Aust. J. Chem. 2006, 59, 508–524 www.publish.csiro.au/journals/ajc
Polymer Chemistry in Diabetes Treatment by EncapsulatedIslets of Langerhans: Review to 2006
Igor LacíkA
A Polymer Institute of the Slovak Academy of Sciences, Dúbravská cesta 9, 842 36 Bratislava,Slovak Republic. Email: [email protected]
Polymeric materials have been successfully used in numerous medical applications because of their diverse prop-erties. For example, development of a bioartificial pancreas remains a challenge for polymer chemistry. Polymers,as a form of various encapsulation device, have been proposed for designing the semipermeable membrane capableof long-term immunoprotection of transplanted islets of Langerhans, which regulate the blood glucose level in adiabetic patient. This review describes the current situation in the field, discussing aspects of material selection,encapsulation devices, and encapsulation protocols. Problems and unanswered questions are emphasized to illus-trate why clinical therapies with encapsulated islets have not been realized, despite intense activity over the past15 years. The review was prepared with the goal to address professionals in the field as well as the broad polymercommunity to help in overcoming final barriers to the clinical phase for transplantation of islets of Langerhansencapsulated in a polymeric membrane.
Manuscript received: 25 May 2006.Final version: 14 August 2006.
Introduction
Diabetes
Diabetes is a disease of hormone deficiency, characterizedby hyperglycemia resulting from the body’s inability to useblood glucose for energy.[1] In type 1 diabetes, the pancreasno longer secretes insulin and, therefore, blood glucose can-not enter the cells to be used for energy. In type 2 diabetes,either the pancreas does not provide sufficient insulin or thebody is unable to use insulin correctly. Diabetes is currentlyconsidered as an epidemic disease worldwide[2–5] becauseof strongly increased prevalence, with more than 350 mil-lion patients estimated worldwide by 2030 compared withapproximately 170 million in 2000. This is a consequence ofpopulation aging, diet, obesity, decreased physical activity,stress, and other factors.
The administration of exogenous insulin, most commonlyby multiple daily insulin injections, insulin pumps,[6,7] orinsulin inhalation,[8] does not completely solve the problemof insulin deficiency because the required instant demandof an organism for insulin cannot be achieved, and blood
Igor Lacík graduated in polymer engineering from the Slovak Technical University, Bratislava, in 1986 and received his Ph.D.from the Slovak Academy of Sciences in 1993. This period included a research assistantship with Robert Gilbert (Universityof Sydney, 1990–1991) and was followed by postdoctoral positions with Françoise Candau (CNRS Strasbourg, 1993–1994)and Taylor Wang (Vanderbilt University, 1994–1996). He then joined the Polymer Insitute in Bratislava where he heads thedepartment of special polymers and biopolymers. His main research interests are free-radical polymerization kinetics in theaqueous phase and microencapsulation of bioactive substances for biomedical applications.
glucose concentration differs from the physiological normbetween 3.5 and 7.8 mmol L−1.[9] Low glucose concentra-tions (<3.5 mmol L−1) result in hypoglycemic events, whichmay lead to coma. Untreated, very high glucose levels leadto keto-acidosis and serious health problems, and long-termhyperglycemic conditions not sufficiently controlled withexogenous insulin are connected with secondary compli-cations resulting in diseases of the cardiovascular system,kidneys (nephropathy), blindness (retinopathy), and nervoussystem (neuropathy), thus contributing to shortening the livesof diabetic patients. Therefore, in diabetes treatment, the goalis to provide an amount of insulin to the body so that glucoselevels are continuously as close as possible to physiologicalvalues.
