Note: Descriptions are shown in the official language in which they were submitted.
21'~,311~8
CYTOPROTECTIVE, BIOCOM_ATIBLE~_RETRIEVABL~_MAC~OCAP~UL~
CONTAINMENT SYS_ F~S _QR BIOLOGICALLY Af"rIVE MATF.R~LS
The present invention relates to a new form of
biocompatible materials which envelop encapsulated or free
cells to provide an immune barrier. The resulting
encapsulated material is generally small, but macroscopic,
so that it is retrievable in situ. More specifically the
present invention relates to a composition and system for
treatment of diabetes.
BACKGROUND OF THE INVEI`1TION
Diabetes Mellitus is a serious disease afflicting
over 100 million people worldwide. In the United States,
there are more than 12 million diabetics, with 600,000 new
cases diagnosed each year. Insulin-dependent, or Type I
diabetics, require daily injections of insulin to prevent
them from lapsing into coma.
With the discovery of insulin in 1928, it was
thought that diabetes had been cured. Unfortunately,
despite insulin therapy, the major complications of the
disease caused by high blood sugar levels persist. Each
year, diabetes accounts for 40,000 limb amputations and
5,000 new cases of blindness in the United States. Among
teenagers, diabetes is the leading cause of kidney failure.
Data from the National Institutes of Health show that the
rate of heart disease and stroke is twice as great in
diabe~ics than in the general population.
The diabetic patient faces a 30% reduced
lifespan. Multiple insulin injections given periodically
throughout the day cannot duplicate the precise feedback of
insulin secretion from the pancreas. The only current
method of achieving minute-to-minute glucose control is by
pancreas transplantation.
~`~ "
,')1~9~8
While whole or~3an pancreas trar~sr)lantation
represents a significant adv~nce in cliabetes therclpy, the
operation is technically dif~ic~llt, of` limitecl success and
use because of the problems o~ rejection, and it still
presents a significant risk to the patient. An attractive
alternative would be to extract the insulin-producing cells
(islets) from a donor pancreas and to inject these cells
into the diabetic patient, thus effecting a cure. The use
of such cells, however, would still run the risk of
rejection by the host.
Microencapsulation of islets by an alginate-PLL-
alginate membrane (i.e., an alginate-poly-L-lysine-alginate
membrane) is a potential method for prevention of rejection
by the host's immune system. By this technique,
researchers are able to encapsulate living islets in a
protective membrane that allows insulin to be secreted, yet
prevents antibodies from reaching the islets, causing
rejection of the cells. This membrane (or microcapsule)
protects the islet from rejection and allows insulin to be
secreted through its "pores" to maintain the diabetic in
normal glucose control.
Successful transplants of microencapsulated
islets have not been clinically feasible to date due to
fundamental problems of transplant rejection and/or a
fibrotic reaction to the microcapsule membrane. Lim and
Sun, 1980, reported the first successful implantation of
microencapsulated islets and described normalization of
blood sugar in diabetic rats. However, for
microencapsulated islets to be clinically useful and
applicable in humans, it is important that the
immunoprotective membrane be biocompatible, allow adequate
diffusion for the encapsulated cells to respond
!appropriately to a stimulatory signal, provide the
¦encapsulated cells with necessary nutrients, and be
135 retrievable. Retrievability is desirable for a variety of
21239.~8
--3--
reasons, e.g., so that accumula~ion of the irnplantecl
materials can be avoided, so th~t encapsulclted cells clln be
removed from the recipient when no loncJer ne~ed or desired
(e.g., when the product(s) of the encapsulatecl cells are no
longer needed, if the encapsulated cells fail to perform as
desired, etc.), so that encapsulated cells can be removed
if/when they become non-viable, and the like. Currently
there are no reports of successful reversal of diabetes in
humans by transplantation of encapsulated islets.
Biocompatibility of encapsulated islets remains
a fundamental problem. The term "biocompatible" is used
herein in its broad sense, and relates to the ability of
the material to result in long-term in ~ivo function of
transplanted biological material, as well as its ability to
avoid a foreign body, fibrotic response. A major problem
with microencapsulation technology has been the occurrence
of fibrous overgrowth of the epicapsular surface, resulting
in cell death and early graft failure. Despite extensive
studies, the pathological basis of this phenomenon in
alginate based capsules remains poorly understood.
However, several factors have recently been identified as
being involved in graft failure, e.g., the guluronic
acid/mannuronic acid content of the alginate employed,
imperfections in the microcapsule membrane (allowing
exposure of poly-L-lysine to the in vivo environment),
failure of the microcapsule membrane to completely cover
the cells being encapsulated (thereby allowing exposure of
the cells to the in vivo environment), and the like.
Alginate is a polysaccharide isolated from marine
brown algae including Laminaria hyperborea, Laminaria
digitata, Ascophyllum nodosum and Macrocystis pyrifera.
Alginate forms ionically crosslinked gels with most di- and
multivalent cations. Calcium cations are most widely used,
and give rise to a three-dimensional network in the form of
~ l ~ . ?~
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21 ~3(1~8
an ionically crosslinked gel by in~.er~ in bincling between
G-blocks. (Skj~k-Bræk, 1988).
It has recently be~n ~emonstr~te~ that the
mannuronic acid residues are the ~ctive cytokine inducers
in alginate, and since these cytokines (IL-1 and T~F) are
known to be potent stimulators of fibroblast proliferation
(Otterlei et al., 1991), i~ was deduced that alginate
capsules high in mannuronic acid content (M-content) were
responsible in part for the fibrotic response reported in
the past (Soon-Shiong et al. 1991). More significantiy, it
has been found that this reaction could be ameliorated by
increasing the guluronic acid content (G-content) of the
alginate capsule since guluronic acid appears not to be
immunostimulating. Furthermore, it has been demonstrated
that cyclosporin A resulted in a dose dependent inhibition
of mannuronic acid-induced TNF and IL-1 stimulation of
human monocytes in vitro. ~ased on this information, it
has been hypothesized that the fibrotic reaction of the
microcapsule could be ameliorated in part by an alginate
formulation high in guluronic acid content, as well as a
subtherapeutic course of cyclosporin A to inhibit cytokine
stimulation. By this method, diabetes in the spontaneous
diabetic dog model has been successfully reversed by
transplantation of donor islets encapsulated in high
G-content alginate (Soon-Shiong et al. 1991).
Polyethylene glycols (PEG; also referred to as
polyethylene oxide, PEO) have been investigated extensively
in recent years for use as biocompatible, protein
repulsive, noninflammatory, and nonimmunogenic modifiers
for drugs, proteins, enzymes, and surfaces of implanted
materials. The basis for these extraordinary
characteristics has been attributed to the flexibility of
the polymer backbone, and the volume exclusion effect of
this polymer in solution or when immobilized at a surface.
The solubility of PEGs in water, as well as a number of
2123~8
common organic solvents, ~cilit.ltes moclific~ltion by a
variety of chemical re~ctions. ~ reccn~ r~vi~w (~ rris,
1985) describes the synthesis o~ numero-ls derivatives of
PEG and the immobilization thereo~ to s-lrf~ces, proteins,
and drugs.
PEG bound to bovine serum al~u~in has shown
reduced immunogenicity and increased circulation times in
a rabbit (Abuchowski et al., 1977). Drugs such as
penicillin, aspirin, amphetamine, quinidine, procaine, and
atropine have been attached to PEG in order to increase
their duration of activity as a result o~ slow release
(Weiner et al., 1974; Zalipsky et al., 1983). PEG
covalently bound to poly-L-lysine (PLL) has been used to
enhance the biocompatibility of alginate-PLL microcapsules
used for the encapsulation of cells. PEG has been
covalently bound to polysaccharides such as dextran (Pitha
et al., 1~79, Duval et al., 1991), chitosan (Harris et al.,
1984) and alginates (Desai et al., 1991). These
modifications confer organic solubility to the
polysaccharides.
Surfaces modified with PEG were found to be
extremely nonthrombogenic (Desai and Hubbell, l991a;
Nagoaka and Nakao, 1990), resistant to fibrous overgrowth
in vivo (Desai and Hubbell, 1992a) and resistant to
bacterial adhesion (Desai and Hubbell, 1992b). Solutions
containing PEG have also been found to enhance the
~ preservation of organs for transplantation (Collins et al.;
! Zheng et al., 1991). The basis of the preservation
activity is not clearly understood but has been attributed
, 30 to adhesion of PEG to cell surface molecules with a
¦ resultant change in the presentation of antigen so as to
alter the nature of the immune response.
