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약학석사학위논문

Encapsulation of Islets with Clodrosome in

Matrigel in a Xenograft Murine Model

2013년8월

서울대학교대학원

분자의학 및 바이오제약학과

Muhammad Rezwanul Haque

i

ABSTRACT

The purpose of this study is to develop a potent immunosuppressive system to prevent islet

rejection after transplantation for the treatment of type 1 diabetes. This strategy is to

encapsulate islets in matrigel containing liposomal clodronate Clodrosome® to inhibit

activation of macrophages and immune cells in the early stage of transplantation. Liposomal

clodronate, clodronate-encapsulated liposomes, can deplete macrophages, thereby preventing

macrophage activation. The molecular imaging of Cy5.5 labeled liposome in matrigel

demonstrated that liposomal clodronate remained in the matrigel for over 7 days. To evaluate

the therapeutic efficacy of transplanted islets, four groups of islet transplanted mice (n=6 in

each group) were prepared, such as below 1) Islet group, 2) Islet-Matrigel group, 3) Islet-

Clodrosome group, and 4) Islet-Matrigel-Clodrosome group, where 2000 IEQ islets were

subcutaneously transplanted and the dose of clodronate was 6.25 mg/kg. When islets were

transplanted in matrigel containing liposomal clodronate (Islet-Matrigel-Clorosome group),

the median survival time (MST) of transplanted islets was significantly increased (> 60 days)

when compared to other groups. Immunohistochemical staining of islet-grafted tissues in

matrigel demonstrated that locally delivered liposomal clodronate in matrigel effectively

inhibited the activation of macrophage after islet transplantation. In addition, liposomal

clodronate was effective on inhibiting immune cell migration and activation, and pro-

inflammatory cytokine secretion was significantly down-regulated. In conclusion, locally

delivered liposomal clodronate in matrigel effectively improved the grafted survival time of

grafted islets.

Keywords: pancreatic islets; liposomal clodronate; matrigel; islet transplantation;

macrophage depletion

ii

Student ID: 2011-24242

Thesis advisor: Professor Youngro Byun

iii

TABLE OF CONTENTS

ABSTRACTS………………………………………………………………………….... i

LIST OF CONTENTS…………………………………………………………………... iii

LIST OF TABLES……………………………………………………………………..... v

LIST OF FIGURES…………………………………………………………….............. vi

ABBREVIATIONS………………………………………………………………………

viii

1. INTRODUCTION

1.1 Diabetes Mellitus…………………………………………………………….……1

1.2 Type 1 Diabetes (T1D)…………………………………………………………....3

1.3 Treatments of T1D………………………………………………………………...5

1.3.1 Pancreatic islet cell transplantation……………………………………….…7

1.4 Role of macrophages on rejection of transplanted islets………………………....10

1.5 Targeting macrophages by liposomal clodronate………………………………...12

1.6 Subcutaneous route of transplantation…………………………………….……..15

1.7 Extracellular matrix (ECM) based hydrogel as transplant scaffold……….……..17

1.8 Rationale………………………………………………………………….………19

2. MATERIALS AND METHODS

2.1 Animal………………………………………………………………..……..…..20

2.2 Pancreatic islet isolation……………………………………………...…………21

2.3 Optical imaging of liposomal clodronate……………………….………………21

2.4 Islet transplantation……………………………………………………………...22

2.5 Intraperitoneal glucose tolerance test (IPGTT)…………………………..………25

2.6 Quantification of insulin and cytokine levels in serum…………………………25

iv

2.7 Quantification of insulin and cytokine levels in matrigel………………………25

2.8 Immunohistochemistry………………………………………………………....26

2.9 Statistical analysis……………………..………………………...........................27

3. RESULTS

3.1 Releasing profiles of Cy5.5 labeled liposome from the transplanted matrigel in

nude mouse ……………………………………………………………..………..27

3.2 Survival time of transplanted islets………………………………………………29

3.3 Inhibition effects of liposomal clodronate and matrigel on inflammation and

immune cell activation…………………………………………………………...32

4. DISCUSSION………………………………………………………………………..37

5. CONCLUSION………………………………………………………………………38

6. REFERENCES……………………………………………………………………….41

v

LIST OF TABLES

Table 1. Properties of clodronate

Table 2. Composition of growth factor reduced matrigel

Table 3. Groups for transplantation with transplant composition

vi

LIST OF FIGURES

Figure 1. Schematic representation of the types of diabetes mellitus

Figure 2. Illustration of daily schedule of a Type 1 diabetic patient

Figure 3. Devices for treating Type 1 diabetes and the side effects of exogenous insulin

therapy

Figure 4. Different cell types in pancreas

Figure 5. Islet isolation and transplantation: from donor to recipient

Figure 6. Role of macrophages in rejection mechanism of transplanted islets

Figure 7. Targeting macrophages by liposomal clodronate

Figure 8. Common experimental and clinical islet transplant sites, with features of most

common sites.

Figure 9. Loss of extracellular matrix during isolation

Figure 10. Work scheme: Isolation to transplantation

Figure 11. In vivo bio-distribution of Cy5.5 labeled liposome. (a) Balb/c nude mice were

subcutaneously injected matrigel with Cy5.5 labeled liposome. (b) Photon counts of

injected sites in comparison with untreated group were quantitated at 14 day by in vivo

imaging system. (c) Ex vivo bio-distribution image of matrigel containing Cy5.5 labeled

liposome extracted from Balb/c nude mice after 14 days of transplantation. (d) Photon

counts of extracted matrigel containing Cy5.5 labeled liposome. Data were expressed as

mean ± SD (n=3). *Significantly lower (p < 0.001) compared to Day 0 group.

Figure 12. Non-fasting blood glucose level after islet transplantation subcutaneously into diabetic

mice (a) Islet group (n=6), (b) Islet-Clodrosome group (n=6) (Subcutaneously; 6.25 mg/kg)

(n=6), (c) Islet-Matrigel group (n=6), (d) Islet-Matrigel-Clodrosome group (n=6) (e) Graft

survival rate of matrigel encapsulated islet recipients with (black circle) or without (black

square) liposomal clodronate.

vii

Figure 13. The IPGTT of normal mouse (black circle; n=7), diabetic mouse (black square; n=3) and

Islet-Matrigel-Clodrosome group (black triangle; n=5, at 60 day of transplantation). Data

were expressed as mean ± SD.