Diabetes Treatment
The most appropriate means to restore the physiologicallevel of insulin may be whole organ replacement. However,because of donor shortage, major surgery issues, and perma-nent need for immunosuppression, pancreas transplantation
transplant rejection cannotenter to the capsule interiorGlucose, nutrients, O2
freely enter capsule interiorthrough the membrane
to the islets
Islets respond to the glucoseconcentration when higher
than physiological one, thenrelease insulin
MembraneIslets
Cells of immune systemdo not adhere to the
capsular surface and donot modify membrane
diffusion properties
Fig. 1. Semipermeable membrane for immunoprotection of encapsulated islets.
is not realistic for diabetes treatment even though the successrate of pancreas transplantation as a whole organ is rela-tively high.[10,11] This result is particularly true in infantswhere the risks related to transplant and immunosuppressionmust be balanced against exogenous insulin delivery. How-ever, the transplantation of insulin-producing cell clusters,called islets of Langerhans, represents technical simplicityand has the potential to restore glucose homeostasis andprevent secondary complications.[12] This effect was clearlydemonstrated by the breakthrough method for transplantationof islets of Langerhans, known as the Edmonton protocol,[13]
in which human islets are transplanted into the portal veinof diabetic patients. A carefully selected combination ofimmunosuppressive drugs safer for transplanted islets is usedto prevent their rejection by the recipient’s immune system.This[13] and follow-up[14–17] work proved that transplantedislets fully function on the average time scale of more thanone year when brittle-diabetic patients were free of exoge-nous insulin. In spite of these promising results, the mostrecent up-date by Ryan et al.[17] calls for further progressin the availability of transplantable islets, improving isletengraftment, preserving islet function, and reducing toxicimmunosuppression.
Polymeric Membranes in Islet Transplantation:No Need for Immunosuppression
For much of the past decade, research has focussed on (a) theidentification of additional cell sources,[18] primarily stemcells[18,19] and animal cells,[20,21] and (b) the transplantationof insulin-producing cells without using the immunosup-pressive drugs. The latter principle, termed a bioartificialpancreas, is based on separating insulin-producing cells fromthe immune system by using a semipermeable membrane(Fig. 1). The semipermeable membrane has specific func-tions that allow free passage of nutrients, oxygen, glucose,and insulin, and prevent exposing the cells to the immune sys-tem, which would destroy the cells after recognizing them asalien. At this point, macromolecular chemistry has a criticalrole since, apart from most recently appearing membranesbased on microfabricated technologies[22] and metallic[23]
and sol–gel[24,25] materials, polymers are the most prevail-ing materials developed for immunoprotection of islets ofLangerhans. Polymers are suitable because they are a group
of materials that will not provoke an immune response, andbecause the properties of polymers are highly diverse and canbe adjusted as desired. Thus, the design of a long-term func-tional semipermeable membrane represents another exampleof the application of polymer chemistry in the biomedicalfield.[26]
Semipermeable Membranes for Immunoprotectionof Islets of Langerhans
The concept of microencapsulation in polymeric semiper-meable membranes was established by Chang in 1964.[27]
Artificial cells were created by emulsification of collodion ornylon, and enzymes were contained within the polymers. Thefirst attempts to create an immunoprotective barrier for trans-planted islets of Langerhans to treat diabetes in the 1970swere reported by Lim and Sun,[28] who designed a micro-capsule based on polyelectrolytic complexation betweenanionically charged polysaccharide sodium alginate and syn-thetic polycations poly(l-lysine) and poly(ethylene imine).The encapsulated rat islets survived in vitro for thirteen weeksand in vivo for two weeks, and demonstrated the feasibil-ity of controlling blood glucose levels by insulin releasedfrom transplanted viable islets encapsulated in a poly-meric semipermeable membrane. To the author’s knowledge,a detailed justification for using sodium alginate and poly(l-lysine) as the main components for the capsules was notprovided. However, in retrospect, this work[28] was ingeniousand influential, since the combination of alginate with poly(l-lysine) provides the basis for work of several cell encapsula-tion groups even today. As a collective criticism to the field,although this initial publication[28] was so promising, muchof the basic work which was required was never carried out,and many groups began testing this encapsulation system andalternate ones. Unfortunately, there has been a general lackof standardization, which will be addressed below.
