Accepted Manuscript

An in situ forming biodegradable hydrogel-based embolic agent for interven‐

tional therapies

Lihui Weng, Nassir Rostambeigi, Nicole D. Zantek, Parinaz Rostamzadeh, Mike

Bravo, John Carey, Jafar Golzarian

PII: S1742-7061(13)00304-8
DOI: http://dx.doi.org/10.1016/j.actbio.2013.06.020
Reference: ACTBIO 2775

To appear in: Acta Biomaterialia

Received Date: 7 February 2013

Revised Date: 5 June 2013
Accepted Date: 10 June 2013

Please cite this article as: Weng, L., Rostambeigi, N., Zantek, N.D., Rostamzadeh, P., Bravo, M., Carey, J., Golzarian,
J., An in situ forming biodegradable hydrogel-based embolic agent for interventional therapies, Acta
Biomaterialia (2013), doi: http://dx.doi.org/10.1016/j.actbio.2013.06.020

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 An in situ forming biodegradable hydrogel-based embolic agent for
interventional therapies

Lihui Wenga, Nassir Rostambeigia, Nicole D. Zantekb, Parinaz Rostamzadeha,

Mike Bravoc, John Careyc, Jafar Golzariana,*

a
Department of Radiology, University of Minnesota, Minneapolis, MN 55455
b
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455
c
North American Science Associates Inc., Brooklyn Park, MN 55443

*
Corresponding author. Address: Department of Radiology, University of Minnesota. Mayo B228, 420 Delaware Street SE,
Minneapolis, MN 55455. Phone: 612-625-5147. Fax: 612-626-5580. Email: jafar@umn.edu.

1
 An in situ forming biodegradable hydrogel-based embolic agent for
interventional therapies

ABSTRACT

We present herein characteristics of an in-situ forming hydrogel prepared from carboxymethyl chitosan

and oxidized carboxymethyl cellulose for interventional therapies. The gelation, owing to the

formation of Schiff base, occurred both with and without the presence of a radiographic contrast. The

hydrogel exhibited a highly porous internal structure (pore diameter: 17 ± 4 µm), non-cytotoxicity to

human umbilical vein endothelial cells, hemocompatibility with human blood, and degradability in

lysozyme solutions. Drug release from hydrogel loaded with a sclerosant, tetracycline, was measured

at pH 7.4, 6 and 2 at 37 oC, and showed that tetracycline was more stable at acidic conditions with a

lower release rate observed at pH 6. Moreover, an anticancer drug, doxorubicin, was loaded into the

hydrogel and a cumulative release of 30% was observed over 78 h in PBS at 37 oC. Injection of the

hydrogel precursor through a 5-F catheter into a fusiform aneurysmal model was feasible, leading to

complete filling of the aneurysmal sac, which was visualized under fluoroscopy. Level of occlusion

was compared between hydrogel precursors (1.8% and 2.1%) and standard microspheres (100-300 µm)

in a rabbit renal model. Embolization with hydrogel precursors was performed without clogging and

the hydrogel achieved effective occlusion in more distal arteries than calibrated microspheres. In

conclusion, this hydrogel possesses promising characteristics potentially beneficial for a wide range of

interventional vascular procedures that involve embolization and drug delivery.

Keywords: In situ forming; hydrogel; carboxymethyl chitosan; embolization; drug delivery

2
1. Introduction

Interventional vascular therapies offer minimally invasive methods to treat patients, allowing for

faster healing processes compared with open surgeries. Currently, the treatment of endoleaks after

endovascular stent-grafting, tumor chemoembolization, and endovascular drug-delivery methods are

rapidly gaining interest due to their safety and efficacy [1-3]. Several embolic materials are now on

the market, such as coils, liquid embolics, and particles. Coils have been extensively used to treat

aneurysms, but only 30% of the sac can be occupied by the coils and a significant portion of the sac

will be occluded by unorganized thrombi [4, 5]. Hence, recurrence of endoleak has been observed

frequently. In contrast, liquid embolic agents such as cyanoacrylates and Onyx® (ev3, Irvine, CA) are

shown to achieve more complete filling [6, 7]; however, both types of these liquid agents have proven

to be problematic with regard to the migration of embolic agents out of the aneurysmal sac and

cytotoxicity due to the presence of organic solvents [5, 8]. Moreover, these materials are not resorbable

or loadable. Another shortcoming of currently available embolic agents lies in the flexibility in usage

for various clinical implications. For example, coils cannot be used to treat hypervascular tumors while

particles cannot be used to treat large aneurysms. Thus, it is very appealing to explore a new multiple-

potent embolic agent formulated with biocompatible components that can fill the aneurysm sac,

embolize vessels, and deliver drugs.

In situ forming hydrogels are a class of hydrogel materials that solidify from liquid forms in the

application sites. Among all the materials used to fabricate in situ forming hydrogels, polysaccharides

are of great interest due to their biocompatibility and biodegradability compared to synthetic materials

[9]. Strategies used to achieve in situ forming hydrogels generally involve physical crosslinking or

chemical crosslinking. Physical crosslinking usually takes a longer time with a weaker gel formed,

where the gelation is driven by hydrophilic-hydrophobic interactions [10] or inclusion complex

formation [11]. In contrast, chemical crosslinking may have a faster gelation with a higher gel

3
mechanical strength where the gelation is driven by Schiff base reactions [12, 13], Michael addition

reactions [14, 15], ionic interactions [16] or photocrosslinking [17].