Figure 14. Immunohistochemistry analysis after transplantation (Islet-Matrigel-Clodrosome, at 60

day; Islet-Matrigel, at 10 day). Grafts were stained for H&E, insulin, glucagon and

somatostatin. Asterisk: transplanted islets. Magnification X400

Figure 15. Immunohistochemistry analysis after transplantation (Islet-Matrigel-Clodrosome group, at

60 day; Islet-Matrigel group, at 10 day). (a) CD4, CD8, CD20 and CD11b positive cell

staining. Asterisk: transplanted islets. Magnification X400 (b) Evaluation of immune cell

infiltrations into transplanted matrigel. (CD8, CD20 and CD11b positive cell staining).

T=Tissue, M=Matrigel. Magnification X200

Figure 16. Insulin and pro-inflammatory cytokine levels in recipient’s serum and injected matrigel

(Islet-Matrigel-Clodrosome group, at 60 day; Islet-Matrigel group, at 10 day). (a) insulin

(n=5) (b) IL-1β (n=3) (c) TNF-α (n=3), Data were expressed as mean ± SD. (*P<0.05 and

***P<0.001)

Figure 17. Illustration of immunoprotection of locally delivered liposomal clodronate for successful

islet transplantation.

viii

ABBREVIATIONS

T1D Type 1 diabetes

T2D Type 2 diabetes

IBMIR Instant Blood mediated inflammatory reactions

APC Antigen presenting cell

ECM Extracellular matrix

ELISA Enzyme-linked immunosorbent assay

1

1. INTRODUCTION

1.1 Diabetes Mellitus

As defined by American Diabetes Association (ADA), diabetes mellitus is ‘a group

of metabolic disorders characterized by hyperglycemia resulting from defects in insulin

secretion, insulin action or both. The chronic hyperglycemia is associated with long-term

damage, dysfunction and failure of various organs, especially the eyes, kidney, nerves, heart

and blood vessels.’ The pathogenic processes involved in diabetes development range from

autoimmune destruction of pancreatic β cells with absolute deficiency of insulin to

abnormalities that result in insulin resistance. Both impaired insulin secretion and insulin

resistance may coexist in the same diabetic patients, and it often remains uncertain that which

reason is the primary cause of hyperglycemia. The majority of cases of diabetes fall into two

broad categories, type 1 diabetes (T1D) and type 2 diabetes (T2D). Commonly visible

symptoms in both types of diabetes include polyuria, weight loss, polydipsia, late

responsiveness to infections and blurred vision. T2D is the most abundant form of diabetes

accounting for ~90-95% of those with diabetes [1]. This form of diabetes is mostly the

consequence of insulin resistance in which the muscle and adipocytes do not respond

properly to insulin. On the other hand, T1D results from cell mediated autoimmune

destruction of the β-cells of the pancreas (Fig. 1).

2

Type 1 Diabetes: Insufficient insulin Type 2 Diabetes: Insulin resistance

Figure 1. Schematic representation of the types of diabetes mellitus

(Jul. 4 (2013) from website http://dtc.ucsf.edu/types-of-

diabetes/type1/understanding-type-1-diabetes/what-is-type-1-diabetes/ and

http://www.made4ll.com/disease/type-2-diabetes-medication/c)

3

1.2 Type 1 Diabetes (T1D)

T1D, previously known by the terms insulin dependent diabetes mellitus (IDDM) or

juvenile- onset diabetes, accounts for only 5-10% cases of total diabetic patient. T1D patients

need to maintain a tight balance among food intake time and menu, insulin administration

and a strict monitoring of blood glucose level for leading a sound and healthy life (Fig. 2).

Genetic susceptibility is one of the major causes of T1D; whereas, environmental insults also

play role to unleash autoimmunity. Clinical symptoms of T1D become visible following

several silent immune events. In response to autoantibodies, self reactive lymphocytes

become activated and migrated to the pancreas to destruct insulin producing beta cells. This

persistent and targeted destruction may go undetected for years until a majority of beta cells

have been rendered dysfunctional. As a result, the individual becomes dependent on insulin

for survival.

4

Figure 2. Illustration of daily schedule of a Type 1 diabetic patient

5

1.3 Treatments of T1D

Present therapies for treating T1D are 1) exogenous insulin therapy and 2) pancreas

transplantation. T1D was a fatal disease with death occurring shortly after diagnosis before

the insulin was discovered. The discovery of insulin by Paulescu, and then Banting and best

let it understandable that the deficiency of hormone needs to be replaced which is caused by

the autoimmune destruction of pancreatic islet β cells [2]. Even though exogenous insulin

therapy has been the standard therapy since the discovery of insulin, it has never been a cure

for the diabetic patient. Moreover, it has severe drawbacks including poor patient compliance,

sudden rise of body weight and the risk of hypoglycemia (Fig. 3). Pancreas transplantation

might be the ‘cure’ available for diabetes. However, this procedure is highly invasive and

requires lifelong immunosuppression [3]. In quest for a cure of T1D, islet transplantation

promises a better choice, as effective as pancreas transplantation with much less invasiveness.

6

Needle and Syringe

Insulin pen

Insulin pump

Side effects

Injection site - Itching, mild pain, redness, or swelling at the injection site

Systemically - Hypoglycemia, weight gain, insulin oedema,

Figure 3. Devices for treating Type 1 diabetes and the side effects of exogenous

insulin therapy

7

1.3.1 Pancreatic islet cell transplantation

Whole pancreas transplantation is very effective in maintaining long-term

normoglycemia in T1D patient. Concurrent pancreas and kidney transplantation is considered

to be the standard therapy for T1D patients with end-stage renal failure. Although more than

80% patients achieve insulin independence for 1 year, pancreas transplantation still retains

the risks related with any major surgical and long-term immunosuppressive drug therapy.

Comparatively, islet transplantation is less invasive and does not need general anesthesia.

Moreover, islet transplantation is free of the complications related to exocrine secretions of

the pancreas as experienced in whole pancreas transplantation (Fig. 4). The advantages of

islet transplantation over pancreas transplantation including the potential of modifying

immunogenicity through gene therapy, tissue encapsulation for immunoisolation and

potential for engraftment in immuneprivileged site.

Great advances have done in the field of islet transplantation in recent years. In the

year 2000, Shapiro et al. published results of a successful case of islet transplantation where

seven out of seven patients remained insulin independent at the end of 1 year using a steroid

free immunosuppressive drug regimen [4]. However, clinical application of islet

transplantation is limited by the need for lifelong immunosuppressive drug therapy and donor

deficiency.