History and Presence of Encapsulationof Islets of Langerhans
Research since Lim and Sun’s[28] work has covered all pos-sible aspects of cell encapsulation for diabetes treatment.In spite of developments during these years, a suitablematerial for clinical application has not been identified, andthe field is lagging behind the optimistic promises of the
510 I. Lacík
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Fig. 2. Progress in cell encapsulation for diabetes treatment.
late 1990s.[29,30] The field may not have developed becausethe questions asked in pioneering papers (types of materialand device, properties, transplantation site, and islet source)have not been answered, so progress has not advanced to theextent expected. However, the progress achieved so far rep-resents the natural development of this multidisciplinary andextremely difficult field, which combines materials engineer-ing with endocrinology, immunology, cell physiology, andother medical disciplines.
In the author’s opinion, the historical development of thecell encapsulation field in diabetes treatment can be describedby a typical logistic growth[31] as shown schematically inFig. 2. After a relatively quiet period following Lim andSun[28] (Milestone 1), the beginning of the 1990s was char-acterized by intense development that resulted in severalencapsulation devices and transplantation studies using var-ious large animal models[32] (Milestone 2). These studiesresulted in the first clinical trial by Soon-Shiong et al.,[33]
who claimed diabetes treatment by human islets encapsulatedin alginate–poly(l-lysine) microcapsules (Milestone 3). Thiswork proved the principle and, consequently, raised optimismfor a clinically approved technology to be ready for treat-ing diabetes. Several groups and companies were establishedduring this period, producing data with different materials.The main feature of those studies was poor inter- and intra-laboratory consistency, and the encapsulation communityrealized the complexity, difficulties, and number of steps thatneeded to be documented before claiming success with a spe-cific encapsulation device. This period can be visualized asreaching a saturation point (Fig. 2) towards 2000. Interest wasfor a short time even more depressed after the announcementof the Edmonton protocol (Milestone 4),[13] which was ini-tially deemed sufficient for islet transplantation even withoutthe need for a semipermeable membrane.
However, it was soon realized that the Edmonton pro-tocol was not the best solution, especially for children,because of the permanent need for immunosuppression.
Thus, islet immunoprotection by encapsulation remains themost promising solution at least for forthcoming years beforeother strategies, for example stem cells, develop to the clin-ical stage. A fifth milestone may be the demonstration ofintercontinental logistics where tissue donors, encapsulation,and recipients can be separated by several time zones whilemaintaining the viability of islets.
The most recent advances would place the bioartificialpancreas in the promising and rapidly advancing categories.Past experience, and the proven quality of islets from theEdmonton protocol in different centers, may mean the cur-rent era is called the period of ‘focussed strategies’ witha high potential to bring islet encapsulation to pre-clinicaland clinical stages. This possibility is also demonstrated byreviews published in the past four years covering aspects ofislet encapsulation. Nastruzzi et al.[34] described the gen-eral principles of a bioartificial pancreas, with an overviewand critical discussion of the main approaches and materi-als used in the late 1990s. The alginate-based microcapsulesare considered to have the highest potential as a mate-rial for islet encapsulation, and there are several reviewsof alginate-based microcapsules and alginate beads used incell immobilization.[35–39] We described the situation withthe most commonly used polymer systems for encapsulationbased on polyelectrolyte complexation, and provided recom-mendations for capsule formation.[40] Further information onpolyelectrolytes used for capsule formation with a focus onmechanical properties, cell adhesion, and oxygen supply aregiven in Bhatia et al.[41] Hunkeler et al.[42] gave a view to thethird millennium for bioartificial organ grafts, and envisionedjust a few years were needed for implementation of bioarti-ficial organs based on allografts without the need to pass thexenograft stage. Hunkeler et al.[43] attempted to justify themetrics for assessing the properties of bioartificial organswith the aim to have objective tools for inter-laboratorycomparison of data obtained with different materials and pro-tocols, and improve consistency in islet encapsulation data.The call for consistency has become a main task for newactivities of the Bioencapsulation Research Group[44] withinthe EU-COST project[45] launched at the beginning of 2006.