In situ forming hydrogel can be potentially administered straightforward through a catheter prior to

gelation. Different in-situ-forming hydrogels have been investigated for the treatment of cerebral

aneurysms [18], cerebral arteriovenous malformations [15], hemorrhage [19], endoleaks after

endovascular repair [20], and as tissue sealants [14]. However, none of them has been well adapted as a

multi-potent embolic agent that has the potential to be used in a variety of conditions. The constellation

features of in situ forming hydrogels with the potential to deliver drugs, exert embolic properties, and

eventually resorb in tissue milieu without any toxicity or hemostasis alteration may enhance efficacy,

and reduce complications or reintervention rates reported with the use of previous agents [2, 21].

The purpose of this study was to comprehensively evaluate the physicochemical properties of a new

in situ forming embolizing hydrogel prepared from water-soluble natural polysaccharides, namely

carboxymethyl chitosan (CCN) and carboxymethyl cellulose (CMC). Although the safety of using

these two polymers for biomaterials has been established individually, the combination of two low-cost

polymers for fabricating an in-situ forming hydrogel has not been reported. We aimed to demonstrate

the non-cytotoxicity, degradability, impact on hemostasis, and drug release characteristics of this new

material and to demonstrate its embolization and immediate occlusive effects in a rabbit renal model.

2. Materials and methods

2.1. Preparation of OCMC, CCN and CMC/CCN hydrogel

Sodium carboxymethyl cellulose (CMC) (Mw = 700,000 Da), and chitosan (Mw = 190,000~310,000

Da) were purchased from Sigma-Aldrich Corp. (St Louis, MO). All the other chemicals and solvents

were of highest purity commercially available. Oxidized CMC was prepared by oxidizing CMC with

sodium periodate. Briefly, in a 250 mL flask, 1 g of sodium CMC and 80 mL distilled water were

added. After CMC dissolved completely, 25% molar equivalent of sodium periodate in 20 mL distilled
4
water was added into the flask. The reaction was allowed to proceed for 24 h at 25 oC. Then 0.21 mL

ethylene glycol was added into the flask to stop the reaction. After 30 min the mixture was poured into

a dialysis tube (MWCO 3500, Fisher Scientific, Pittsburgh, PA) to dialyze thoroughly against distilled

water. Dry product was then obtained by lyophilizing the solution. The oxidation degree of OCMC was

defined as the percentage of CMC structural units that were oxidized and it was determined by

measuring the aldehyde content in OCMC by iodometry [22], and the molecular weight was measured

with GPC (Waters, Milford, MA). CCN was synthesized with Chen’s method [23]. The substitution

degree of chitosan (carboxymethylation) was determined by the back-titration method as previously

described [24].

The hydrogel was prepared by blending OCMC and CCN aqueous solutions (1.5%, 1.8%, 2%, or

2.1%, w/v) in 1:1 ratio. Then the well mixed precursors were added into the 24-well non-tissue culture

plates and incubated at 37 °C for 2 hours to reach full gelation.

2.2. Gelation time

The gelation time of the CCN/OCMC precursor in different solvents (water, 0.9% saline, 0.01M

PBS, and citrated human plasma) with or without radiographic contrast (Optiray 320, Mallinckrodt

Inc., Hazelwood, MO) was determined by the following protocol [25]. Human plasma was collected by

centrifuging the citrated whole blood donated by one study investigator who was known to have

normal results for the assays tested. In a Petri dish (BD, Franklin Lakes, NJ), 100 µL of OCMC

solution and 100 µL CCN solution were mixed with a magnetic stir bar at 155 rpm using a

hotplate/stirrer (Isotemp 11-100, Fisher Scientific, Pittsburgh, PA). The gelation time was determined

when the mixture formed a globule. The experiments were repeated four times per sample.

2.3. Morphology of the hydrogel

The morphology of lyophilized swollen hydrogel was evaluated by scanning electron microscopy

(SEM) (JEOL 6700, Japan) after spray-coated with gold. Swollen gel pieces were snap-frozen in liquid

5
nitrogen and then lyophilized. A section of the dried gel was mounted on a metal stub coating a layer of

conductive adhesive. SEM images were obtained at a 2.0 KV acceleration voltage in a deceleration

mode under a nitrogen atmosphere.

2.4. Cytotoxicity

Primary human umbilical vein endothelial cells and medium (MCDB–131 Complete medium) were

purchased from VEC Technologies, Inc. (Resselaer, NY). The endothelial cells were seeded in 35 mm

plastic dishes (an area of 8.55 cm2, BD, NJ) at a density of 200,000 cells per well. In order to avoid the

cell damage merely caused by weight of the hydrogel on top of the cells, one circular plastic was put at

the center of each dish before seeding. Hydrogels were prepared with 2% OCMC and 2% CCN

combination. The resulting hydrogels were further conditioned with PBS solution (10×, pH 7.4, Life

Technologies, NY) while shaking at 50 stroke/min for 30 min. Thereafter they were sterilized with

70% ethanol followed by thorough PBS rinsing. Then they were conditioned with standard endothelial

cell medium to remove PBS trapped inside the hydrogel. The circular plastic in the study group was

removed after 24 h and the area was replaced with a piece of hydrogel. After 24 h of culture of cells

with and without hydrogel, cells were rinsed twice with HBSS (Hank's Balanced Salt Solution, Life

Technologies Corp., NY). Then cells were incubated for 30 min at 37 °C in 1 mL 1 mg/mL MTT

reagent (Sigma-Aldrich Co., St. Louis, MO) dissolved in phenol free 20% Roswell Park Memorial

Institute medium (RPMI). After that, the MTT solution was removed from the cell culture dish, and the

dish was air dried. Subsequently, 1 mL of DMSO was added into the dish to dissolve all the MTT. The

absorbance of the MTT/DMSO solution was measured via a microplate reader (Gen5, BioTek®

Instruments, Inc., Winooski, VT) at 570 nm.