The process of islet transplantation involves isolation of islets from a donor pancreas;

assess purity, purify islets from the exogenous cells and culture if needed. Afterwards,

transplant the isolated islets into a diabetic patient. Isolation techniques of the endocrine

portion of human pancreas have developed rapidly over past few decades. An overview of

human pancreatic islet isolation to transplantation process is illustrated on Fig. 5 [5].

8

Figure 4. Different cell types in pancreas

(Nabeel Bardeesy, Ronald A. DePinho. Pancreatic cancer biology and genetics. Nature

Reviews Cancer 2, 897-909 (December 2002))

9

Figure 5. Islet isolation and transplantation: from donor to recipient

(Shaheed Merani and A. M. James Shapiro. Current status of pancreatic islet

transplantation. Clinical Science (2006)110, 611–625)

Reviews Cancer 2, 897-909 (December 2002))

10

1.4 Role of macrophages on rejection of transplanted islets

As soon as islet cells are transplanted the host recognizes foreign antigens, which

leads to the process called ‘antigen presentation’ to the host immune cells. Antigen presenting

cells (APC) like macrophages and dendritic cells both of host and donor origin present the

foreign antigens to the host immune cells to reject the unfamiliar body. Allotransplantation,

which is mediated by a direct pathway of antigen presentation, involves migration of donor

APCs from the implanted site to the host T-cells resulting in the development of CD4+

helper

Th1 cells. Th1 cells turn out a set of cytokines that help activation of cytotoxic CD8+ T-cells

[6]. On the other hand, xenotransplantation of islets activate the indirect pathway in which the

donor antigens are eaten up by host APCs and make a complex with MHC II molecules. This

complex consequently leads to the activation of cytotoxic CD8+ T-cells [7].

Among the APCs the macrophages play the most important role on transplanted graft

rejection. Other than activating T-lymphocytes, NK cells and B-lymphocytes [8],

macrophages damage the transplanted graft by producing oxygen free radicals and other

cytokines including IL-1β, TNF-α, and IFN-γ. TNF-α potentiates the destruction of islet cells

by IL-1. It has been demonstrated that the cytotoxic effect of TNF-α, IFN-γ, IL-6 on islets is

cumulative. Another important mediator for islets graft rejection is the production of reactive

oxygen molecules by macrophages. The transplanted islet cells are very sensitive to free

radicals like oxygen radicals, hydroxyl radicals and NO species because of the lacking of the

scavenging activity. IL-1β and TNF-α-stimulated endogenous iNOS activity accelerate NO

production in islet β cells, which directly contributes cell injury. Reactive oxygen species

break down DNA strands which induces the DNA repair enzyme poly(ADP) ribose

polymerase (PARP). This PARP, In turn, uses and diminishes nicotinamide adenosine

dinucleotide (NAD). As a result, cell death occurs (Fig. 6) [9].

11

Figure 6. Role of macrophages in rejection mechanism of transplanted islets

12

1.5 Targeting macrophages by liposomal clodronate

Macrophages comprise the primary defense against foreign body invasion. They

are attracted to the transplanted site by complement components, immune complexes, and

collagen fragments. As macrophages have phagocytic capability towards foreign antigens, it

is a hard task to target them. Nowadays, liposomes are extensively used carriers for

delivering drugs to macrophages. Almost all types of drugs for macrophage involved

disorders have been tested using liposomal carrier. The multilamellar liposomal form of

clodronate has revealed as an effective macrophage depleting agent in improving allogeneic

graft survival time [10]. Clodronate belongs to a class of pharmaceutical compounds called

biphosphonates with a small molecular weight and high hydrophilic properties (Tab. 1). As

soon as liposomal clodronate has delivered to the recipient, the liposome will come into

contact with macrophages and other APCs and being recognized as foreign particles. The

macrophages engulf these foreign bodies to destroy them, which form an internal vesicle

called phagosome. Cellular lysosomes fuse with the phagosomes to form phagolysosome.

The internal pH of the phagolysosome was lowered down by the lysosomal membrane proton

pumps. The lysosomal digestive enzymes including phospholipases along with the low pH

lead to destroy the liposomal membrane, thus releasing the encapsulated clodronate. The low

internal pH of the phagolysosome conduct the released clodronate into the cytosol, which is

mistakenly recognized as cellular pyrophosphate to produce a non-hydrolyzable ATP analog

adenosine 5’-(β, γ- dichloromethylene) triphosphate (AppCCl2p) by several Class ll

aminoacyl-tRNA synthetases [11-13]. Cytosolic AppCCl2 cross the mitochondrial outer

membrane and irreversibly bind to the ATP/ADP translocase to inhibit the enzyme, which

starts pore openings of the mitochondrial inner membrane and the membrane loses its

integrity. It results in depolarization and molecular signals are allowed to be released out

from the mitochondrion. Finally, cell dies (Fig. 7) [14]

13

Molecular Weight 288.9 (Clodronate Disodium)

359.9 (Clodronate Disodium Tetrahydrate)

Formula CH10Cl2Na2O10P2 (Tetrahydrate form)

Common names

Clodronate disodium; Dichloromethylene diphosphonate, disodium

salt; DMDP; Cl2MDP

IUPAC

disodium tetrahydrate hydrogen [dichloro(hydrogen phosphonato)

methyl]phosphonate

CAS Number 22560-50-5

Pubchem ID 23724874

Table 1. Properties of clodronate

(Jul. 5 (2013) from website http://www.clodrosome.com/products/standard-

clodrosome-reagents/clodrosome/)

14

Figure 7. Targeting macrophages by liposomal clodronate

15

1.6 Subcutaneous route of transplantation

Lacy and colleagues suggested liver as an optimal transplantation site for islet cells

[15]. By the year 1980s, islet graft was successfully transplanted into human liver by infusing

islet cells through portal vein. Later, the report published on insulin dependence in diabetic

patients after islet infusion through the portal vein revealed liver as the site of choice for islet

cell transplantation in clinical trials [16, 17]. According to the International Islet Transplant

Registry, 90% of clinical islet transplantations have been performed to liver as a site of

transplantation. However, most of the patients resume insulin therapy following islet

transplantation into the liver, since the result is not everlasting. The major factor involve in

such poor outcome is the loss of as many as 50% to 75% islets during engraftment in the

liver. As a result, huge numbers of islet cells are required to achieve insulin independence.