Scope of this Review
In a single review it would be difficult to refer to all significantliterature. Nevertheless, the author feels from own and peers’experience that some issues in islet encapsulation are stillinsufficiently covered and might not be publicly addressed.Therefore, it may be difficult to realize which directions mightbe the optimum in designing materials for islet encapsulation.Hopefully some information will be found useful, at least toopen discussion on some highlighted and likely controversialpoints in the text.
The main purpose for this review is to provide guidelinesfrom the point of view of polymer chemistry on (a) the cur-rent status of encapsulation devices, (b) how to visualize theentire protocol for encapsulation of islets of Langerhans fordiabetes treatment with a focus on microcapsule formationby ionic interactions, (c) the main unsolved problems in thisfield, (d) how to avoid, or minimize, previous mistakes, and
Fig. 3. Principal types of encapsulation devices used for immuno-protection of islets of Langerhans. Arrows point to the semipermeablemembrane.
(e) to attract polymer chemists to enter this area to help resolveparticular issues, and to apply this technology to the treatmentof diabetes in human patients.
Categorization of Encapsulation Devices
Immunoprotective polymeric membranes will be categorizedwith a focus on those which remain either in development,or pre-clinical testing. Primary encapsulation devices, shownin Fig. 3, include intravascular membranes, macrocapsules,and microcapsules. Each device has its long history and goodreasons for testing and, for some, for discarding, as summa-rized in several reviews from more than ten years ago[46,47]
to the present.[34,48]
Intravascular Chambers
Until the early 1990s, the intravascular chambers connectedto main veins represented a promising way of islet immuno-protection behind the semipermeable membrane.[49–51] Theadvantage of this type of bioartificial pancreas was the closeproximity of islets to the blood stream, separated by thesemipermeable membrane, which is the ideal condition forislet survival and insulin release to the blood stream whenblood glucose levels increase. Alternatively, risks connectedmainly with surgical complications and thrombosis resultedin a lack of interest for using intravascular chambers, andthere is no new literature data on this islet encapsulationdevice.
Macrodevices
The therapeutic load of islets placed inside macrodevicesrepresents another possibility. Since they are extravascular,the surgical risk is minimized compared to the intravascularchambers, and the design towards their surface vasculariza-tion should provide sufficient contact between islets and theblood stream. The macrodevices are of different geometriesand a literature survey indicates that macrocapsules (or diffu-sion chambers) and hollow fibers are no longer used, mainlybecause of their failure caused by islet necrosis (diffusiondistance for oxygen and nutrients from ranges from severalmillimetres to centimetres), insufficient biocompatibility, andmechanical instability. Therefore, macrodevices have mostly
been used in the form of tailor-designed planar membranes ofdifferent geometries with a thickness of a few millimetres atmost. Planar membranes are polymer-based multilayer sheetswhere each layer has specific functions for permeability,mechanical support, biocompatibility, and vascularization.Typical products of this type are Encellin XP[52–54] andTheraCyte.[55] Encellin XP may be useful for xenotransplan-tation of islets, as announced in the late 1990s.[54] However,there seems to be no recent research in this area. Thera-Cyte has been continuously used for immunoprotection ofencapsulation islets of Langerhans, most recently in xeno-transplantation of porcine islets to non-obese diabetic (NOD)mice and monkeys.[21] Immunoprotection, vascularization,and biocompatibility are claimed for these planar devices. Inaddition, the possibility of replenishment with fresh islets,and retrieval, are medically sound advantages. However, itis unknown why there are delays in clinical tests in isletencapsulation for these planar membranes.