2.5. In vitro degradation

Eighteen pieces of hydrogel were prepared from 0.5 mL 2 w/v% CMC solution and 0.5 mL 2 w/v%

CCN solution in a 24-well plate for a 21-day period. The hydrogels were incubated with 1 mL of

6
lysozyme PBS solutions (10 µg/mL and 4 mg/mL) at 37 oC and the medium was changed every other

day. At predetermined intervals, three samples were retrieved and rinsed thoroughly with distilled

water. The resulting hydrogel residues were then freeze-dried. The percentage of weight loss was

calculated based on the following equation: Percent weight loss = [(Wt – W0)/ W0]×100%, where Wt

and W0 were the dry weight of the hydrogel at day t and day 0, respectively.

2.6. Thromboelastography (TEG)

Human whole blood was collected from one study investigator who was known to have normal

results for the assays tested. Blood was collected into a 3.2% sodium citrate vacutainer (Becton

Dickinson, Franklin Lakes, NJ) using routine veno-puncture techniques.

The blood was kept at room temperature prior to use and testing was initiated within 30 minutes to 2

hours after collection. Testing was performed on a TEG® Hemostasis System 5000 (Haemoscope

Corporation, Niles, IL) following a reported protocol [19] with slight modifications. All reagents used

were from Haemoscope Corporation (Stoughton, MA) unless noted. Clear disposable TEG cups and

pins were pre-warmed to 37 °C. One mL of blood was added to a vial of kaolin and mixed gently by

inverting the vial. The reagents for the reaction were placed into the cup in the following order: 320

µL blood from the kaolin vial, 20 µL saline (0.9% sodium chloride injection USP, Baxter Healthcare

Corp., Deerfield, IL) or hydrogel precursor, and 20 µL 0.2 M calcium chloride. The hydrogel precursor

was added using a positive-displacement pipette (Microman®, Gilson, WI). Complete gelation of the

hydrogel precursor did not occur prior to its addition to the reaction. The reaction was allowed to run

until all variables had reached their endpoint.

2.7. Release profile of tetracycline and doxorubicin from the hydrogel

For tetracycline loading and release study, 3 mL of 2% w/v CCN aqueous solution, 1 mL 1.2% (w/v)

tetracycline hydrochloride (Sigma-Aldrich Corp., St Louis, MO) aqueous solution, and 2 mL of 3%

OCMC aqueous solution, were added into a vial to give a drug payload of 10%. After the solutions

7
were mixed well, 2 mL aliquots were removed from the vial and introduced into 20 mL glass vials in

triplicates. Then the vials were kept at 37 oC for 2 h to ensure complete gelation. The release studies

were performed at three different pH conditions: 2, 6, and 7.4. Fifteen milliliter of the solution at

desired pH was added and then the vials were incubated at 37 ± 0.5 oC with mild shaking (100

strokes/min). At predetermined time-points, aliquots of 200 μL were withdrawn from each vial and

replaced with an equal volume of fresh medium of similar pH. Absorbance of the released tetracycline

was read at a predetermined λmax nm with UV-Vis spectrophotometer (DU650, Beckman Coulter Inc.,

Brea, CA) for each pH condition. The calibration curves were prepared for a series of tetracycline

solutions with known concentrations for each pH prior to the conduct of each release study. For

doxorubicin loading and release study, 1 mL 2 % CCN aqueous solution, 0.5 mL 2 mg/mL doxorubicin,

and 1 mL 2% OCMC aqueous solution were mixed in a 20 mL glass vial. The vial was then placed in

the refrigerator (5 oC) for 20 h for full gelation of the mixture. Into each vial containing the

doxorubicin loaded hydrogel, 15 mL PBS (0.01M) was added. The vial was then placed on a shaker

(50 stroke/min) in the incubator (37 oC). At predetermined time points, 0.5 mL aliquot was taken out

from the vial and replaced with 0.5 mL fresh PBS. The doxorubicin concentration was measured with

the UV-Vis spectrophotometer at a wavelength of 482 nm. All the releasing studies were done in

triplicate.

2.8. Injection in an in vitro aneurysmal model

The injectability of the hydrogel precursor (2%) was tested with a 5-F catheter (Terumo, Glidecath,

ID=0.038") after mixing 5 times through a 3-way stopcock. The hydrogel solution containing 20% (v/v)

radiographic contrast was injected into an aneurysmal sac of a fusiform aneurysmal model mimicking

the endovascular treatment. Fluoroscopy was performed during the procedure depicting the filling of

the aneurysmal sac.

2.9. Arterial embolization in a rabbit renal model

8
 The study protocol was approved by the Institutional Animal Care and Use committee. Four adult

rabbits (New Zealand, white) that weighed 3-4 kg were used. Anesthesia was induced by administering

ketamine hydrochloride (35 mg/kg), with xylazine (5 mg/kg) and acepromazine (0.75 mg/kg)

intramuscularly. Rabbits were intubated and general anesthesia was maintained with 1.5%-2%

isoflurane and 1 L/min oxygen, with approximately 100-110 mL tidal volume and 10-15 breaths/min.

