shorter digestion time and an improper collagenase composition would yield impure islets mixed with exocrine cells. On the other hand, a longer digestion time would increase the exposure of islets to the enzymes, resulting in low islet integrity due to disrupted cell-to-cell adhesion and cell-to-matrix interaction.1-2 Islets are miniature organs having numerous vasculature intracellularly.4 The capillaries are essential for transporting glucose, nutrients and oxygen into the core of the islets, and for distributing insulin out of the islets into the blood stream.5 Enzyme digestion, which is responsible for rupturing the blood vessels, therefore results in poor islet viability after transplantation. Several reports have shown, however, that islets regrow their vasculature after transplantation,6-7 and attempts have been made to promote islets to regrow their vasculature prior to transplantation by VEGF gene delivery or co-encapsulating VEGF protein with islets.8-9 Besides causing poor islet viability, enzyme digestion poses another problem, the loss of periislet basal membrane, which induces islet apoptosis.10-11 To overcome this problem, the function and viability of ß-cells in the islet had to be enhanced by re-establishing cell-to-matrix


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relationship before transplantation. This was done by using collagen and fibronectin in islet culture, which accelerated insulin release and reduced apoptosis.12 However, the complete regrowth of vascular basal membrane before transplantation in vitro has been barely demonstrated, indicating that the process might be time consuming. This also contradicts the Edmonton protocol which proposed that the cold ischemic time should be reduced before transplantation.13 The peri-islet basal membrane, which helps cells to migrate, differentiate, and survive, is also found to act as a barrier against leukocyte infiltration,14 as indicated by higher levels of leukocytes found at the ruptured peri-islet basal membrane along with a loss of insulin positive cells during the progression of insulitis.15 One of the ideas proposed by the Edmonton protocol was to transplant freshly isolated islets to minimize cold ischemia induced injury.13 However, freshly isolated islets might have a greater chance of immune rejection due to the loss of periislet basal membrane. The use of glucocorticoid free immunosuppressive drug protocol, also proposed in the Edmonton protocol, advanced the transplant outcome associated with immunogenicity, although it did not yield an impressive success rate in a long-term.13, 16 A great amount of research has been conducted in pursuit of islet immunoprotection that can be applied at the precarious initial stage of transplantation without using immunosuppressive drugs. In our previous study, we demonstrated that immunoisolated NHP islets with polyethylene glycol (PEG) employing layer-by-layer (LbL) nano-encapsulation method prolonged survival time in a mouse model when compared with naked islets. The addition of a glucocorticoid free immunosuppressive drug protocol synergistically improved the survival rate up to 100% for 150 days with no rejection, in contrast to the 50% survival rate of naked islets.17 We further improved the nano-encapsulation efficiency by incorporating heparin into the



encapsulation composition. LbL nano-encapsulation with PEG and heparin showed reduction in instant blood mediated inflammatory reactions (IBMIR) and immunogenesis, resulting in better survival outcome in NHP allo-recipients. In this study, we investigated islet microenvironment in regulating BGL in the context of vascular basal membrane, cell-to-matrix relationship, and cell-to-cell interaction in a NHP-tomouse xenotransplantation model. NHPs are a model of interest in preclinical studies as they are the phylogenetically and immunologically closest species to humans and can provide an understanding about human islets.18 Here we propose a polymeric nano-encapsulation system composed of hb-PEG and heparin to protect islets from the host’s humoral and cell-mediated immune activation during the typically vulnerable early stage of transplantation. Nanoencapsulation is non-toxic to islets, can be done within a brief time duration and immediately after isolation without prolonging the cold ischemic time.

■ RESULTS Loss of islet microenvironment during isolation delays engraftment after transplantation. Islet isolation caused the basement membrane collagen in islets to rupture (Figure 1a), leading to the loss of islets’ native microenvironment. IHC staining of the transplanted grafts showed hampered insulin production after one week of transplantation (Figure 1b). However, the transplanted islet restored its insulin production capability within a month. Insulin staining after 4 weeks of transplantation showed a significant increment (p

Effects of Transplanted Islets Nano-Encapsulated with Hyperbranched

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