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Crosslinked PEG gels h~ve been prepare-l and
utilized for immobilization of enzymes and microbial cell.s.
Fukui and Tanaka (1976) and Fukui e~ al., (19~7) have
prepared polymerizable derivatives o~ PEC (such as the
dimethacrylate) and photocrosslinked them with W licJht in
the presence of a suitable initiator to form a covalently
crosslinked gel. Kuma~ura and Kaetsu (1983) have reported
the polymerization and crosslin~.ing of diacrylate
derivatives of PEG by gamma radiation for the purpose of
immobilizing microbial cells. Due to the mild nature of
the photopolymerization, i.e., absence of heating, without
shifting pH to extreme values, and without the use of toxic
chemicals, the Fukui and Tanaka (1976) publication suggests
that this technique is desirable for the entrapment not
only of enzymes, but also for cells and organelles.
Dupuy et al. (1988) have recently described a
photopolymerization process for the entrapment of agarose
embedded pancreatic islets in microspheres of crosslinked
acrylamide. However, this reference describes entrapment
of individual microspheres, but does not describe further
entrapment of these already entrapped cells. Visible light
was used as the initiating radiation in the presence of a
photochemical sensitizer (vitamin B2, i.e., riboflavin),
and a cocatalyst (N-N-N ' N ' -tetramethylethylene diamine).
A high pressure mercury lamp was used as the source of
visible radiation and the islets were demonstrated to
maintain a good viability in vitro following the
polymerization step.
Visible radiation between wavelengths of 400 -
700 nm have been determined to be nontoxic to living cells(Karu, 1990; Dupuy et al., 1988). A recent review (Eaton,
1986) describes a variety of ciyes and cocatalysts that may
be used as polymerization initiators in the presence of
appropriate visible radiation.
.
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In recent years consiclerable in~eres~ has been
expressed in the use o~ lasers ~r po~ymeri%cltiorl p~ocf3s.~es
(Wu, 1990). These polymerizations are e~tremely fast an~
may be completed in millisecon~c-, (Dec~er an~ ~oussa, 1989;
Hoyle et al., 1989; Eaton, 1~6). The use of coherent
radiation often results in the polymerization being
innocuous to living cells. This arises from the use of
wavelength specific chromophores as the polymerization
initiators, and these chromophores are typically the only
species in the polymer/cell suspension that absorb the
incident radiation.
SUMMARY 0~ THE INVENTION
It has previously been demonstrated that Type I
insulin-dependent diabetes can be reversed in rats and dogs
by implantation of micro-encapsulated pancreatic islets in
the peritoneal cavity of these animals using both allograft
(dog islets to dog recipients) and xenograft (dog islets to
rat recipients) models (Soon-Shiong et al., 1991). The
alginate microcapsules employed in these studies provide
immunoprotectivity, and result in graft survival of
allografts for several months in the absence of
immunosuppression. The alginate gels, however, are
ionically crosslinked and, therefore, are subject to
breakage and resorption as a result of ionic equilibration
in vivo. In addition, free microcapsules are difficult to
retrieve due to their small size (200 - 600 ~m) and
nonlocalization within the peritoneal cavity.
While it is ~nown that the mannuronic acid -
guluronic acid (M-G) ratio of the alginate employed plays
an important role in preventing fibrosis, in accordance
with the present invention, other factors involving various
defects in prior art microcapsules have been identified
which play an important role in preventing long term
viability of the transplanted graft. Photomicrographs
~ 3 '3 9 8
--8--
demonstrate these heretofore unrecogni~ed clefects in prior
art microcapsules.
In accordance with the present invention, a
number of factors relating to capsule failure, i.e.,
likelihood of inducing an immune response, likelihood of
loss of function due to death of the encapsulated cells,
the degree of protection afforded encapsulated cells, and
the like, have been identified, i.e., that:
(i) Both mechanical and chemical stability of the
microcapsule play a critical role in fibrous
overgrowth. It has been discovered that the
water-soluble membrane dissolves over a prolonged
period of in vivo exposure, eventually exposing
the encapsulated material to the host's immune
system, initiating a rejection response;
(ii) The "roughness" of the capsule membrane, as
well as defective microcapsules, result in
' macrophage activation, cell adherence, cellular
~ overgrowth and eventual fibrosis;
i 20 (iii) Disruption or breakage of microcapsules,
' with cracks in the capsule membrane, results in
, fibrosis;
i~ (iv) While alginate, specifically high G-content
alginate, is relatively biocompatible, it has
~ 25 been discovered that poly-L-lysine (PLL) is a
;~ potent stimulator for fibrous overgrowth in vivo.
~d Indeed, the prior art describes attempts to cover
the outer coat of PLL with an outer layer of
alginate to prevent this overgrowth problem.
However, according to the present invention, it
has been discovered that any disruption of the
membrane, or any imperfection of coating of the
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~ 1239!~8
membrane results in ~ibrous overgrowth when
polymer materials ~uch as PLL ~re exposed.
Furthermore, current methods of covering Pl,L with
an outer layer of alyinate are ineffective to
fully prevent some exposure of PLL over time, in
vivo; and
(v) Exposure of biologically active material on
the surface of the microcapsule (because such
material is not adequately entrapped in the gel
forming the microcapsule) e~poses such material
to the host's immune system, initiating an immune
response.
In accordance with the present invention, it has
been further discovered that individual spherical
microcapsules provide a large exposed surface area,
facilitating transport of nutrients through the membrane,
while on the other hand, increasing the probability of cell
adherence and fibrous overgrowth. It has been found that
by reducing this exposed microcapsule sur.ace area and/or
by reducing exposure of any unbound, positively chargedpolylysine, while maintaining the critical diffusion
capacity of the immuno-protective membrane, increased
biocompatibility will ensue. By reduction of exposed
microcapsule surface area, the percentage of "roughness"
associated with each individual microcapsule would be
reduced, resulting in improved biocompatibility. In
addition, in accordance with the present invention, it has
been recognized that improving mechanical integrity of the
capsule is an important step in achieving long term graft
function.
In accordance with the present invention, it has
been found that long term graft functiGn can be achieved by
entrapping or encasing biologically active material,
optionally contained within a microcapsule (e.g.,
, .. ,.. ~.:
21.2~9g8
--10-
individual microencapsulated cells) in a macroc~psule which
is biocompatible, thereby (i) increasin-J the
cytoprotectivity of the entrappecl individually encapsulated
cells, (ii) reducing exposure of unhound positively charged
polylysine to the host in vivo environment, (iii) enhancing
the mechanical stability of the capsular membrane, (iv)
reducing the exposed microcapsule surface area roughness;
and (v) reducing the exposure of cells adhering to the
surface of the microcapsule to the host in vivo
lo environment; all of the above advantages are obtained while
(vi) maintaining the diffusion capacity of the polymeric
material used for encapsulation, thereby allowing the
entrapped encapsulated cells to be nourished and respond to
a stimulatory signal.
Additionally, macrocapsules of the present
invention provide a system of rapid but sustained release
of the material made by and secreted by the encapsulated
cell(s), which in turn provides for more regulated control
of physiological processes (e.g., blood glucose levels in
the case of encapsulated islets). Specifically, in
response to an intravenous glucose stimulus, insulin
release occurs more rapidly from encapsulated islets
entrapped within a macrocapsule than from free floating
mieroencapsulated islets, as demonstrated by the in vivo
intravenous glucose stimulation studies described in
Example 9, below. In addition, insulin release from these
gel entrapped microcapsules is sustained over a longer
period of time, as demonstrated by the in vitro glucose
stimulation studies described in Example 8, below.
The present invention also permits retrievability
of the implant because of its macroscopic size and its
grouping of the various microencapsulated islets into a
single package or a plurality of macroscopic packages.
Retrievability is desirable for a variety of reasons, e.g.,
so that accumulation of the implanted materials can be
,'
~1239~8
-- 1 . --
avoided, so that encapsulated cells can be removecl from the
recipient when no longer needecl or desired (e.cJ. ~ wtlfln the
product(s) of the encapsulated cells are no longer needed,
if the encaps~lated cells fail to perform as desired,
etc.), so that encapsulated cells can be removed if/when
they become non-viable, and the like.