Each animal was continually monitored with an electrocardiogram, oxygen saturation levels, body

temperature and assessment of depth of anesthesia during the procedure. The right common femoral

artery was surgically exposed and a 4-F sheath was placed. Using fluoroscopy, the renal artery was

catheterized with a 4-F Cobra catheter (ID = 0.038", Length: 65 cm, GlidecathTM, Terumo Medical

Corp., Somerset, NJ). A 2.8-F microcatheter (ID=0.027", ProgreatTM, Terumo Medical Corp., Somerset,

NJ) was placed in the renal artery and the 4-F catheter was pulled back into the aorta for free flow

delivery of microspheres at a constant flow rate of 2 mL/min. For each kidney, embolization was

performed until the total occlusion was achieved. The right kidneys were embolized with the hydrogels

while the left kidneys received calibrated bioresorbable microspheres (100-300 µm, 10 mg/mL in

normal saline) for comparison. The calibrated bioreosorbable microspheres were prepared according to

an inverse emulsion method [26]. Two of the four rabbits received the 1.8 w/v% hydrogel precursor

while the other two were embolized with the 2.1 w/v% hydrogel precursor. For each kidney,

angiography was performed before, immediately after, and 10 min after embolic procedure. The

volumes of the hydrogel precursor and microsphere suspension required were recorded. The ease of

injection was subjectively evaluated by an interventional radiologist (J. G.) with greater than 20 years

of experience.

Immediately after the final angiogram, these rabbits were given heparin at 150 units/kg and 5 min

later euthanized with an overdose of Euthasol®. The kidneys were surgically removed and fixed in

10% buffered formalin. 4-µm coronal sections were obtained from each kidney and stained with H&E.

9
The size and location of all embolized vessels (i.e., vessels containing embolic agent) were determined

as previously shown [27]. For the distribution of hydrogel/microspheres in kidneys, the following

parameters were assessed: 1) The arterial location of the hydrogels/microspheres. 2) The numbers of

glomeruli were counted for each kidney to obtain the percentage of glomeruli containing gels from 6

representative slides. 3) The vessel diameter occluded by microspheres. To measure the vessel sizes,

the histological slides were scanned with a whole slide scanner (Aperio ScanScope XT) using a

tracking feature. Then the vessel size was measured with a calibrated ruler in Aperio ImageScope

viewing and annotation software.

2.10 Statistical analysis

Data were summarized using mean and standard deviation for numeric variables, and counts and

proportions for categorical variables. Student’s t test and ANOVA were used for the comparison of

means. Difference was considered significant if p was less than 0.05.

3. Results and discussion

Bioresorbable materials are currently of increasing interest in interventional radiology with respect

to their temporary occlusion characteristics and biodegradable nature that overcomes the persistency in

the body of the permanent materials [26]. The first report on the use of bio-resorbable materials

appeared in 1980s [28] with subsequent reports on the use of various resorbable materials such as

living tissue. Injectable in situ forming bioresorbable hydrogels have received extra attention because

they can be used as an embolic agent through a coaxial system. Nonetheless the loco-regional impact

in the homeostasis of the organs, drug delivery and embolization features are largely unknown. We

presented a new in situ forming bioresorbable hydrogel composed of CCN and OCMC. Different

characteristics of this material indicated that its application in a variety of clinical conditions is feasible.

The indications such as the treatment of tumors, arteriovenous malformations, and arterial

hemorrhages as well as adjunctive use with stent graft for endovascular treatment of aortic aneurysm

10
are very likely applications that this hydrogel can be successfully used for.

3.1. Modification of CMC and CN

CMC was partially oxidized with sodium periodate. Periodate turned the vicinal hydroxyl groups

into two aldehyde groups, and a number of the sugar rings on CMC were thus open, leading to a more

flexible polymer backbone [29] with functional groups. The oxidation degrees of OCMCs were 17.01

± 0.78% as determined by the iodometry titration method. The corresponding weight average

molecular weights were 109370 Da, as measured by GPC. Although chitosan is a biocompatible and

biodegradable polymer, it is not soluble at physiological pH that is necessary for materials designated

for biological applications. Carboxymethyl groups were thus introduced into the structure of chitosan.

The substitution degree of chitosan (carboxymethylation) was 1.03 ± 0.03 as verified by the back-

titration method. The modified chitosan was readily soluble at physiological pH.

3.2 Hydrogel formation and gelation time

The mixture of CN and OCMC solutions underwent a sol-gel transition and gelled into a clear

hydrogel at ambient temperature (23 oC) or 37 oC. The gelation is due to a self-crosslinking process

based on Schiff base formation [26]. The gelation time was adjustable via both the concentration of the

individual polymers and composition of the precursors as shown in Fig. 1. Incorporation of iodine

contrast delayed the gelation process, but less effect was observed at higher polymer concentrations.

The presence of 20% (v/v) contrast in a 2% hydrogel precursor produced a radiopaque gel within 2.55

± 0.05 min (mean ± SD). Moreover, the gelation time was both temperature and solvent dependent.

Elevation in temperature from 23 oC to 37 oC accelerated the gelation process, where at 23 oC, the

gelation time decreased from 4.81 ± 0.35 to 1.92 ± 0.22 min (mean ± SD) with an increase in the

polymer concentration from 1.5% to 2% (Fig. 1a). The gelation can occur in different media, such as

distilled water (DI), 0.01M PBS, 0.9% normal saline and human plasma (Fig.1b). At 37 oC, gelation in

PBS (1.30 ± 0.06 min) occurred slightly earlier than those in DI (1.53 ± 0.02 min) or saline (1.35 ±

11
0.09 min). In comparison, the gelation was slower in citrated human plasma (4.28 ± 0.36 min). The

slower gelation process is likely linked to the interactions between the proteins in the plasma and

individual polymers in the hydrogel precursor. Meanwhile, it is worthwhile mentioning that gelation in

this situation occurred with the plasma as the solvent for both polymers, whereas in vivo studies were

performed with hydrogel polymers dissolved in saline and then the precursor was introduced to blood

flow at approximately 1 min after mixing. In the latter situation gels formed inside the arteries within 1

min, which was evidenced by the rapid disappearance of the distal arteries during fluoroscopy in

animal studies that we conducted.