Moreover, liver site is associated with haemorrhage, thrombosis and instant blood mediated

inflammatory reactions (IBMIR). An ideal site for islet transplantation, from the

immunologic point of view, should minimize early graft rejection by keeping inflammatory

reactions suppressed even after transplantation. Reduced graft-blood interaction minimizes

activation of complement and blood coagulation cascade known as the instant blood mediated

inflammatory reactions (IBMIR). From the surgical standpoint, the ideal site would be easily

accessible and non-invasive to meet patient compliance. In addition, good access of

immunosuppressive drugs, ease of monitoring make a site closer to the ideal site for

transplantation. A subcutaneous site might be a choice, which closely meets the requirements

of being an ideal site. It is easily accessible and can be screened whenever there is a need.

Sadly, the reports been published on this site for islet transplantations are not satisfactory.

One of the reasons may be the subcutaneous harsh condition play a negative impact on the

viability of transplanted graft (Fig. 8).

16

Figure 8. Common experimental and clinical islet transplant sites, with features of

most common sites.

(S. Merani, C.Toso, J. Emamaullee, A.M.J.Shapiro. Optimal implantation site for

pancreatic islet transplantation. British Journal of Surgery 2008; 95: 1449–1461)

17

1.7 Extracellular matrix (ECM) based hydrogel as transplant scaffold

Islet cells are usually surrounded by a thin capsule made up of a layer of fibroblasts

and the collagen they produce. This capsule layer is closely related to the periinsular

basement membrane (BM), which is a specific type of extracellular matrix (ECM) composed

of laminin and nonfibrillar collagen linked by interactions with entactin/nidogen. Islets are

heavily influenced by cell-ECM interactions. This interaction regulates cell survival, insulin

secretion, proliferation, and helps restoring islet morphology. ECM proteins and

polysaccharides encode extracellular signals necessary for cell that interact directly with cell

membrane receptors. ECM also binds and store growth factors, cytokines and other soluble

signaling molucles, thus, modulates cellular activities [18]. However, during isolation, islets

undergo mechanical stresses and expose to cell digestive enzymes (Fig. 9). Consequently,

they lose their ECM and internal vascularization. This is why, islet transplantations are

associated with lower engraftment efficiencies than the whole pancreas transplantation. β-cell

culture demonstrated that ECM based materials improve cell survival, proliferation, and

insulin secretion [19]. The use of matrigel, a basement membrane protein based hydrogel, can

provide islet cells a three-dimentional support structure similar to those in the tissue of origin

during the posttransplantation period. Matrigel is extracted from Engelbreth-Holm-Swarm

(EHS) mouse sarcoma, a tumor rich in ECM proteins. It is mainly composed of laminin,

collagen iv, heparan sulfate proteoglycan and entactin with trace amount of various kinds of

growth factors (Tab. 2). For this composition, matrigel is effective for the attachment and

differentiation of different cell types.

18

Composition Percentage %

bFGF (pg/ml) 0-0.1

Epidermal growth factor (ng/ml) <0.5

Insulin-like growth factor-1 (ng/ml) 5

Platelet-derived growth factor (pg/ml) <5

Nerve growth factor (ng/ml) <0.2

Transforming growth factor beta (ng/ml) 1.7

Proteins: Laminin, Collagen IV, Heparan sulfate proteoglycan, Entactin 83

Isolation

Enzyme

digestion

ECM layer

disrupted

Islets seeded into

Hydrogel Scaffold

Figure 9. Loss of extracellular matrix during isolation

Table 2. Composition of growth factor reduced matrigel

(Modified July, 07 (2013) from website

http://www.bdbiosciences.com/ptProduct.jsp?ccn=356231)

19

1.8 Rationale

Successful islet transplantation represents a promising method for the treatment of type

1 diabetes. However, among type 1 diabetic patients received an islet transplant followed by

long-term cocktailed immunosuppressive drug therapy, only 50% of patients maintained

insulin independence for 5 years [20]. The main cause of graft failure is due to the activation

of host’s immune system and subsequent release of antigen from the transplantation site. The

innate immune system is triggered by activation of macrophage and neutrophil, causing

inflammation and immune cell infiltration into the transplanted site [21]. The activation of

macrophages and neutrophils release the inflammatory cytokines and reactive oxygen

species, thereby activating antigen-presents cells (APCs), helper T cells (CD4) and cytotoxic

T cells (CD8) [22, 23]. Various secreted cytokines and growth factors from macrophage are

regulated by immune reaction and the activation of the immune reactions subsequently

damage the transplanted islets [24]. Macrophages have several additional functions such as,

antigen presentation and phagocytosis. Thus, macrophages play a key role in the initiation

and maintenance of immune reactions and inflammatory reactions in the microenvironment

of transplanted islets. [22, 25].

Therefore, depletion of macrophage activation is a crucial strategy to inhibit islet graft

rejection. Bottino et al. reported the effect of macrophage depletion on graft survival and

microenvironment activation. It has been reported that liposomal clodronate improved the

survival time of grafted islets, which helped to inhibit the initiation of immune reaction [10,

24, 26-29]. In addition, Wu et al. demonstrated that xenografted porcine islets rejection was

delayed in macrophage-depleted mice [27]. However, systemically delivered liposomal

clodronate was not effective to induce macrophage depletion because of its short retention

time in blood. Also, systemically administered liposomal clodronate could cause many side

effects [30, 31]. Therefore, it is important to find appropriate methods for sustaining delivery

20

of liposomal clodronate. Many studies have investigated the portal vein infusion as an

alternative site of delivery since current clinical pancreatic islet transplantations are being

performed by the intraportal infusion. However, since the liver is known to have a very

strong immune response and inflammation reaction (e.g. IBMIR), intraportal delivery of

liposomal clodronate is not being seen as the optimal route of delivery [32]. Currently, many

investigations have demonstrated that the subcutaneous delivery could be a proper site using

injectable hydrogel owing to the minimal invasiveness and easy access [33-37].

In these respects, we expected that locally delivered liposomal clodronate using

injectable hydrogel would reduce the side effects and maximize the drug action. In this study

we have demonstrated that subcutaneous delivery of pancreatic islets embedded within

matrigel containing liposomal clodronate would be significantly enhance the survival time in

T1D mice model.

2. MATERIALS AND METHODS

2.1. Animal

Sprague-Dawley (SD) rats (male, 8 weeks old) were used as islet donors and inbred

C57BL/6 mice (male, 7-8 weeks old) were used as recipients. They were purchased from

Orient Bio Inc. (Seongnam, South Korea) and were housed under a specific pathogen-free

condition at our institution. T1D was induced chemically in recipient C57BL/6 mice by a

single intraperitoneal injection of 180 mg/kg of streptozocin (STZ; Sigma, St. Louis, MO).