The present invention overcomes the problems of
the prior art by improving the cytoprotectivity and
biocompatibility of implanted biological systems. The
present invention also provides a gel entrapment system
which provides a rapid response of entrapped cells to a
stimulatory signal. The present invention further provides
a sustained release system made from microencapsulated
cells which are further packaged in a macrocapsule. The
present invention also provides a system that localizes
microcapsules in a particular region (e.g., a region of
high vascularization such as the omentum), as well as a
system which minimizes the breakage of microcapsules, and
facilitates their ready retrieval.
As will be readily apparent to a person of skill
in the art, the macrocapsules of the present invention may
be used to entrap not only encapsulated or unencapsulated
living cells, but also any chemical reagents which may have
a pharmacological or physiological effect upon sustained
release by the system disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing of one embodiment of a
macrocapsule of the present invention containing
encapsulated biological material.
~ 30 FIG. 2 is a graph uf the blood glucose of rats
¦ implanted with entrapped encapsulated islets according to
the present invention (filled circle ), free-floating
~'1
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~ 1~3!~98
-12-
encapsulated islets (~illed triangle) ancl ~nerlc~psulat~d
islets (open circle O) over time, when the islft:s ~r~
xenografts tcanine islets).
FIG. 3 is a grapl~ o~ the serum glucose response
to an intravenous glucose challenge (I~lG~T) in rats treated
with macroencapsulated canine islets according to the
present invention, compared to the response in rats
transplanted with free floating microencapsulated canine
islets 7 days after implantation of the microcapsules and
macrocapsules.
FIG. 4 is a graph of the serum glucose response
to an intravenous glucose challenge (IVGTT) in rats treated
with macroencapsulated canine islets according to the
present invention, compared to the response in rats
transplanted with free floating microencapsulated canine
islets.
FIG. 5 is a graph of blood glucose levels of rats
implanted with entrapped encapsulated islets according to
the present invention (filled circle ) over time, when the
islets are xenografts (canine islets). On day 14, the
entrapped encapsulated islets were retrieved. The diabetic
state recurred within 24 hours following retrieval of the
entrapped encapsulated islets, proving the viability of the
islets entrapped within the macrocapsule of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
The following specification describes materials
and methods of macroencapsulating biologically active
materials, such as living cells, to provide an enhanced
biocompatible, retrievable system which is useful, for
example, for cell containment and transplantation. In the
following description, numerous details such as specific
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2~ 23~!)8
materi.als and methods are set forth in order to provicle a
more complete understancling o~ the presen~ inv~n~ion. Lt
is understood by those skilled in th~ ~rt that the present
invention can be practlced witho-lt these specieic details.
S In other instances, well known materials and methods are
not described in detail so as not to obscure the present
invention. In addition, the following description is yiven
with particular reference in many instances to islets and
diabetes treatment. This description is given as a
presently preferred application of the present invention.
Nonetheless, it is apparent that the present invention is
not limited to the treatment of a single disease state.
The present invention can be used equally as well for other
cell types and treatment of other disorders or for other
physiological purposes and could be used with
microencapsulated cells or unencapsulated cells of any
type.
As part of the work carried out pursuant to the
present invention, photomicrographs were taken of prior art
alginate-PLL-alginate microcapsules in efforts to identify
the faults and defects therein [see, for example, Clayton,
in J. Microencapsulation 8:221-233 (1991) and Wijsman et
al., in J. Transplantation 54:588-592 (1992) for a
description of prior art microcapsules]. The resulting
micrographs readily reveal the faults and defects in prior
art microcapsules. For example, a photomicrograph (40x) of
an empty alginate-PLL-alginate microcapsule (retrieved from
the peritoneal cavity of a rat) demonstrates that cellular
'i overgrowth (appearing as a darkened area) occurs in areas
, 30 of the exposed microcapsule gel or in areas of exposed
, polylysine when capsular membrane integrity is lost. A
1~ photomicrograph (lOOx) of an alginate-PLL-alginate
¦ encapsulated islet shows areas of "roughness" or "stress
marks" associated with individual microcapsules. The
, 35 surface area of such individual microcapsules is large and
! thus in turn, the exposure of "rough" surfaces is huge,
.,
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.
2123~98
increasing the risk of cellular overcJrowt~. rn .~ddi~ion,
imperfect covering of polylysine by an outer coa~ Oe
alginate results in potential over(Jrowth.
A photomicrograph (40x) o~ a defective empty
microcapsule, amongst intact empty alginate microcapsules,
all retrieved at the same time from the peritoneal cavity
of a rat, demonstrates cellular overgrowth (which appears
as darkened areas). Vigorous overgrowth is observed to be
associated only with the de~ective capsule. This provides
further evidence that minor imperfections invoke a foreign
body response to defective capsules and to exposed capsular
material, especially PLL.
A photomicrograph (40x) of disrupted alginate
microcapsules containing canine islets (transplanted free
floating into the peritoneal cavity), retrieved from the
peritoneal cavity of a diabetic dog when reversal of
diabetes failed, shows the presence of disrupted capsules,
providing evidence that loss of mechanical stability plays
an important role in graft failure. .hese
microencapsulated canine islets successfully reversed
diabetes, but only for a short period.
As used herein, the term "macrocapsule" means a
capsule of gel material surrounding biologically active
material, optionally contained within a microcapsule (e.g.,
one or more microcapsules, where each microcapsule contains
at least one living cell, such as islets or other
pharmacological agent producing cells, or certain drugs or
physiologically active agents). The term "macrocapsule"
may include "macro-membranes," "macrogels," "gel entrapped
microcapsules," "lace," "noodles," "teabags," "threads,"
"worms," and the like, which a person of skill in the art
would understand refers to the general class of
compositions described herein. While the actual dimensions
of the various components of the invention particles are
2123~8
-15-
not critical, the term "microcapsules" i5 yenerally used to
refer to particles wherein the laryest dimensions thereof
fall in the range of about 5 up to ~000 microns, while the
term "macrocapsules" is genera]ly used to refer to
particles wherein the largest ~imensions thereof fall in
the range of about 500 microns up to about 50 cm. ~hat is
important according to the present invention is that the
contents of a microcapsule be f~rther encapsulated into a
macrocapsule, thereby affording the added benefits of the
macroencapsulating polymer, as described herein.
In accordance with the present invention,
biologically active material, e.g., a plurality of cell-
containing microcapsules, are covered with a thick layer of
gelled material, forming a microcapsule-containing
macrocapsule. A schematic showing a cross-section of such
a macrocapsule is provided in Fiqure 1. Inspection of the
Figure reveals that the macrocapsule itself contains no
polycation or other fibrogenic surface; instead, the
immunoprotective membrane (i.e., polycation such as PLL) is
localized at the surface of the microcapsules, which are
buried deep within the macrocapsule (and protected from
exposure to the in vivo environment by thousands of
molecular layers). The core of the microcapsules (as well
as the macrocapsule itself) are maintained in the gelled
state (i.e., crosslinked or insoluble form), in contrast to
prior art encapsulation systems, wherein the core material
is liquified.
Generally the layer of gelled material in
invention macrocapsules has a thickness of at least about
l micron, with a thickness of at least about 20-40 microns
being preferred, and a thickness of at least about 50
microns or greater being especially preferred. This
provides more effective masking of the polycation layer on
the microcapsule than the fe~l molecule thickness (of
alginate outer layer) achieved in the preparation of prior
~''' ' .
1: . .
21239~8
-16-
art microcapsules. The layer of gellf?cl m~lteri.~l in
invention macrocapsules prevent.ci dire(t e~posure of arly
immunogenic agents at the microcapsule ;ur~ace (e.q.,
polycations, unencapsulated cells, ancl the like~ to the in
5 vivo environment, thereby preventiny immune response
triggered by prior art microcapsules. In addition, the
layer of gelled material is e~ceptionally stable to long-
term exposure to physiological conditions since the yelled
material is ionically and/or covalently crosslinked to
lO itself, and does not depend on an interaction ~ h the
incorporated material for strength and/or stability. This
is in direct contrast with alginate outer layers employed
in prior art microcapsules, wherein the sole means of
anchorage of the alginate outer layer to the capsule is the
15 the formation of a charge complex (i.e., ionic interaction)
between the alginate and the polycation immunoprotective
layer.