3.3. Morphology of the hydrogel

SEM showed interconnecting pores in the hydrogel prepared from a 2% OCMC/CCN precursor (Fig.

2) and pore size analysis gave an average pore diameter of 17 ± 4 µm. In addition, it was also observed

that the pore size distribution depends on the swollen state of the hydrogel and polymer concentration

(data not shown). The porous structure of the hydrogel may facilitate the drug release, which has been

similarly shown for other hydrogels [12].

3.4. Cytotoxicity

This aqueous in situ gel-forming matrix is completely devoid of residual toxic solvents as compared

with many preformed bulk gels. Both CCN and CMC are biocompatible polymers [11, 26] and

cytotoxicity is not expected from the combination of these two polymers. Endothelial cell line was

selected for co-culture since they bear similarity to the endothelial layering of the arterial surface. Up

to 24 h, there was no significant difference in the cell density, morphology and distribution between the

cells co-cultured with hydrogels (Fig. 3a) and control (Fig. 3b). In addition, the standard MTT assay

based on the number of viable cells in the culture flask showed no significant difference between

endothelial cells cultured with and without hydrogels (optical density 1.17 vs. 1.19, p = 0.8, n = 4) (Fig.

3c), indicating the hydrogels were nontoxic to endothelial cells. In contrast to the conventional liquid

12
embolics such as Onyx that have angiotoxicity [20], this hydrogel therefore appears to be more

desirable for in vivo usage.

3.5. In vitro degradation

This hydrogel underwent degradation under physiological pH in the presence of lysozyme. The dry

weight of the hydrogel showed a consistent decrease over the degradation period of incubation with

lysozyme (Fig. 4). In the 4 mg/mL lysozyme solution, there was a significant drop in the hydrogel

weight between day 0 and day 10 (p = 0.01). The starting dry weight of the hydrogel was 20 mg on day

0, and decreased to 15.2 mg (day 3), 7.4 mg (day 5), 5.1 mg (day 7), and 1.2 mg (day 12)

demonstrating a consistent decrease in the weight of hydrogel with greater than 20% decline every two

days. The change after the tenth day was less remarkable although remained significant (p = 0.01). In

comparison, the degradation in the 10 µg/mL lysozyme solution occurred at a slower pace. On day 7,

the weight percentage of the hydrogel remained in this solution was 57.8%, which was over 2 times

higher than that (25.5%) in the 4 mg/mL lysozyme solution (p = 0.004). Moreover, there was still

22.1% of the hydrogel left on day 21, indicating that decreasing the concentration of the lysozyme

solution delayed the degradation process. The degradation can be attributed to two mechanisms: 1) The

main crosslinks in the hydrogel, Schiff bases, are well known to be subject to hydrolysis; 2) Chitosan,

part of the hydrogel, can be degraded by lysozyme, an enzyme that is ubiquitously found in the body

[30].

Hypothetically, the aneurysmal sac or an arteriovenous malformation vascular bed will be ideally

filled over time with fibrotic tissue while hydrogel is being resorbed and delivery of sclerosing agent

(i.e. tetracycline) happens. Therefore, by having a biodegradable embolic material there will be no

persisting foreign body in the vessel. There will be, instead, an organized solidified tissue structure as

opposed to a multi-particle apparatus. Additionally, transient hypoxia due to biodegradable materials

may potentially reduce hypoxia-induced angiogenesis in the malignant tumors [31]. Moreover, the

13
vessels would be more accessible for potentially later interventions if the embolic agent is resorbed.

Also there is a chance that the resulting small pieces produced during the degradation of the hydrogel

might enter the main stream circulation. Embolization in the case of transcatheter arterial

chemoembolization (TACE) is performed in end-arteries and therefore embolization of degrading

pieces to more distal arterioles would not be troublesome. Indeed, resorbable materials (e.g. Gelfoam)

have been used in many embolization cases and distal migration has not been an issue. However, the

potential untoward effect of this biodegradable material could be that the detached hydrogel materials

during degradation embolize into undesired areas. Thrombus formation would help to prevent the

migration of the degraded material. In vivo studies are needed to test these potential effects.

3.6. Thromboelastography

For biomaterials used for vascular applications, their interactions with blood determined their

intended functions in vivo. A number of tests have been reported for assessing the hemocompatibility

of biomaterials, such as partial thromboplastin time (PTT), prothrombin time (PT), thrombin clotting

time (TT) [32, 33]. However, thromboelastography (TEG) is a global assay of blood coagulation based

on quantitative measurement of the elasticity of whole blood, from the beginning of coagulation

through clot formation towards the final fibrinolysis [34]. It provides additional information compared

to traditional lab tests (PT or PTT) which focus only on the initiation of the coagulation cascade. Fig. 5

presents representative thromboelastographs for citrated whole human blood with normal saline (black)

or hydrogel precursor (green) to elucidate their gelation effects on blood coagulation, and the data was

summarized in Table 1. The presence of hydrogel did not inhibit or enhance the onset of clot formation

or coagulation (Fig. 5 and Table 1) (i.e., R time, 7.1 min vs. 6.7 min, p = 0.34; K, 2.4 min vs 2.3 min, p