Mice with the blood glucose level over 350 mg/dl for two consecutive days were selected as

diabetic recipients for transplantation. All experimental and surgical procedures were

conducted by following the guide-lines of the Institute of Laboratory Animal Resources,

Seoul National University (IACUC no. SNU-070822-5).

21

2.2.Pancreatic islet isolation

Pancreatic islets were isolated from the pancreases of outbred male SD rats. Briefly,

SD rats were anaesthetized with intraperitoneal injection of ketamine (90 mg/kg) and

xylazine (10 mg/kg) mixture, and pancreases were exposed by laparotomy. The common bile

duct was ligated, cannulated with a 25-gauge-needle, and then 10 ml of Hank’s balanced salt

solution (HBSS; Sigma) containing 0.8 mg/ml Collagenase P (Roche, Indianapolis, IN) was

injected. Distended pancreases were removed and incubated at 37 °C for 20 min. After

incubation, digested tissues were washed with cold HBSS and filtered through a tissue-

collecting sieve (Sigma; 40 mesh). Islets were then purified by centrifuging in the solution

having discontinuous Histopaque (Sigma) density gradient at 2400 rpm for 18 min. Isolated

islets were cultured for 3 days in RPMI-1640 (Sigma) containing 10% fetal bovine serum

(FBS; Sigma) at 37 °C in a humidified 5% CO2 atmosphere.

2.3. Optical imaging of liposomal clodronate

Noninvasive imaging of the transplanted liposomal clodronate was carried out using

Cy5.5 labeled liposome (Encapsula NanoScience, Nashvile, TN) in male Balb/c nude mice

(Orient Bio Inc.) over an extended period of time. Cy5.5 labeled liposome was added to

growth factor reduced Matrigel® (BD Biosciences, Franklin Lakes, NJ), mixed

homogeneously using pre-cooled pipet tips and injected immediately through the

subcutaneous part on the scruff region of the pre-anesthetized mice. They were, then, laid

down in prone position, fixed on scanning plate and placed inside Optix acquisition system

(Optix MX3TM

, ART Advanced Research Technologies Inc., Montreal, Canada). Cy5.5

labeled liposome localization on the transplanted mice was assessed for 14 days by using

OptiScan™ software (ART Advanced Research Technologies Inc.). After 14 days, the

transplanted matrigel containing Cy5.5 labeled liposomes were retrieved and the fluorescence

22

intensity was compared with the matrigel excised right after transplantation. The intensity

profiles were obtained using OptiViewTM

software (ART Advanced Research Technologies

Inc.) on all images.

2.4. Islet transplantation

To evaluate the therapeutic effect of transplanted islets, four groups of islet

transplanted mice (n=6 in each group) were prepared as below; 1) 2000 IEQ islets were

subcutaneously transplanted without matrigel (Islet group), 2) 2000 IEQ islets were

subcutaneously transplanted in matrigel (Islet-Matrigel group), 3) 2000 IEQ islets were

subcutaneously transplanted with liposomal clodronate (Clodrosome®, Encapsula

NanoSciences, Nashville, TN) without matrigel (Islet-Clodrosome group), 4) 2000 IEQ islets

were subcutaneously transplanted in matrigel containing liposomal clodronate (Islet-

Matrigel-Clodrosome group) (Tab. 3).

Matrigel was stored in -20 °C and thawed overnight at 4 °C in ice before use. After

matrigel reaches to its liquid jellylike state, 500 µl of matrigel was added to 2000 IEQ of

islets suspended in 20 µl PBS (Life technologies, Grand Island, NY) with or without 25 μl of

liposomal clodronate at 6.25 mg/kg dose. The mixture was homogenized by pipetting several

times using pre-cooled pipette tips in microtubes. This process is conducted on ice to prevent

matrigel gelation as it rapidly forms gel at 22 °C to 35 °C. Diabetic mice were anesthetized

by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (16 mg/kg). Pre-cooled 1 ml

syringe with 26-gauge-needle was used to load the matrigel with islets with or without

liposomal clodronate and the mixture was subcutaneously injected into the scruff of the mice.

Matrigel was rapidly gelated just after injection at body temperature (Fig. 10).

The transplantation procedure was considered as successful if the blood glucose level

decreased less then 200 mg/dl within 3 days after transplantation. Non-fasting blood glucose

23

level was monitored everyday by drawing blood from the tail veins using glucometer (Super

glucocard II, Arkray, Kyoto, Japan) and body weight was also checked as well. The

transplanted islets were considered graft rejection if the blood glucose concentration was

higher than 300 mg/dl for three consecutive days.

24

Groups Matrigel µl Islet cell

(PBS) µl

Clodrosome

(5 mg/ml) µl

PBS µl

SC 2000 IEQ Islet - 100 -

SC 2000 IEQ Islet + Clodrosome

6.25 mg/kg

- 100 25

SC 2000 IEQ Islet + Matrigel 500 20 25

SC 2000 IEQ Islet + Matrigel +

Clodrosome 6.25 mg/kg

500 20 25 -

Blood glucose

measurement;

IPGTT

Islet isolation

Enzyme digestion:

Collagenase P;

Density gradient: Histopaque SD rats Isolated islets

Mix islets

w/ or w/o MG

w/ or w/o

Clodrosome

Transplantation

2000 IEQ islet

subcutaneous

Gel retrieval

-ELISA

-IHC

Isola

tion

Tra

nsp

lan

tati

on

Figure 10. Work scheme: Isolation to transplantation

Table 3. Groups for transplantation with transplant composition

25

2.5. Intraperitoneal glucose tolerance test (IPGTT)

Intraperitoneal glucose tolerance testing (IPGTT) was performed at the 60th

day of

transplantation to evaluate the glucose responsiveness of the transplanted islets and compared

the effect with healthy and diabetic mice. Mice were administered 20% glucose solution

(Sigma) at a dose of 2 g/kg into the peritoneal cavity following overnight fasting. Blood

glucose levels were measured at 0, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. At the end of

this experiment, transplanted matrigel was retrieved and histology and cytokine

concentrations were analyzed.