In a preferred embodiment of the present
invention, when microcapsules are incorporated into
20 macrocapsules, the microcapsules (and the macrocapsules
themselves) are maintained in the gelled state (i.e.,
ionically and/or covalently crosslinked). This is contrary
to the teachings of the prior art, wherein the
encapsulating matrix of the microcapsule is liquified prior
25 to implantation. In accordance with the present invention,
it has surprisingly been found that the diffusion of
nutrients in, and products of the encapsulated cells out of
invention macrocapsules is highly efficient.
s
Macrocapsules of the invention can be produced in
30 a variety of shapes, i.e., in the shape of a cylinder
s (i.e., a geometrical solid generated by the revolution of
a rectangle about one of its sides), a sphere (i.e., a
~ solid geometrical figure generated by the revolution of a
5s semicircle around its diameter), a disc (i.e., a generally
q 35 flat, circular form), a flat sheet (i.e., a generally flat
, ~
212399~
-17-
polygonal form, pre~erably square or rectarlgulclr)~ a waf~r
(i.e., an irregular flat sheet), a dog-bone (i.e., a shape
that has a central stem and t~o ~nds which are larqer in
diameter than the cen~.ral s~em, such as a ~lumbell), or the
like.
The materials used to provide such entrapment
could be alginate (preferably high G-content alginate), or
a modification of such alginate to improve its
biocompatibility and stability, e.g., a polymerizable
alginate allowing covalent crosslinkage, or crosslinkable
or polymerizable, water soluble polyalkylene glycol, or
combinations of these materials. The process to cause gel
entrapment of such materials can be accomplished either by
ionic or covalent crosslinkage.
Macrocapsules contemplated for use in the
practice of the present invention contain therein
biologically active material, wherein said biologically
active material is optionally contained within
microcapsules (i.e., a plurality of microcapsules within
said macrocapsule). The invention macrocapsule can be
prepared from a variety of polymeric materials, such as,
for example, covalently crosslinkable or polymerizable
linear or branched chain PEG, mixtures of different
molecular weight covalently crosslinkable or polymerizable
linear or branched chain PEGs, ionically crosslinkable
alginate, combinations of alginate and covalently
crosslinkable or polymerizable PEG and modified alginate
that is capable of being covalently and ionically
crosslinked, and the like.
Macrocapsules prepared in accordance with the
present invention comprise biologically active material
I encapsulated in the above-described biocompatible
¦ crosslinkable material, wherein the macrocapsule has a
, volume in which the largest physical dimension is greater
~,''"' ~''
~ l~3'i~
than 1 mm. Macrocapsules can cont.lin ~free~' ~i.e.,
unmodified by any coating) eells or groups o~ c~115
therein. Alternatively, macrocapsules m~y contain cells or
groups of cells which are themselves ~ncapsulated within
microcapsules.
~ iologically active materials contemplated for
encapsulation (to produce microcapsules and/or
macrocapsules) according to the present invention include
individual living cells or groups of living cells,
biological materials (for diagnostic purposes, e.g., for in
vivo evaluation of the effects of such biological materials
on an organism, and conversely, the effects of the orqanism
on the materials), tumor cells (for evaluation of
chemotherapeutic agents), human T-lymphoblastoid cells
sensitive to the cytopathic effects of HIV;
pharmacologically active drugs; diagnostic agents, and the
like. As employed herein, the term "living cells" refers
to any viable cellular material, regardless of the source
thereof. Thus, virus cells, prokaryotic cells, and
eukaryotic cells are contemplated. Specifically
contemplated cells include islets of Langerhans (for the
treatment of diabetes), dopamine secreting cells (for the
treatment of Parkinsons disease), nerve growth factor
secreting cells (for the treatment of Alzheimer's disease),
hepatocytes (for treatment of liver dysfunction),
adrenaline/angiotensin secreting cells (for regulation of
hypo/hypertension), parathyroid cells (for replacing
thyroid function), norepinephrine/metencephalin secreting
cells (for the control of pain), hemoglobin (to create
artificial blood), and the like.
I Covalently crosslinkable and/or polymerizable
I polyethylene glycols (PEGs) contemplated for use in the
! practice of the present invention include linear or
! branched chain PEGs (including STAR PEGs) modified with a
~ 3S substituent X which is capable of undergoing free radical
i
,
:
~ ~
.,-,
~1239~8
-19-
polymerization (X is a moiety cont~lining a carbon-~arbon
double bond or triple bond capable of free radic~l
polymerization; and X is linked covalently to said PEG
through lin~ages selected from ester, ether, thioether,
disulfide, amide, imide, secondary amines, tertiary amines,
direct carbon-carbon (C-C) lin~ages, sulfate esters,
sulfonate esters, phosphate esters, urethanes, carbonates,
and the like). Examples of such covalently crosslinable
polyethylene glycols include vinyl and allyl ethers of
polyethylene glycol, acrylate and methacrylate esters of
polyethylene glycol, and the like.
PEGs having a wide range of molecular weights can
be employed in the practice of the present invention, thus
mixtures of different molecular weights of covalently
crosslinkable PEGs contemplated for use in the practice of
the present invention include PEGs having a MW in the range
of about 200 up to 1,000,000 (with ~olecular weights in the
range of about 500 up to 100,000 preferred, and PEGs having
molecular weights in the range of about 1000 to 50,000
being the presently most preferred). Such PEGs can be
linear or branched chain (including STAR PEGs). STAR PEGs
are molecules having a central core (such as divinyl
benzene) which is anionically polymerizable under
controlled conditions to form living nuclei having a
predetermined number of active sites. Ethylene oxide is
added to the living nuclei and polymerized to produce a
Xnown number of PEG "arms", which are quenched with water
when the desired molecular weight is achieved.
Alternatively, the central core can be an ethoxylated
oligomeric glycerol that is used to initiate polymerization
of ethylene oxide to produce a STAR PEG of desired
molecular weight.
Alginates contemplated for use in the practice of
! the present invention include high G-content alginate, high
M-content alginate, sodium alginate, and the like;
~,"'
.: ' '' ` ;
.
~1~3~
-20-
covalently crosslinkable alginates contemplated for usq in
the practice of the present invention include alqinates
modified with a substituerlt X which is capable of
undergoing free radical poly~erization (X is a moiety
S containing a carbon-carbon double bond or triple bond
capable of free radical polymerization; and X is linked
covalently to said alginate through linkages selected from
ester, ether, thioether, disulfide, amide, imide, secondary
amines, tertiary amines, direct carbon-carbon (C-C)
linkages, sulfate esters, sulfonate esters, phosphate
esters, urethanes, carbonates, and the like). Examples of
covalently crosslinkable algina~es include allyl and vinyl
ethers of alginate, acrylate and methacrylate esters of
alginate, and the like.
Combinations of alginate (ionically and/or
covalently crosslinkable) and covalently crosslinkable PEG
contemplated for use in the practice of the present
invention include combinations of any two or more of the
above-described alginates and PEGs.
A small amount of a comonomer can optionally be
added to the crosslinking reaction to increase the
polymerization rates. Examples of suitable comonomers
include vinyl pyrrolidinone, acrylamide, methacrylamide,
acrylic acid, methacrylic acid, sodium acrylate, sodium
methacrylate, hydroxyethyl acrylate, hydroxyethyl
methacrylate (HEMA), ethylene glycol diacrylate, ethylene
glycol dimethacrylate, pentaerythritol triacrylate,
pentaerythritol trimethacrylate, trimethylol propane
triacrylate, trimethylol propane trimethacrylate,
tripropylene glycol diacrylate, tripropylene glycol
dimethacrylate, glyceryl acrylate, glyceryl methacrylate,
and the like.
''123~9~
Free radical polymerization of the above-
described modified materials can be carrie~ out in a
variety of ways, for example, initiated by irradiation with
suitable wavelength electromagnetic radiation (e.g.,
visible or ultraviolet radiation) in the presence of a
suitable photoinitiator, and optionally, cocatalyst and/or
comonomer. Alternatively, free radical polymerization can
be initiated by thermal initiation by a suitable free
radical catalyst.