= 0.8; α angle, 59.1 degree vs 59.4 degree, p = 0.9). In addition, there was no significant difference in

the clot strength (as measured by the maximum amplitude, MA, 70.2 mm vs 68.5 mm, p = 0.1), and

fibrinolysis at 30 min (both were 0%) and 60 min (0.1% vs 0.2%, p = 0.2). Peng et al [21] investigated

14
the blood compatibility of a hydrogel made of polyethylene glycol and polyallamine hydrochloride

designated for hemorrhage control and found that the presence of their hydrogel enhanced the overall

procoagulant effects. In our study, the presence of the OCMC/CCN hydrogel did not show significant

impact on the blood clotting time, clot strength, or fibrinolysis as compared to normal saline control.

The difference in observations between our study and Peng’s [21] may be due to the addition of kaolin

in the reaction or the components of the hydrogel. Despite the addition of kaolin, a marked inhibitory

or prothrombotic effect by the hydrogel would be expected to be detected by the method used here, as

kaolin is routinely used in the test performed in clinical laboratories where it is specifically used to

detect abnormalities in hemostasis. However, kaolin is a strong activator of coagulation and a weak

inhibitory or prothrombotic effect of the hydrogel may not be detected with significant power due to

the short reaction time of the assay. A material for embolization that does not alter hemostasis may be

ideal for embolization purposes. A product that had an anticoagulant effect, i.e. inhibits thrombus

formation, would have the potential to worsen bleeding. While a product that has a prothrombotic

effect may locally promote thrombus formation and decrease local bleeding, if any of the product

enters into the more general circulation may set up an undesirable prothrombotic state elsewhere

perhaps leading to a more systemic effect such as disseminated intravascular coagulation. This

OCMC/CCN hydrogel does not appear to inhibit or enhance coagulation which allows its general use

in different conditions within blood vessels.

3.7. Release profile of tetracycline and doxorubicin from the hydrogel

Tetracycline is an antibiotic that is used as a sclerosing agent and is reported to provoke sclerosis and

tissue fibrosis, which will in turn lead to shrinkage of cavities such as lymphoceles or pleural space

[35]. This effect can also be utilized in the aortic sac via injection of tetracycline loaded hydrogels after

stent graft implantation to treat abdominal aortic aneurysm. Loading tetracycline hydrochloride into

the OCMC/CCN hydrogel was performed as the hydrogel matrix was negatively charged (-COO-)

15
while tetracycline was positively charged (-+NH(CH3)2). In addition to neutral pH, we tested

tetracycline release in two other conditions: PH = 6 was chosen to replicate more acidic conditions in

clinical situations such as infectious milieu; PH = 2 was chosen merely for experimental purposes to

show the trend of release and stability of tetracycline in extreme conditions. The tetracycline release

followed a consistent increasing pattern over the first 24 h, where the initial drug release speed at pH =

6 was lower than those at both pH = 7.4 and pH = 2 (Fig. 6a). Within the first hour, the cumulative

tetracycline released was 29.2%, 8.6%, and 23.4% in the medium of pH = 2, pH = 6 and pH = 7.4,

respectively. In the medium of pH = 7.4, the highest cumulative tetracycline release was 65.8% at 24 h.

Subsequently, a decline in the tetracycline concentration was observed after 24 h, which was attributed

to the instability of tetracycline in the medium of pH = 7.4 [37]. On the contrary, in the pH 6 solution

and more so with the pH 2 solution, the amounts of drug release leveled off after 24 h without a decline

until the end of the study period at 72 h since tetracycline was more stable in acidic conditions [36].

The final cumulative tetracycline release was 77.0% and 58.6% in the medium of pH = 2 and pH = 6,

respectively. This hydrogel showed a relatively sustained tetracycline release at pH = 6 as compared to

pH = 7.4 and pH = 2. This suggested that this hydrogel would have some unique in vivo releasing

profiles because the extracellular pH is slightly acidic (about 6 ~ 7) under ischemic conditions [37]

which also happen in the embolization procedure.

To further explore the possibility of using this hydrogel as a drug eluting vehicle for TACE,

doxorubicin, was incorporated into the hydrogel before the gelation occurred. Fig. 6b shows the 78 h

release profiles of doxorubicin from the hydrogels in 0.1 M PBS at 37 oC. The hydrogel released

approximately 6% of its drug content within 1 h. The release leveled off around 50 h with a final

cumulative percentage of approximately 31.3%. Compared to the tetracycline release, the cumulative

release of doxorubicin was lower at pH = 7.4. This difference can be possibly explained as follows:

First, the initial payload of tetracycline was 10% while that of doxorubicin was 2.5%, and thus there

16
was likely a portion of tetracycline that remained in the hydrogel by simply physically trapping inside

the hydrogel network instead of ionic binding, and during releasing, this trapped portion of tetracycline

was released first. Secondly, the ionic interaction between doxorubicin and hydrogel (-+NH3 and -

COO-) [38] was stronger than that between tetracycline and hydrogel (-+NH(CH3)2 and -COO-). Thus,

relatively higher percentage of doxorubicin was retained inside the hydrogel through ionic binding.

TACE using drug-loaded microspheres has shown less systemic toxicity and drug-related side-

effects compared to the conventional TACE. However, the only commercially available drug-eluting

bead (DC bead, Biocompatibles, UK) in USA is not biodegradable, and it is reported that long-term

presence of these microspheres containing potentially harmful drug in the body provoked chronic

inflammation and thus caused more tissue damage [39]. In contrast, the biodegradable hydrogel may

be resorbed and the loaded drug completely released, reducing the chances of protracted tissue damage

related to chronic inflammation.