2.6. Quantification of insulin and cytokine levels in serum

After anesthetized the mice by diethyl ether (Sigma), blood was collected by retro-

orbital sinus puncturing. The whole blood was allowed to clot by leaving it at room

temperature for 30 min. Clot was removed by centrifugation at 2000 g for 10 min in a

refrigerated centrifuge. The supernatant was immediately transferred into another tube,

aliquoted and stored at -70 °C. Insulin level was measured using a rat/mouse insulin ELISA

kit (Millipore Corp., Billerica, MA), cytokine IL-1ß (R&D Systems, Minneapolis, MN) and

TNF-α (BioLegend, Inc., San Diego, CA) concentration were also measured in accordance

with the manufacturer’s instructions.

2.7. Quantification of insulin and cytokine levels in matrigel

Transplanted matrigel was retrieved from the transplanted site, followed by

homogenizing in hypotonic lysis buffer (1 ml of 1% RIPA buffer, Sigma) and centrifuging at

5000 g for 5 min. The supernatant was collected, aliquoted and stored at -70 °C. Insulin

concentration was measured by using a rat/mouse insulin ELISA kit (Millipore Corp.).

Cytokine IL-1ß (R&D Systems) and TNF-α (BioLegend) concentrations were also measured

26

by ELISA.

2.8. Immunohistochemistry

At 60th

day after islet transplantation, matrigel was retrieved and fixed in 10%

formalin for 2 days at room temperature. To evaluate immune cell infiltration and hormone

secretion, the matrigel was embedded in paraffin and sectioned as 4 µm. The sections were

deparaffinized by heating in dry oven for 1 h and washing vigorously in xylene. Slides were

then rehydrated serially in 100%, 90%, 80% and 70% alcohol. The antigens were retrieved by

heating in 10 mM citrate buffer (pH 6.0, Sigma) using microwaves (5 min, 3 times, 700 W),

and then cooling down to room temperature for 20 min. Citric acid was neutralized by

immersing the slides in 3% H2O2 (Sigma) consisted in 70% methanol (Sigma) for 15 min.

After washing in PBS, the slides were incubated overningt at 4 °C with mouse monoclonal

anti-insulin (1:50; Abcam Inc., Cambridge, MA), anti-somatostatin (1:50; Biomeda, Foster

City, CA), anti-glucagon (1:200; DAKO, Carpinteria, CA), anti-CD31 (1:50; Santa Cruz

Biotechnology Inc., Santa Cruz, CA), anti-CD11b (1:10; eBioscience, San Diego, CA), anti-

CD20+ (1:40; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-CD4

+ (1:100; Abcam

Inc., Cambridge, MA), and anti-CD8+ (1:25; BioLegend, San Diego, CA) in a humidified

chamber, respectively. Next day before further treatments, the slides were kept at room

temperature for 1 h. After washing, the tissue sections were observed with a peroxidase

labeled polymer conjugated to goat anti-mouse immunoglobulins in Tris-HCl buffer

(Envision plus System-HRP labeled polyer; Dako, Glostrup, Denmark), which was incubated

for 2 h at room temperature. Slides were washed, and the chromogen was developed for 15

min with liquid 3, 30-diaminbenzidine (Dako). The slides were counterstained with Mayer

hematoxylin. Negative controls were treated similarly with the exception of primary

antibodies. All slides were gradually dehydrated using 70%, 80%, 90% and 100% alcohol.

27

Finally, tissue slides were fixed using mounting medium (Dako) with a glass coverslip.

2.9. Statistical analysis

The survival time was analyzed as the median ± SEM. In vivo optical intensity, IPGTT

of the transplanted islets, insulin and cytokine concentrations were expressed as the mean ±

SD. Statistical analysis was carried out using the unpaired t-test or ANOVA one-way test. The

p value of less than 0.05 was considered to be statistically significant.

3. RESULTS

3.1. Releasing profiles of Cy5.5 labeled liposome from the transplanted matrigel in nude

mouse

The localization and release rate of encapsulated liposomal clodronate from the

matrigel in nude mice was evaluated using molecular imaging technique for 14 days after

transplantation. Cy5.5 labeled liposome loaded in matrigel was injected subcutaneously (Fig.

11a) and the fluorescence images were taken at different time points. The fluorescence

intensity at the transplanted site was gradually decreased with time. The fluorescence

intensity (95.60 ± 7.33 photons) was remarkably decreased at day 14 of transplantation,

compared with the fluorescence intensity of first time point (497.33 ± 88.57 photons),

indicating that the liposome was gradually released out from the matrigel (Fig. 11b).

Afterwards, the matrigel was retrieved, and the fluorescence intensity was measured (Fig.

11c). It was also confirmed that 98% of the Cy5.5 labeled liposome was released out from the

matrigel after 14 days of transplantation (Fig. 11c,d).

28

(c) Ex vivo Imaging

0 h

8

h

24 h

72 h

5 d

ay

7 d

ay

9 d

ay

12 d

ay

14 d

ay

Matrigel Matrigel+ Drug

(a) In vivo Imaging (b) Intensity profile for In vivo Imaging

0 h

14 d

ay

(d) Intensity profile for Ex vivo Imaging

Figure 11. In vivo bio-distribution of Cy5.5 labeled liposome. (a) Balb/c nude mice

were subcutaneously injected matrigel with Cy5.5 labeled liposome. (b) Photon

counts of injected sites in comparison with untreated group were quantitated at 14

day by in vivo imaging system (c) Ex vivo bio-distribution image of matrigel

containing Cy5.5 labeled liposome extracted from Balb/c nude mice after 14 days of

transplantation. (d) Photon counts of extracted matrigel containing Cy5.5 labeled

liposome. Data were expressed as mean ± SD (n=3). *Significantly lower (p <

0.001) compared to Day 0 group.

29

3.2. Survival time of transplanted islets

In order to evaluate the therapeutic potential of liposomal clodronate as an

immunosuppressive drug and also to evaluate the efficacy of matrigel in protecting

transplanted islets, 2000 IEQ of islets were transplanted into STZ-induced diabetic C57BL/6

mice. Non-fasting blood glucose levels of the recipients were measured to determine the

viability of islets after transplantation. For Islet group and Islet-Clodrosome group, none of

the mice achieved normoglycemia, as shown in Fig. 12a and 12b. However, the mice in Islet-

Matrigel group showed that the blood glucose level was returned to normoglycemia after islet

transplantation (MST: 5.50 ± 0.22 days, Fig. 12c). This result proposed the importance of

matrigel as the extracellular matrix to maintain the viability of transplanted islets. To improve

the survival time of transplanted islets in matrigel, we further evaluated the combination and

immunosuppressive effects of matrigel along with liposomal clodronate (Islet-Matrigel-

Clodrosome group, Fig. 12d). In case of this group, all recipient mice had achieved

normoglyemia within 24 h after the transplantation and 83.33% of recipient mice maintained

normoglycemia for more than 60 days (Fig. 12e). The combination system of matrigel and

liposomal clondronate was significantly enhanced the survival time of transplanted islets.