A variety of free radical initiators, as can
readily be identified by those of skill in the art, can be
employed in the practice of the present invention. Thus,
photoinitiators, thermal initiators, and the like, can be
employed. For example, suitable UV initiators include 2,2-
dimethoxy-2-phenyl acetophenone and its water soluble
derivatives, benzophenone and its water soluble
derivatives, benzil and its water soluble derivatives,
thioxanthone and its water soluble derivatives, and the
like. For visible light polymerization, a system of dye
(also known as initiator or photosensitizer) and cocatalyst
(also known as cosynergist, activator, initiating
intermediate, quenching partner, or free radical generator)
are used. Examples of suitable dyes are ethyl eosin,
eosin, erythrosin, riboflavin, fluorscein, rose bengal,
methylene blue, thionine, and the like; examples of
suitable cocatalysts are triethanolamine, arginine,
methyldiethanol amine, triethylamine, and the like.
Microcapsules contemplated for use in the
practice of the present invention can be formed of a
variety of biocompatible gel materials, such as, for
example, alginate, covalently crosslinkable alginate (i.e.,
modified alginate that is covalently and ionically
crosslinkable), covalently crosslinkable PEG, combinations
of any of the above-described alginates and any of the
.1 .
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~-~:` ~:,, ~'` ` `' '~' '' '
~ . . !
21239!J~
-22-
above-described PEGs, as well as the optional presence of
one or more comonomers, as describecl above.
Microcapsules employed i~ the pr~ctice of the
present invention are optionally treated with an
immunoprotective coating, as described by Lim in U.S.
Patent No. 4,352,~83, incorporate~ by reference herein (Lim
refers to this immunoprotective coating as a "permanent
semi-permeable membrane"). I~munoprotec~ive mat2rials
contemplated for use in the practice of the present
invention include polycations (such as polyamino acids
[e.g., polyhistidine, polylysine, polyornithine, and the
like]; polymers containing primary amine groups, secondary
amine groups, tertiary amine groups or pyridinyl
nitrogen(s), such as polyethyleneimine, polyallylamine,
polyetheramine, polyvinylpyridine, and the like. Treatment
with such immunoprotecting materials can be carried out in
a variety of ways, e.g., by crosslinking surface layers of
an alginate gelled core containing encapsulated cells with
polymers having acid-reactive groups such as amine or imine
groups. This is typically done in a dilute solution of the
selected immunoprotecting polymer. ~ithin limits,
semipermeability of the immunoprotecting coating can be
controlled by proper selection of the molecular weight of
the immunoprotecting polymer, its concentration, and the
degree of crosslinking with the underlying gel. Molecular
weight of the immunoprotecting polymer can also vary,
depending on the degree of permeability desired. Typically
molecular weights will fall between about 1,000 and 100,000
or higher. Presently preferred immunoprotecting polymers
employed in the practice of the present invention fall in
the range of about 10,000 up to 50,000. Optional treatment
with immunoprotective coating can be avoided especially
when the macrocapsular or microcapsular material is
¦covalently crosslinkable, so that ~he desired porosity (or
¦35 immunoprotectivity) may be achieved through judicious
Iselection of the polymerizable macromonomers employed
21239~8
-23-
(i.e., polymeri~able alginate, polymeri~ble PEC, ~nd the
like), as well as mixtures of such monomers.
In a presently preferred embodiment of the
present invention, mul~iple alginate-PLL-alginate
S microcapsules are entrapped in an alginate macrocapsule.
The microcapsules employed preferably have solid gel cores.
The alginate employed can be ionically and/or covalently
crosslinkable. With modified, covalently crosslinkable
alginate, the introduction of polycations to control
microcapsule porosity and/or to provide an immunoprotecting
barrier is optional because the desired porosity (or
immunoprotectivity) may be achieved by using suitable
concentrations of modified alginate or by using modified
alginate having different degrees of substitution with the
moiety X, or combinations of such modified alginate.
Another presently preferred embodiment of the
invention involves the photopolymerization of an aqueous
physiological covalently crosslinkable linear or branched
chain PEG (e.g., a PEG-diacrylate solution, or
polymerizable equivalent) containing suspended alginate
microcapsules (e.g., alginate-PLL-alginate). The
microcapsules employed preferably have solid gel cores.
The appropriate free radical initiators and cocatalysts are
used with visible light sources such as a laser or a
mercury lamp. A solid (i.e., non-liquified) gel of PEG is
formed around the alginate microcapsules which in addition
to providing mechanical support and retrievability, forms
a nonfibrosing, cell nonadherent, and immunoprotective
coating around the microcapsules. In this embodiment, the
polycation layer is optional because the PEG macrocapsule
can readily provide immunoprotectivity by selection of
appropriate molecular weights and concentrations of
crosslinkable PEGs.
~: "
21239!38
-24-
In yet another presently pre~rred emhodlmerlt,
multiple alginate-PLL-alginate microcapsules are entrapped
in a macrocapsule comprising ionically and/or covalently
crosslin~able alginate, and covalently crosslinkable PEG.
In this embodiment as well, the polycation layer is
optional because the PEG macrocapsule can readily provide
immunoprotectivity by selection of appropriate molecular
weights and concentrations of crosslin~:able PEGs.
In a still further presently preferred
embodiment, multiple alginate-PLL-alginate microcapsules
are entrapped in an (ionically and/or covalently
crosslinkable) alginate macrocapsule, followed by a
covering of PEG such that PEG and alginate are in intimate
contact, and the PEG is exposed on the surface of the
macrocapsule. In this embodiment as well, the polycation
layer is optional because the PEG outer coat can readily
provide immunoprotectivity by selection of appropriate
molecular weights and concentrations of crosslinkable PEGs.
In still another presently preferred embodiment,
biologically active material is entrapped directly in a
macrocapsule comprising ionically and/or covalently
crosslinkable alginate, and/or covalently crosslinkable
linear or branched chain PEG.
The invention will now be described in greater
detail with reference to the following non-limiting
examples.
Example 1
PreParation Of PEG Diacrvlate Usinq Acrvlovl Chloride
PEG of molecular weight 18500 (abbreviated 18.5k,
also available commercially as PEG 20M) was modified
' chemically using the following procedure to incorporate
acrylate functionalities into the molecule which rendered
'
2~'~39'J8
~` -25-
it polymerizable. Other PEGs r~nqing in moleclllclr waigh~s
from as low as 200 to as hiyh as (hut not limited to) 35000
could also be modified by the sa~e proceclure.
PEG 18.5k was dried thorouf~hly by heating in a
vacuum oven at 80C for 2~ hours. Alternately, the PEG
could be dissolved in toluene and the solution distilled
wherein any moisture could be re~oved as an azeotrope with
toluene. 20g of dry PEG were dissolved in 200-250 ml of
dry toluene (acetone, benzene, and other dry organic
solvents may also be used). A twofold molar excess of
acryloyl chloride was used (0.35 ml) and a base, triethyl
amine (0.6 ml) was added to remove HCl upon formation.
Before addition of acryloyl chloride, the solution was
cooled in an ice bath. The reaction was carried in a round
bottomed flask under argon with constant reflux for 24
hours. The reaction mixture was filtered to remove the
insoluble triethyl amine hydrochloride while the filtrate
was added to an excess of ether to precipitate PE
diacrylate. The product was redissolved and precipitated
twice for further purification and any remaining solvent
removed in vacuum. Other purification schemes such as
dialysis of a PEG-water solution against deionized water
followed by freeze drying are also acceptable. Yield:
17g.
Examole 2
Alternate Methods for Preparation of Polymerizable PEG
Several other methods may be utilized to prepare
a modified PEG that is polymerizable or crosslinkable by
introducing unsaturation at the ends of the PEG chain.
Some of these methods are briefly outlined below.
` An esterification reaction between PEG and
acrylic acid (or higher homologue or derivative thereof)
may be carried out in an organic solvent such as toluene.
~, ' :' ' ' - `.' ~
~t23~38
A small amount of acid s~lch as p-~oluene sul~c)rlic acLd may
be used to catalyze the reaction. Exc~s~ o~ one of ~he
reactants (acrylic acid) will clrive the reac~ion towards
the products~ The reaction is refl~l~e~ for several hours.
Since water is formed as a prcduct of ~he reaction, in
order to drive the equilibrium touards the products, it may
be continuously withdrawn by distillation of the azeotrope
formed with toluene. Standard purification schemes may be
utilized. Methacrylic acid or methacrylate may be reacted
in a similar fashion with PEG to obtain the polymerizable
derivative (Fukui and Tana~a, 1976).
Alternatively, the method of Mathias et al.
(1982) which involves the reaction of the PEG al~oxide with
acetylene gas to produce the vinyl ethers of PEG may be
employed to produce a polymerizable PEG derivative.