3.8 Injection in an in vitro aneurysmal model

Endovascular repair of aortic aneurysms (EVAR) may fail if it leads to the development of

endoleaks [2, 40]. It is reported that aneurysmal sac expansion and endoleak formation may lead to

reintervention in up to 27% of procedures [21]. Thus the use of preventive measures to reduce

endoleaks appears to be critical in the treatment of these patients. In this study, the injection of

hydrogel precursors was attempted before the sol transformation into gel in a static model without

considering the blood flow. However, in a real clinical situation, a stent graft will be deployed before

filling the sac, and the rapid pulsatile flow in the aorta will have less effect on the separated sac area as

compared to an open system without the stent graft. Thus, the gelation of the precursor after injection

is more important. This process was performed under fluoroscopic guidance (Fig.7). The contrast

media (Ioversol) incorporated in the hydrogel precursor allowed visualization of the infusion process.

The OCMC/CCN sol traversed through the 5-F catheter easily without any clogging, and formed a gel

17
after infusing into the occluded aneurysm sac in the presence of Ioversol as expected from results

mentioned previously (Fig.1). More importantly, upon gelation the gel was also able to effectively fill

the aneurysmal sac (Fig.7f), which was observed previously for Embogel and Ultragel [5]. With

additional drug loading/releasing ability demonstrated above, the hydrogel has the potential to be

mixed with sclerosing agents such as tetracycline and be used during the for EVAR procedure to

prevent endoleak formation.

3.9. Level of occlusion in the rabbit kidney

Another application of this hydrogel with its drug-delivery feature involved is tumor embolization.

Liquid and particulate embolic materials are effective in penetrating small vessels, and meticulous use

of these materials is essential in preventing unintended occlusions [41]. To address the level of

occlusion of the OCMC/CCN hydrogels in the vascular network, renal embolization was performed

with microspheres as comparison. In all kidneys of four rabbits, the injection of hydrogel and

microspheres through the microcatheters was performed and embolization was done without any

clogging or aggregation. When the concentration of the hydrogel increased from 1.8% to 2.1%, the

force needed for injection increased but it was not difficult.

Before embolization, the renal arteriogram showed a normal vascular anatomy and homogeneous

renal parenchyma (Fig. 8a). Angiogram immediately after embolization with hydrogel revealed

occlusion of the renal artery (Fig. 8b). The volumes of microsphere suspension needed to achieve total

occlusion were between 5.5 and 7 mL, and the volumes of 1.8% hydrogel precursor needed were

comparable to those of microspheres (Table 2). However, the injection volume dramatically decreased

when the concentration of hydrogel precursor increased from 1.8% to 2.1% (7-8.5 mL vs.1-2 mL).

A follow-up angiogram performed 10 min after embolization confirmed the persistence of the renal

artery occlusion, suggesting the embolization was well maintained (image not shown).

Fig. 9 depicts the histological sections of the rabbit kidneys after embolization. The hydrogel was

18
found more distally than the microspheres in the renal artery. For both 1.8% and 2.1% hydrogel

precursors, hydrogels were observed in the glomeruli of the kidneys embolized. Owing to the

functional groups (-COO-) on the hydrogel matrix, hydrogels appeared to be dark pink under H&E

staining (Fig. 9a and 9c) surrounded with white areas. The white spaces were likely attributed to the

dehydration of the hydrogel (i.e. shrinking) during the staining process procedure [27]. Alternatively it

is also possible that the liquidity of hydrogel precursors allowed them to pass through the arteries to

that level. Composed of polysaccharides, the hydrogels exhibited a purple color under PAS staining

(Fig. 9b and 9d). This was the distinguishing feature of hydrogels from red blood cells which showed

similar pink color in H&E staining but negative response to PAS staining. For the kidneys embolized

with 1.8% hydrogel precursor, hydrogel was observed mainly at the glomerular level with a percentage

of 49.2% of 1869 counted glomeruli although the embolization endpoint was a total occlusion as

confirmed by angiogram (Fig. 8). In the kidneys embolized with 2.1% hydrogel precursor, hydrogels

were found at all levels from interlobar arteries to glomeruli (Fig. 9c, 9d, and 9e). Also 74.9% of the

1907 glomeruli counted contained gels. There were two possible reasons for the different percentages

of glomeruli with hydrogel content when we compared kidneys embolized with 2.1% and 1.8%

hydrogel: First, the 1.8% precursor was diluted by the blood and the gelation was not completed, i.e.,

the formed hydrogel was too weak so that it was partially flushed away by the blood flow. Second, the

weak hydrogel was washed out during the histological processing, as evidenced by lack of hydrogels in

large arteries although a total occlusion was performed. In all histological samples, the microspheres

were seen in round or oval homogenous structures with a pink-purple color at H&E staining in tissue.

They were found either completely blocking the arteries or surrounded by white spaces inside the

arteries (Fig. 9f). Apart from the dehydration of the microsphere, the cutting that was not through the

cross-section of the diameter could be another reason of the white areas, which has also been observed

with Embospheres [27]. The microspheres mainly landed in interlobar and arcuate arteries in 77.8%

19
and 22.2% of the arterioles. The mean (±SD) diameter of the vessel occluded by the microspheres was

224.0 ± 70 µm. There was no microsphere observed in glomeruli. These findings suggest that these

hydrogels can penetrate more distally than microspheres in the vascular network, allowing a better

penetration into the tumors and therefore a better drug delivery.