At 60th

day after the islet transplantation of Islet-Matrigel-Clodrosome group, IPGTT

was performed to evaluate the glucose responsiveness in vivo (Fig. 13). After administration

of a high dose of glucose, the normal mice could maintain blood glucose level rapidly to the

normal level within 2 h. While, the diabetic mice could not maintain; blood glucose level

remained over 500 mg/dl after 5 min of the glucose injection. Comparing to the diabetic

group, the blood glucose level profile of Islet-Matrigel-Clodrosome group was well

maintained, indicating that islet transplanted recipient had normal glucose sensitivity until 60

days of transplantation.

30

Time (day)

0 2 4 6 8 10

Non fasting b

lood g

lucose (

mg/d

l)

0

200

400

600

800

0 20 40 600

20

40

60

80

100

120

Time (day)

Gra

ft s

urv

ival ra

te (%

)

Time (day)

0 2 4 6 8 10

Non fasting b

lood g

lucose (

mg/d

l)

0

200

400

600

800

Time (day)

0 2 4 6 8 10

Non fasting b

lood g

lucose (

mg/d

l)

0

200

400

600

800

Time (day)

0 10 20 30 40 50 60

No

n f

astin

g b

loo

d g

luco

se

(m

g/d

l)

0

100

200

300

400

500

600

Figure 12. Non-fasting blood glucose level after islet transplantation

subcutaneously into diabetic mice (a) islet group (n=6), (b) Islet-Clodrosome group

(Subcutaneously; 6.25 mg/kg) (n=6), (c) Islet-Matrigel group (n=6), (d) Islet-

Matrigel-Clodrosome group (n=6), (e) Graft survival rate of matrigel encapsulated

islet recipients with (black circle) or without (black square) liposomal clodronate

(a) Islet group

(b) Islet-Clodrosome group

(c) Islet-Matrigel group

(d) Islet-Matrigel-Clodrosome group

(e) Survival Day

31

0 30 60 90 1200

200

400

600

800

Time (min)

Blo

od g

lucose level (m

g/d

l)

Figure 13. The IPGTT of normal mouse (black circle; n=7), diabetic mouse (black

square; n=3) and Islet-Matrigel-Clodrosome group (black triangle; n=5, at 60 day of

transplantation). Data were expressed as mean ± SD.

32

3.3. Inhibition effects of liposomal clodronate and matrigel on inflammation and immune cell

activation

To further investigate the functionality of transplanted islets and immune cell

invasion to the graft, histological analysis was carried out on two groups, such as, Islet-

Matrigel group and Islet-Matrigel-Clodrosome group. Immunohistological analysis for Islet-

Matrigel-Clodrosome group was conducted on 60th

day of transplantation and for Islet-

Matrigel group right after rejection at different time points ranging from day 4 to day 10 of

transplantation. Hematoxylin and eosin (H&E) staining demonstrated that islet morphology

was disrupted when islets were transplanted in matrigel; but when liposomal clodronate was

loaded in the matrigel, the structure of transplanted islets was remained intact (Fig. 14).

Insulin, glucagon and somatostatin were released from well-structured islets in Islet-Matrigel-

Clodrosome group, on the other hand, those hormones in Islet-Matrigel group were stained

irregularly.

One possible reason for the destruction of islets structure in Islet-Matrigel group

might be due to the infiltration of lymphatic cells to the transplanted graft since significantly

great number of CD4+, CD8

+, CD20

+ and CD11b

+ positive cells were detected in this group.

However, much less infiltration of CD4+, CD8

+, CD20

+ and CD11b

+ positive cells were

found in Islet-Matrigel-Clodrosome group (Fig. 15a,b).

Furthermore, we measured insulin and pro-inflammatory cytokine concentration

quantitatively both in serum and matrigel in order to confirm the functionality of transplanted

islets and immune reactions. In case of Islet-Matrigel-Clodrosome group, insulin

concentrations in both matrigel and serum (14.35 ± 11.19 µg/ml and 1.24 ± 0.56 ng/ml) was

higher than those of Islet-Matrigel group (0.46 ± 0.34 µg/ml and 0.41 ± 0.33 ng/ml), which

were statistically significant (p < 0.05) (Fig. 16a). These results indicate that the liposomal

clodronate did not interfere with the insulin secretion of the transplanted islets and the islets

33

in the matrigel containing liposomal clodronate were well functioned.

To assess whether macrophage-depleting agent could reduce the macrophage

activation and the related cytokine expression in the transplanted site, the concentrations of

IL-1β and TNF-α have been determined. As shown in Fig. 15b, the IL-1β concentration in the

matrigel was significantly decreased as low as 0.5% by the liposomal clodronate in the

matrigel, that is, the IL-1β concentrations in matrigels of Islet-Matrigel group and Islet-

Matrigel-Clodrosome group were 582.75 ± 269.49 pg/ml and 3.25 ± 6.50 pg/ml (p < 0.05),

respectively. In addition, the TNF-α concentration in the transplanted matrigel was also

significantly decreased by the liposomal clodronate in the matrigel such that the TNF-α

concentrations in matrigels of Islet-Matrigel group and Islet-Matrigel-Clodrosome group

were 72.33 ± 10.40 pg/ml and 6.80 ± 4.73 pg/ml (p < 0.001), respectively. Likewise, the

serum concentrations of IL-1β and TNF-α were also decreased by liposomal clodronate in the

matrigel such as IL-1β (202.33 ± 73.08 pg/ml to 138.67 ± 49.51 pg/ml) and TNF-α (20.54 ±

11.42 pg/ml to 15.59 ± 12.14 pg/ml), as shown in Fig. 15c. These results indicated that

macrophage-depleting agent significantly reduced the secretion of IL-1β and TNF-α from

macrophage and inflammatory reaction. In addition, the concentration of these pro-

inflammatory cytokines at the site of transplantation was higher than in the serum, which

justifies the co-delivery of liposomal clodronate along with islets implanted within matrigel

instead of systemic delivery of the drug.

34

H&E Insulin

Isle

t-M

atri

gel

Is

let-

Mat

rigel

-Clo

dro

som

e

Figure 14. Immunohistochemistry analysis after transplantation (Islet-Matrigel-

Clodrosome, at 60 day; Islet-Matrigel, at 10 day). Grafts were stained for H&E,

insulin, glucagon and somatostatin. Asterisk: transplanted islets.