A reaction between PEG and allyl chloride in a
dry solvent catalyzed by small amounts of stannic chloride
also results in a polymerizable PEG.
Several other techniques may be utilized to
obtain polymerizable PEG derivatives. Harris (1985)
describes a number of protocols involving PEG chemistry
from which alternative synthetic schemes are provided.
Exam~le 3
Visible Liaht Photopolymerization To Produce PEG Gels
PEG derivatives prepared by the techniques
outlined in examples 1 and 2 were dissolved in aqueous
bicarbonate buffered saline (such as 5-40 wt%, or other
buffer) at pH 7.4. The photosensitizer, ethyl eosin
(O.Ol~M up to O.lM), a cocatalyst, triethanolamine (O.Ol~M
up to O.lM), and comonomer, vinyl pyrrolidinone (0.001 to
10%, but not essential) were added to the solution which
was protected from light until the photopolymerization
23!3~8
reaction. In the alternative, other initiators
cocatalysts, comonomers and w~velerlcJths of laser radiation
may be used, the selection of ~hich is well known in the
art.
A small quantity of the prepared solution was
taken in a test tube and e~posed to visible radiation
either from an argon ion laser at a wavelength of 514 nm at
powers between 10 mW to 2 W, or a 100 watt mercury arc lamp
which has a fairly strong emission around 514 nm. The
gelling time was noted and found to be extremely rapid with
the laser (on the order of milliseconds) and fairly rapid
with the mercury lamp (on the order of seconds) and varied
with the concentrations of polymer, initiator, cocatalyst,
and comonomers in the system.
Example 4
UV Liqht Photopolymerization To Produce PEG Gels
A different initiating system from the one above
was used to produce PEG gels. A Uv photoinitiator, 2,2-
dimethoxy-2-phenyl acetophenone was added to a solution of
polymerizable PEG in aqueous buffer at a concentration of
1000 - 1500 ppm. This solution was exposed to long wave W
radiation from a 100 watt W lamp. The time required for
gellation was of the order of seconds and was a function of
the concentrations of initiator and addition of other
polymerizable monomers such as vinyl pyrrolidone (0.001 to
10%). A UV laser may also be used for the
photopolymerization. Other UV photoinitiators may also be
used (e.g., benzoin ethyl ether).
i
. i;r: A~., ' ' ' ''-' ' ~ '' ,, '
. ~,~ ~ ' .,. ' "" ' '' ' '' , ,
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2123'J~
-28-
Examp lQ_S
_ometries o~ Microcapsule-Conrcl nln~_PEG Gels eOr
Implantatlon
Microcapsules of alginate or any other material
containing cells or enzymes or drugs may De delivered and
retrieved from an implant using varying c3eometries of the
microcapsule-containing PEG gels. A larqe number of
individual microcapsules may be localized to a preferred
region in the peritoneal cavity (or other implantation
site) by embedding these microcapsules in a crosslinked PEG
gel. Various geometries may be considered for implantation
such as cylindrical rod, circular disk, flat plate, dog
bone, and long cylindrical "laces" or "threads" or "worms."
Regardless of the geometry, the formation of the
final implantable products required similar processing
techniques. The cell containing microcapsules were
suspended in a physiological solution of PEG 18.5~
diacrylate (30 wt%; PEGs of varying molecular weights may
be used) containing triethanolamine (O.Ol~M up to O.lM),
ethyl eosin (O.Ol~M up to O.lM), and vinyl pyrrolidinone
(0.001 to 10%). This suspension was exposed to visible
light from a laser or mercury lamp which caused rapid
crosslinking of PEG-diacrylate and resulted in
microcapsules embedded in the PEG gel.
In order to produce a cylindrical "lace," a
suspension is disposed in a hypodermic needle having a
suitable gauge needle or cannula and the emergent stream is
expelled into a buffered solution (which may have
multivalent cations if alginate is used in the suspension)
and simultaneously exposed to laser radiation from a
suitable lamp source if photopolymerization is required for
the reaction. The lamp could be a laser light source or a
W light source depending upon the material to be
polymerized and the photoinitiator used, as is known in the
: ,: ` ''
~, .,
~ ' '' ' ' . ~. ,: ,
~ -
` 21239~
-29-
art. Crosslinking is inscantaneous, i.e., the emerCJing
stream is photocrosslinked as tast as it is extruded,
resulting in the formation o~ a "lace" or "noodle." Gels
or other geometries can be produced from c~ppropriat~ molds
fabricated for the purpose.
E~am~le 6
Alqinate Gel Entrapment O~ Microenca~sulated Islets
Canine islets ~ere isolated ~rom donor pancreata
by collagenase digestion and purified using a physiological
islet purification solution. Islets were then encapsulated
in an alginate-polylysine alginate microcapsule by the
following process: Ten thousand purified islets were
suspended in 1.8% solution of Na alginate (G content 64%)
and via an air-droplet generating device, islets were
lS entrapped in alginate gel beads by crosslinking alginate in
0.8% CaCl2 solution. A polylysine membrane was formed
following suspension of these alginate encapsulated islets
in a 0.1% polylysine solution for 4-8 minutes. The
encapsulated islet was then coated with an outer layer of
alginate by suspension in 0.2% alginate for 5 minutes.
A cylindrical tube (i.e., a lace, approximately
1-5 mm in diameter) of alginate encasing these
microencapsulated islets was then produced as follows:
The microencapsulated islets were suspended in
1.8% alginate in the ratio of 1 volume microcapsule pellet
to 4 volumes of alginate, and extruded into a solution of
divalent cations containing CaCl2alone, or a combination of
barium and calcium chloride. The ratio of barium and
calcium chloride as a combination of divalent cations
crosslinking agent could vary from 1:10 to 1:100
(barium:calcium), and the preferred ratio is in the range
of 1:50 to 1:100. By combining barium and calcium, a gel
of greater strength results. Thus, by this process of
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2.1~3~
-- , o--
ionic crosslinking, approximat~ly ~000 mi~roencapsul.lted
islets are entrapped in a 16 cm lon~ cylinclrical tube Oe
alginate gel.
Alternatively, spherical macrocapsulescontaining
microencapsulated islets can be prepared by introducing
droplets of the above-described solution (i.e.,
microencapsulated islets in alginate solution) into a bath
containing polyvalent cations (e.g., Ca ), as described
above.
Example 7
Variations in Composition of Retrievable Systems
A number of variations in compositions and
materials used in the design of retrievable systems are
acceptable and advantageous. The crosslinked alginate or
PEG spheres (or lace) containing alginate microcapsules may
be replaced in the appropriate situations by an ionically
and/or covalently crosslinked alginate spheres.
Combinations of alginate and PEG may also be used. The
following variations, among others, are possible:
(i) PEG alone, crosslinked as described above;
mixtures of different molecular weight
crosslinkable PEGs (linear or branched
chain) may be used to adjust permeability of
the resultant gels;
(ii) Alginate alone, ionically crosslinked;
~, (iii) Alginate alone, modified to be both
ionically and covalently crosslinkable;
(iv) A combination of ionically crosslinkable
alginate and covalently crosslinkable PEG
(linear or branched chain);
(v) A combination of modified alginate (i.e.,
ionically and covalently crosslinkable) and
~ covalently crosslinkable PEG (linear or
¦ branched chain);; and
,~-i.. ,. -.,-.- .. , . .
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(vi) An ionically anc~/or covalently crosslinked
alginat~ macrocapsllle ~e.g., a sphere or
lace) covered with a coatirlg of covalently
crosslin~ed PEG (linear or branched chain)
S covering the exterior such that the PEG and
alginate are in intimate contact.
Ionic crosslin~ing of alginate can be
accomplished with either Ca alone or a combination of Ba
and Ca in a ratio of 1:10 to l:lO0 (Ba:Ca).
The alginate referred to in the preceding
paragraph may be replaced with a modified alginate that is
chemically crosslin~able in addition to its ionic gelling
capabilities. The modified alginates mentioned herein are
described in detail in copending patent application Serial
No. 07/784,267, incorporated by reference herein in its
entirety.
The PLL referred to above may also be replaced
with a modified PLL that is covalently crosslinkable. The
modified PLL mentioned herein is described in detail in
copending patent application Serial No. 07/784,267,
incorporated by reference herein in its entirety.