Microcapsules
The encapsulation of either individual or a few islets indevices called microcapsules has become the most com-mon way for immunoprotection of transplanted cells. Themain drawbacks foreseen in the past have been identifiedand the field has moved forward substantially. Table 1 com-pares the advantages and disadvantages based on viewsprovided in the very early period of islet encapsulation,[49]
and recently.[29,40,48] Table 1 documents that the field ofislet microcapsule immunoprotection has advanced based onproven advantageous features, and identifies tasks for thefuture. It is likely that the main reason for the high popularityof microencapsulation is the relative simplicity of encap-sulation principles, which initially does not require a deepexpertise in related fields of polymer chemistry, membranes,hydrogels, and networks. However, oversimplification wasresponsible for the irreproducible results and informationnoise during the 1990s, and this situation has to be avoidedin current developments in islet encapsulation.
Various types of materials have been investigated becauseof the positive features of immunoprotection of islets ofLangerhans in microcapsules (Table 1), and recent reviewscontain detailed descriptions.[34,40,48] Here, we only brieflycomment that microcapsules made by electrostatic inter-actions, including polyelectrolyte complexation (interactionbetween two polyelectrolytes) and ionotropic gelation (inter-action between polyelectrolyte and gelling ion), dominate thefield of islet encapsulation. Other types of materials havealso shown their potential, for example (a) agarose beads pre-pared by temperature-induced gelling,[56] (b) hydrogels madeof copolymers of hydroxyethyl methacrylate with methylmethacrylate (HEMA–MAA) prepared by solvent extractionand precipitation,[57] and (c) poly(ethylene glycol) (PEG),used predominantly for conformal coating of islets.[58–60]
No recent literature was found on novel developments inHEMA–MAA microcapsules for islet encapsulation. Thereis scarce information on using agarose-based beads[61] andresearch on using the PEG coating of islets is ongoing.[62,63]
512 I. Lacík
Table 1. Advantages and disadvantages of encapsulation of islet of Langerhans in microcapsules
Initial period of islet encapsulation (1980s) Current period (2000–2005)
Positive features To be solved Positive features To be solved
Easy transplantation to multiple sites Stability in vivo Numerous materials Standardization (materials, protocols)No vascular interface Fibrotic response Mass transport ReproducibilityRapid diffusion Delay to blood stream Resistance to mechanical stress Ultimate materialProtects against rejection Gentle process Islet source
Transplantation to various sites Site of transplantationVarious techniques for encapsulation Up-scaling
RetrievabilityRegulatory issues
The latter capsules appear promising because of the biocom-patibility of PEG.[64] However, it is not proven that theirperformance is superior compared to microcapsules withsemipermeable membranes formed by electrostatic interac-tions. Therefore, microencapsulation and immunoprotectionof islets of Langerhans in microcapsules made by poly-electrolyte complexation and/or ionotropic gelation seemsto represent the most promising encapsulation principle.However, it is possible that a type of device other than micro-capsules, which has not been described in the literature, mayemerge as the final solution for the clinical transplantation ofencapsulated islets.
Prior to providing more details on the semipermeablemembrane in the form of microcapsules, the general prop-erties of semipermeable membranes will be discussed in thenext section.
General Properties of Semipermeable Membranes
A semipermeable membrane has to provide long-term protec-tion to encapsulated islets able to produce insulin accordingto the blood glucose concentration. For this, the design ofa semipermeable membrane has to account for propertiessummarized in Table 2. The properties of a semipermeablemembrane are generally categorized into biological, physical,chemical, and process-related properties. Most properties areknown and are given in recent reviews.[29,34,35,40] Therefore,the discussion of Table 2 aims to provide a different perspec-tive to these reviews based on most up-to-date developmentin the field and the author’s experience.
Optimum Window
All properties listed in Table 2 must be within their optimumwindow in order to create the optimum semipermeablemembrane for encapsulation of islets of Langerhans in dia-betes treatment. However, the optimum properties have notbeen quantified as metrics because there does not exist a func-tioning benchmark semipermeable membrane.Therefore, thetask for the near future is to develop the benchmark materialwith defined properties and flexibility to adjust them in orderto gain the necessary information on the optimum windowfor immunoprotection of islets of Langerhans. This strategyof defining the relevant ranges and targets is a similar goalto creation of metrics for microencapsulation as proposed byHunkeler et al.[43] However, the suggested steps for estab-lishing the optimum window properties represent a simpler,
bottom-up approach to learn about suitable properties fromsuccessful transplantation cases.