Magnification X400

Isle

t-M

atri

gel

Is

let-

Mat

rigel

-Clo

dro

som

e

Glucagon Somatostatin

* *

* * *

* *

* *

35

Isle

t-M

atri

gel

Is

let-

Mat

rigel

-Clo

dro

som

e

CD4+ CD8+ CD20+ CD11b

Isle

t-M

atri

gel

Is

let-

Mat

rigel

-Clo

dro

som

e

CD8+ CD20+ CD11b

(a)

(b)

Figure 15. Immunohistochemistry analysis after transplantation (Islet-Matrigel-

Clodrosome group, at 60 day; Islet-Matrigel group , at 10 day). (a) CD4, CD8,

CD20 and CD11b positive cell staining. Asterisk: transplanted islets. Magnification

X 400 (b) Evaluation of immune cell infiltrations into transplanted matrigel. (CD8,

CD20 and CD11b positive cell staining). T=Tissue, M=Matrigel. Magnification

X200

36

Matrigel+Islet+Drug

Insu

lin c

on

c. in

Seru

m (

ng

/ml)

0.0

0.5

1.0

1.5

2.0

Matrigel+Islet

Matrigel+Islet+Drug

IL-1

b c

on

c. in

Seru

m (

pg/m

l)

0

100

200

300

Matrigel+IsletMatrigel+Islet+Drug

IL-1

b c

onc. in

Matr

igel (p

g/m

l)

0

200

400

600

800

1000

Matrigel+Islet

Matrigel+Islet+Drug

TN

F-

con

c. in

Ma

trig

el (p

g/m

l)

0

20

40

60

80

100

Matrigel+Islet

Matrigel+IsletMatrigel+Islet+Drug

Insu

lin c

onc. in

Matr

igel (u

g/m

l)

0

10

20

30

Figure 16. Insulin and pro-inflammatory cytokine levels in recipient’s serum and

injected matrigel (Islet-Matrigel-Clodrosome group, at 60 day; Islet-Matrigel group,

at 10 day). (a) insulin (n=5) (b) IL-1β (n=3) (c) TNF-α (n=3), Data were expressed

as mean ± SD. (*P<0.05 and ***P<0.001)

Matrigel+Islet+Drug

TN

F-

con

c. in

Seru

m (

pg

/ml)

0

10

20

30

40

Matrigel+Islet

Insulin (Matrigel) Insulin (Serum)

IL-1β (Matrigel) IL-1β (Serum)

TNF-α (Matrigel) TNF-α (Serum)

(a)

(b)

(c)

37

4. DISCUSSION

In this study, we have developed a new strategy toward improving anti-inflammation

and immunoprotection in islet transplantation for the treatment of diabetes. Simply by

encapsulating the islets with a macrophage depleting agent, clodrosome, in a extracellular

matrix based hydrogel, matrigel, we have improved the transplanted graft survival time for a

long period of time. After transplantation, host immune system is activated by the antigens

released by the transplanted graft. Activated immune cells attack transplanted graft to reject

it. The macrophages and neutrophils are the first line cells to attack the transplanted graft

either to damage directly or initiate further immune inflammatory reactions. Consequently,

other lymphocytes are induced to migrate into the transplanted microenvironment. Cytokines

and reactive oxygen molecules secreted by macrophages damage transplanted islets and

induce other antigen presenting cells (APCs), which in turn, trigger the adaptive immune

reactions. Macrophages remain potent to the transplanted islet for approximately 20 days

after transplantation [22]. Thus, it is important to keep them inactivated within this time

period to stop the progression of adaptive immune reaction.

Our study demonstrated that, co-delivery of liposomal clodronate with islets at the

site of transplantation effectively blocks the activation of macrophage. Molecular imaging

data confirmed the retention of liposomal clodronate in the matrigel scaffold for over 7 days

of transplantation, which is thought to be the probable reason of significantly improved

survival time of Islet-Matrigel-Clodrosome group compared to that of Islet-Matrigel group.

Moreover, 60 days post-transplantation, the blood glucose level of the recipients in the drug

treated group was maintained and a significant decreased macrophage, T-cell and B-cell

invasion was observed along with lower level of pro-inflammatory cytokines.

Although the liposomal clodronate was administered only once by encapsulating in

matrigel along with islets, the survival rate was increased remarkably. This indicates the

38

significance of growing tolerance in the early stage of transplantation to keep the transplanted

graft safe [38]. Locally delivered liposomal clodronate directly inactivate the macrophages at

the transplanted microenvironment, thereby, decrease the therapeutic dose lower than the

previously mentioned effective doses [27, 39]. Furthermore, local delivery of drug with a

small dose increased the probability of lowering down the systemic delivery associated

adverse effects.

Andres et al. reported that macrophage depletion prolongs discordant but not

concordant islet xenograft survival. According to their findings, when gadolinium chloride

(GdCl), a macrophage depleting agent, was injected to recipient 1 day pre-transplantation, the

concordant islet xenograft rejection could not be prevented [40]. Systemically injected GdCl

inhibited activation of recipient’s macrophages but donor macrophages remained active. The

drug was hardly accessible not only to the recipient macrophages but also to the donor

macrophages existed on the transplanted islets. For this reason, systemic macrophage

depletion is not as effective on concordant islet xenograft as discordant islet xenograft. On

the other hand, in our study, since the liposomal clodronate retained in the matrigel scaffold

for a certain period of time, an extended level of therapeutic benefit was achieved by

depleting both host and recipient macrophages. Hence, locally delivered liposomal clodronate

entrapped in scaffold can effectively prolong the grafted islets survival time.

5. CONCLUSION

We established new immunoprevention protocol for improving the survival time of

subcutaneously injected islets by co-encapsulating with liposomal clodronate in matrigel

scaffold. Locally delivered liposomal clodronate acted on depletion of macrophages for a

longer periods. At the same time, pro-inflammatory cytokines secreted from macrophages

were significantly decreased in Islets-Matrigel-Clodrosome group. These findings have an

39

excellent potential for a successful islet transplantation protocol that diminished immune

reaction and prolonged graft survival of islets (Fig. 17).

40

Adaptive immune reaction

Macrophage

Clodronate liposome

MatrigelIslet

Antigen

Helper T cell

Figure 16. Illustration of immunoprotection of locally delivered liposolam

clodronate for successful islet transplantation

41

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