Example 8
Kinetics of Insulin Diffusion from Gel Entrapped
Microenca~sulated Islets: In Vitro Studies
Kinetics of insulin secretion from the gel
entrapped encapsulated canine islets were compared to
individual microencapsulated islets or unencapsulated
canine islets as follows: either free unencapsulated
canine islets (controls) or encapsulated canine islets or
gel entrapped encapsulated canine islets were incubated in
RPMI culture medium containing a basal level of 60 mg%
glucose for G0 minutes, then transferred to medium
~, '.
21 23~8
containing a stimulatory level of ~S0 mCJ% CJl~co5e eOr 60
minutes and returned to basal medium (60 mq% qlucose) for
a further 60 minutes. These tests were perform~cl in
triplicate. The supernatant was collected at the end of
each 60 minute period. Insulin secretion was assayed by
measuring insulin concentration (~Lu/ml per islet equivalent
count) in the supernatant, using RIA.
The study was repeated, but in addition, 10 mM
theophylline was added to the 450 mg% ylucose as an added
stimulus of insulin secretion. The results of these
studies are shown in Table 1.
.,.'~
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239~)8
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2123~8
-34-
One skilled in the art would expect that
entrapping encapsulated canine isle~s in a ~urther outer
coat of polymer material would resu].t in decre~sed response
to a stimulus and reduced diffusion of insulin. 1`hat is,
S entrapping encapsulated islets in yet a second layer of
polymer material ~the microcapsule plus the macrocapsule]
would be expected to reduce the res~onsiveness of the
encapsulated islet to an outside stimulus. ~nexpectedly,
according to the present invention, it has been found that
the rapidity and intensity of the in vitro response Gf gel
entrapped, microencapsulated islets was equal to, if not
better than, the free floating encapsulated islets (as
shown in Table 1).
Following stimulation with 450 mg% glucose alone,
the insulin response (see Table 1) from the
microencapsulated islets immobilized in a macrogel
("entrapped MC") was equal.to, if not better than the free
floating microencapsulated islets ("Free MC") as well as
the free unencapsulated islet controls ("Free Islets").
This phenomenon was also observed in response to 450 mg%
glucose + 10 mg theophylline stimulus. While not wishing
to be bound to any particular theory, it is currently
hypothesized that this surprising result is explained by
the fact that insulin is a negatively charged protein, and
with the release of insulin from the entrapped
microencapsulated cells in response to glucose stimulus,
the negatively charged insulin is electrostatically
discharged from the negatively charged entrapping gel
material, thus accelerating the response time of insulin
release. Enhancement of insulin release from
microencapsulated islets within a macrogel, as compared to
insulin release from individual encapsulated islets, can
also be explained by the fact that the microcapsule is more
neutrally charged than the macrocapsule, due to the
presence of the positively charged PLL in the microcapsule
membrane. This unexpected result was corroborated in the
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in vivo studies (described in Example 9) where the e<31l in
serum glucose in response to L~n intrav~nous ylu~se
injection occurred rapidly in cliabetic rats treated with
gel entrapped microencapsulated canine islets (see FIG. 3),
suggestive that gel entrapment provides an added advantage
of improved insulin response to a systemic glucose signal.
The results shown in Table 1 demonstrate that gel
entrapment of encapsulated canine islets did not impair
insulin secretion in response ~o a glucose stimulus or in
response to glucose plus theophylline. Return of insulin
levels to basal state was however delayed in the
macrocapsule experiment, and is consistent with entrapment
of insulin in the outer gel, resulting in effect a
sustained release drug delivery system. In in vivo
applications, this sustained release of insulin may provide
a more stable homeostatic mechanism.
Thus gel entrapment of microencapsulated islets
provides several unexpected advantages with regard to
insulin secretion:
(i) rapid response of insulin release to a
glucose stimulus, and
(ii) once the insulin is released, a slow return
to basal state, providing a sustained,
continuous release over a longer period.
~5 Both of these responses are important in improving the
homeostatic control of glucose metabolism in the diabetic
recipient.
ExamDle 9
In Vivo Studies of Gel Entrapped Microencapsulated Islets
The efficacy of gel-entrapped microencapsulated
canine islets was compared to free-floating encapsulated
canine islets in the treatment of streptozoticin-induced
(STZ) diabetic Lewis rats. Eight thousand encapsulated
21~3'~
canine islets (either free--floatincJ, or entrapp~d in
cylindrical tubes of alginate) were implanted into the
peritoneal cavities of four STZ-diabetic ~,ewis rats, and
compared with eight thousand unencapsulated canine islets
(controls) implanted in the same way.
To ensure no variability in the viability, purity
and function of the islet preparation, islets from the same
canine pancreatic donor were used in both free floating
microencapsulated and gel entrapped microencapsulated
groups. Furthermore to ensure no variability in the
integrity and formation of the microcapsules themselves,
the islets were all encapsulated in one batch process and
then divided into two -- one half being used for free
floating implantation and the remaining half entrapped in
an alginate macrocapsule (i.e., gel entrapped
microencapsulated islets) prior to implantation.
Immunoprotectivity, biocompatibility, function
and graft survival of the unencapsulated (free) islets
compared to free-floating encapsulated islets (in
microcapsules only) compared to entrapped encapsulated
islets (in macrocapsules with microcapsules therein) were
compared by measuring:
(i) Daily serum glucose levels (see FIG. 2)
Diabetes is induced in rats by STZ injection.
Injected rats are considered diabetic if serum glucose
levels are greater than 200 mg%. Unencapsulated canine
islets (free islets) failed to restore normoglycemia (serum
glucose levels less than 200 mg%) in the diabetic rat. In
contrast, the free floating microencapsulated islets and
the macroencapsulated islets both restored normoglycemia in
these diabetic rats by the 3rd - 4th day. By day 7, the
free floating microencapsulated islets began to fail, with
serum glucose levels rising in these recipients to
~200 mg%. In contrast, rats receiving gel entrapped
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microcapsules (m~crocapsule~) continuecith~ir normoglycemic
sta~e, with serum glucose levels below 100mg% ~FIC. ~).
These ln vivo studies therefore provicle evidence that
(compared to fr~e floating microencapsulated islets) gel
entrapment of microencapsulated islets prolongs yraft
survival, presumably by enhancing cytoprotectivity and
biocompatibility.
(ii) daily urine volume
With onset of diabetes, urine volume increases.
Following STZ injection, urine volume exceeds 30 ccs per
day and is indicative of the diabetic state. With
resolution of diabetes, urine volume falls to normal
levels. Reduction in urine volume coincided with the fall
in serum glucose, providing further evidence that graft
function and survival of macroencapsulated islets was
superior to both free unencapsulated islets and free
floating microencapsulated islets. On day 7, urine volume
in free floating microencapsulated group rose to diabetic
levels (>30mls.), indicating onset of graft failure, while
the rats receiving gel entrapped microcapsules (i.e.,
macrocapsules) maintained a normal level of urine output,
indicating ongoing graft function.
(iii) body weight
Following STZ injections, diabetic rats lose
weight dramatically. Maintenance of weight or weight gain
is an excellent parameter of function of the transplanted
islets. Similar to the correlation between urine volume
and serum glucose levels, changes in body weight
corroborated the above analysis, providing further evidence
that rats receiving macroencapsulated islets retained
normal graft function compared to graft failure in rats
receiving free unencapsulated islets and those receiving
Ifree floating microencapsulated islets. Body weight
iimproved significantly in the animals receiving gel
135 entrapped microcapsules (i.e., macrocapsules), while weight
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loss as a result of diabetes continuec~ in the ~nimal~ with
free unencapsulated islets ~nd fell ~l~ter d~y 7 in th~
animals with free floating encapsulclte~ islets.
(iv) glucose response to systemic glucose
challenge (intravenous glucose tolerance test, IVGTT; see
FIG. 3)
An IVGTT provides an in vivo assessment of the
kinetics of insulin release in response to an intravenous
glucose challenge. 8y calculating the rate of fall of
glucose over time, a numerical value (K-value) can be
ascertained and compared to normal non-diabetic rats. The
more rapid the fall in serum glucose following the glucose
challenge, the higher the K-value. The fall in serum
glucose in response to an intravenous glucose challenge was
studied in both the rats receiving macroencapsulated and
free floating microencapsulated islets. Serum glucose was
measured at l, 4, lO, 20, 30, 60 & 90 minutes following an
intravenous injection of 0.5cc per kg of 50% dextrose. As
can be seen in FIG. 3, following the intravenous glucose
injection, serum glucose rose to 489 mg % in the
macroencapsulated group, and then fell rapidly to normal
levels within lO minutes, indicating an excellent in vivo
insulin response to the systemic glucose challenge.