4. Conclusion

A new in situ forming embolizing hydrogel based on water-soluble natural polysaccharides was

developed and evaluated in regard to its application as an embolizing agent for interventional therapies.

Composed of CMC and CN, this hydrogel is a non-cytotoxic, degradable, and highly porous material

which is capable of loading and releasing drugs. It does not interfere with hemostasis and can

effectively fill the aneurismal sac of the fusiform model with the procedure being fully visualized with

addition of contrast material. Furthermore, this hydrogel successfully embolized kidneys under

fluoroscopy guidance. In addition, various potential therapeutic agents, including cells, drugs and

growth factors, could be mixed into the matrix. Thus, it can be potentially used as an embolizing agent

for different interventional radiology therapies such as endovascular treatment of aortic aneurysm, or

chemotherapy of whole organ tumors, or embolizing bleeding arteries. Further research in the in-vivo

models of these disease processes is required to affirm the findings.

Acknowledgement

We thank Sean L Moen in the Fairview hospital, University of Minnesota Medical Center for his

valuable technical assistance in fluoroscopy.

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24
Figure Legends

Figure 1. Gelation time of the hydrogel precursors: (a) different polymer concentrations with and
without radiographic contrast; (b) different solvents and temperatures.

Figure 2. SEM showing the porous structure of the 2% hydrogel.

Figure 3. Cytotoxicity study of the hydrogels: (a) the cells cultured with hydrogel piece; (b) monolayer
cell control. (c) the cell viability (MTT) assay indicating comparing optical density (OD) of
cells’ reaction to MTT reagent with and without hydrogel.

Figure 4. In vitro degradation of the hydrogels in the lysozyme PBS solutions at 37 °C.

Figure 5. Thromboelastography showing the blood coagulation in the presence of hydrogel (green) as
compared to saline control (black).

Figure 6. Drug release from the hydrogel at (a) Tetracylcine release at pH=7.4 (0.01 M PBS solution),
pH=6 (PBS solution with HCl), and pH=2 (PBS solution with HCl) at 37 oC; (b) Doxorubicin
release in the PBS solution (pH=7.4) at 37 oC.

Figure 7. The stages of filling a fusiform aneurysmal model under fluoroscopic guidance with the
hydrogel showing clear visualization as well as injectability of the hydrogel material through
a 5-French catheter. (a) 0 s, catheter positioned; (b) 15 s, filling starting; (c) 31 s, half filling;
(d) 78 s, complete filling.

Figure 8. Angiogram images of the renal arteries before embolization (a) and after embolization (b)
with hydrogel.

Figure 9. Histological sections of the rabbit kidneys. (a) and (b) Hydrogels in the glomeruli of the
rabbit kidney embolized with 1.8% hydrogel precursor stained by H&E and PAS, respectively.
Green arrow: hydrogel; black arrow: red blood cell. (c) and (d) Hydrogels in the glomeruli of
the kidney embolized with 2.1% hydrogel precursor stained by H&E and PAS, respectively.
Arrow: hydrogel. (e) Hydrogels in the interlobular arteries, H&E staining. Arrow: hydrogel.
(f) A microsphere in the arcuate arteries, H&E staining. Arrow: microsphere.

25
Table 1 TEG measurements of citrated whole human blood in the presence of normal saline or
hydrogel precursor.

Samples R (min) K (min) α (deg) MA (mm) LY30 (%) LY60 (%)

Saline 7.1±0.7 2.4±0.7 59.1±6.3 70.2±1.4 0 0.1±0.1

Hydrogel 6.7 ±0.5 2.3±0.6 59.4±6.4 68.5±1.6 0 0.2±0.3
p 0.34 0.83 0.94 0.17 -- 0.26
Data are expressed as means ± standard deviation (n = 4).

Table 2 Volume of embolic materials needed for total occlusion of the rabbit renal artery.

Concentration of embolic materials Volume (mL)

Rabbit
Left/microspheres Right/hydrogel precursor Left Right
(mg/mL) (w/v)
1 10 1.8 % 7 7
2 10 1.8 % 7 8.5
3 10 2.1 % 5.7 1
4 10 2.1 % 5.5 2

26
Figure 1-3

Figure 1

a 9

8
without contrast
Gelation Time (min) 7 with 20% (v/v) contrast
6

0
1.5% 1.8% 2.0%
Concentration of the hydrogel precursor
b 5

4
Gelation Time (min)

2% hydrogel precursor
3

0 o o o o o

DI, 2
3 C
DI, 3
7 C
ne, 37 C BS, 37 C a, 37 C
Sali m
P Plas
Solvent and temperature
A
Figure 2
Figure 3

a b

c
1.4
1.17 1.19
1.2
Absorbance at 570 nm

1.0

0.8

0.6

0.4

0.2

0.0
Hydrogel Control
Figure 4-6

Figure 4

100

4 mg/mL
Weight percentage (%)

80 10 g/mL

60

40

20

0 3 6 9 12 15 18 21
Time (day)
Figure 5
Figure 6

a 90
Cumulative released Tetracycline (%)

80

70

60

50

40

30

20
pH=2
pH=6
10 pH=7.4

-10 0 10 20 30 40 50 60 70 80

Time (h)
b 40
Cumulative released Doxorubicin (%)

35

30

25

20

15 pH=7.4

10

-5
-10 0 10 20 30 40 50 60 70 80 90

Time (h)
Figure 7-8

Figure 7

d
Figure 8

a b
Figure 9

Figure 9