K-value (which represents the % fall of glucose over time)
in these animals was normal (3.81 + 0.56). This unexpected
rapid insulin response corroborates the in vitro findings
described in Example 8, i.e., that alginate gel entrapped
microcapsules respond rapidly to a glucose stimulus, and
provides further evidence that a potential advantage of
alginate gel entrapment is electrostatic repulsion of
negatively charged insulin from within the capsule, and
hence rapid physiological fall in glucose in response to a
systemic challenge.
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The K values o~ the rats receivinq macrocapsules
were significantly better (K=3.a + 0.56) than the rats
receiving free floating encapsul~ted islets (K=0.6 + 0.73),
indicating loss of graft function in the rats receiving
free-floating encapsulated islets. Since the islets
transplanted into both groups were fro~ the same canine
donor, and the volume of islets emplo~ed were identical,
this difference in response reflects the improved
immunoprotectivity of the macrocapsule.
In a separate e~periment, free floating
encapsulated canine islets were transplanted into diabetic
rats and IVGTT performed in these recipients while graft
function was normal (serum glucose less than 100 mg%). As
can be seen in FIG. 4, the serum glucose fell to normal
levels following the intravenous injection of dextrose only
after 30 minutes (K value 2.4 +0.9) compared to the more
rapid fall of glucose (within lO minutes) in the rats
receiving macroencapsulated islets (K value 3.81 + 0.56).
From the above-described in vivo studies, it can
be concluded that:
(i) gel entrapment of microencapsulated islets
does not decrease the rate of diffusion of
insulin in response to a glucose stimulus
both in vitro and in vivo;
(ii) gel entrapment of microencapsulated islets
in fact enhances insulin release; and
(iii)gel entrapment of microencapsulated islets
prolongs graft function.
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Exam~le 10
Retrieval of Gel Entrap~ed Mlcroenc~lnsu]atecl Islets
Ge] entrapped microencapsulated islets were
implanted into STZ induced diabetic rats and followed for
14 days. Normal serum glucose levels ~"ere achieved within
3 days, and was maintained for the entire observation
period. On the 14th day, the macroqelled encapsulated
islets were retrieved and examined microscopically.
As observed by photomicrograph, viable intact
islets within entrapped microcapsules were found with no
evidence of cellular overgrowth of the outer surface layer
of the macrogel. A photomicrograph (100x) of a
macrocapsule containing canine islets according to the
present invention retrieved from the peritoneal cavity of
an STZ-diabetic Lewis rat 14 days after implantation
demonstrates a smooth surface on the outer exposed layer of
the macrocapsule, with no evidence of cellular overgrowth.
~ Encapsulated viable individual canine islets can be seen
:~! contained within the macrocapsule. At the time of
i 20 retrieval, this diabetic rat was normoglycemic.
i
~, Serum glucose levels were monitored in the rat
:3 following retrieval of the macrogelled encapsulated islets.
As can be seen in FIG. 5, within 24 hours after retrieval
of the macrocapsule serum glucose levels rose to greater
than 200 mg%, indicating that the macrogelled encapsulated
islets was responsible for normalization of the serum
glucose, and providing further evidence of the
immunoprotectivity and biocompatibility of the
macrocapsule.
From these studies, it can be concluded that:
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(i) gel entrapment provides a smoother outer
J surface, a reduced exposed surface area and hence increased
t
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biocompatibility tha~ fre~ floating microencapsulated
islets.
(ii) gel entrapment increases the mechanical
stability of encapsulated islets and provi~es a retaining
matrix even if the individual capsules were to break.
Exa~ple 11
Alleviation of Diabetic Symptoms in a Spontaneous
Diabetic Doq Implanted with Invention Macrocapsules
A spontaneous diabetic dog having the classical
symptoms of Type I Diabetes mellitus at the time of
diagnosis, i.e., clinical signs such as polyuria,
polydipsia, polyphagia, weight loss, persistent fasting
hyperglycemia (i.e., blood glucose >250 mg/dl), persistent
glycosuria, and the need for 1-2 daily injections of
insulin to prevent rapid decompensation, was chosen as a
recipient for transplantation of a macrocapsule of the
invention (containing a plurality of microencapsulated
islets).
Islets from a healthy canine donor were isolated
employing conventional methods (e.g., collagenase
digestion). Islets were then microencapsulated as
described above (see, for example, Example 6), in
conventional alginate-PLL-alginate microcapsules
(approximately 600 microns in diameter). The microcapsules
were then further entrapped (i.e., encapsulated) into
macrocapsules (spherical beads about 4000-aooo microns) of
ionically crosslinked alginate by extrusion of a suspension
of microcapsules in an alginate solution into a bath
containing calcium chloride. Approximately 60-80
microcapsules were entrapped within a single macrocapsule.
Prior to transplantation, the dog had been
maintained on a regimen of 12 Units of insulin per day.
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The transplantation was performecl under genera I anaesthesia
through a 1.5 ventral mi-lli ne i nc ision, an(l th~-
macrocapsules aseptically introduced into the peritonecll
cavity through a stainless steel funnel. The dog rece~ived
a transplant dose of about 20,000 islets/~g body weight.
The incision was then closed by sutures. A regimen of
prednisolone was maintained ~or 1~ days post-transplant as
an anti-inflammatory. Fasting blood glucose, plasma
C-peptide, urine output and body weight were measured daily
for the first 14 days post-transplant, and every 7 days
thereafter. Intravenous glucose tolerance tests (IVGTT)
were performed 7 days prior to transplantation, 14 and 30
days post-transplantation, and every 30 days thereafter.
All measured parameters returned to normal non-
diabetic values within three days after transplantation.
Blood glucose was maintained well below 200 mg/dl, and the
dog remained euglycemic for >150 days following
transplantation, without the need for any exogenous
insulin. IVGTT results were normal and showed rapid
glucose clearance following challenge with a bolus of
glucose.
Example_12
Reversal of Hepatic~ Deficiency by Trans~lantation of
EncaPsulated Hepatocvtes in a Gunn Rat Model
Homozygous Gunn rats having a deficiency of the
liver enzyme uridine diphosphate glucuronyl transferase
(UDPGT) and resultant elevated serum bilirubin levels (a
condition commonly known as jaundice) were chosen as a
model of hepatic deficiency. Heterozygous Gunn rats which
carry the abnormal gene, but are free from jaundice were
chosen as the donors of hepatocytes for cellular
transplant.
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The donor liver was c~nn~la~ed via the portal
vein with silicone tubing (i cl 0 02S inch, o.cl. 0.0~/
inch) and perfused with 100 ml o~ 0 05% collagenase (Si~Jma,
Type IV) in 15 mlnutes. The hepatocytes were then
harvested into a sterile bea~er and ~Jashed three times with
RPMI medium. Viability of the isolated hepatocytes was
tested by acridine orange/propidiu~ iodide staining and
found to be greater than 80%.
The hepatocytes ~ere encapsulated into
conventional alginate-PLL-alginate microcapsules as
described above. These microcapsules were further
entrapped (or encapsulated) in spherical macrocapsules of
ionically crosslinked alginate (as described above.
Approximately 50-100 microcapsules were entrapped in a
single macrocapsule. Each recipient received -10
encapsulated hepatocytes. The macrocapsules were implanted
in the peritoneal cavity of the recipients.
Prior to transplantation, recipient bilirubin
levels were 5.3 + 0.3 mg%. ay the second week post-
transplantation, serum bilirubin levels were reduced to
2.05 + 1.05 mg%, which was significantly lower than pre- -
transplant levels. These reduced levels were maintained
for an average of 73 + 4 days. .~ll recipients received
c-yclosporin A as an anti-inflammatory (10 mg/kg, i.e., at
levels lower than required to prevent immune rejection.
Thus, the feasibility and efficacy of the macrocapsule of
the invention is demonstrated for in vivo transplantation
of hepatocytes.
While the invention has been described in detail
with reference to certain preferred embodiments thereof, it
will be understood that modifications and variations are
within the spirit and scope of that ~hich is described and
claimed.
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Re e_ences
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.1