Biological Properties
Biocompatibility
One of the most intriguing properties in cell encapsula-tion for diabetes treatment is biocompatibility. The goal is toselect a material for the semipermeable membrane that doesnot provoke a strong immune system reaction, resulting information of a thick layer of fibrous tissue overgrowing themembrane surface. This would catastrophically reduce thediffusion of nutrients and oxygen, causing islet necrosis andtransplant failure. On the other hand, the formation of vascu-lar tissue around the semipermeable membrane is a positivefeature because of close contact between islets and blood,higher availability of oxygen and nutrients, and informa-tion on blood glucose concentration. Castner and Ratner[65]
described that the design of the surfaces of medical devicesfor biocompatibility is still not well understood. Implanta-tion of any material to the body evokes an immune systemresponse. The organism reacts similarly to all materials byforming a collagenous layer, including those that are alreadyused for several purposes with reasonable success and arecalled biocompatible.[65] Therefore, the control of the prop-erties of an implant surface is one of the most important issuesin the design of biomedical devices. The concept of biocom-patible material does not follow the idea of the inert material;instead biocompatibility of a biomaterial is understood asthe appropriate reaction generated in the host.[66] This prin-ciple should be followed in designing various types ofsemipermeable membranes for immune-protection of isletsof Langerhans. For example, minimum overgrowth is desiredfor microcapsules transplanted to the peritoneal cavity (‘free-floating’ application) and vascularization with controlledovergrowth of the vascular tissue is the goal for subcutaneoustransplantation of planar semipermeable membranes.[53,67,68]
Non-Cytotoxicity
Non-cytotoxicity to the cells and the host are the primaryprerequisites for materials used for membrane formation. Assoon as islets of Langerhans are in contact with either themembrane, or the membrane components before the mem-brane forms, the conditions must ensure that this contact doesnot have a detrimental effect on the islets. A feasible exper-iment in this respect is to evaluate the cytotoxicity effect of
Polymer Chemistry in Diabetes Treatment 513
Table 2. Properties of semipermeable membrane designed forimmune-protection of transplanted islets of Langerhans
Biological Physical and chemical Process-related
Biocompatibility Permeability ReproducibilityNon-toxicity to cells Mechanical stability UniformityNon-toxicity to recipient Chemical stability CapacityNon-biodegradability Controlled size and shape Availability
membrane-forming polyelectrolytes towards the insulinomacells.[69–71] Semipermeable membranes are usually formedof previously made and characterized polymers. Therefore,islets do not experience harsh conditions of organic synthe-sis. In some cases, however, the conformal membrane may beformed in situ on the islet surface through photopolymeriza-tion of, for example, poly(ethylene glycol) diacrylates.[58–60]
In those cases, mild polymerization conditions have to beselected, and the islets’ viability is carefully checked aftermembrane formation. After transplantation, the membranecannot be toxic to the host and/or toxic membrane compo-nents cannot be released to the organism. This is why themembrane and membrane components must follow strictsafety requirements for the patient.[29]
Non-Biodegradability
The next important property of a semipermeable mem-brane is its non-biodegradability, durability, and stability overyears. Thus, properties set in vitro must remain constant, andthe membrane composition must not change by leaching ofmembrane components.
Physical and Chemical Properties
Permeability
Permeability is the premise of the semipermeable mem-brane for islet immunoprotection. Thus, permeation ofspecies of interest shown in Fig. 1 must be allowed. Muchof the late 1990s was devoted to identifying the molec-ular weight cut-off (MWCO),[72–74] defined as the lowestmolecular weight of a selected solute, most commonlypolysaccharides and/or proteins, which does not permeate themembrane.At that stage, encapsulation research was aimed atexclusion of immunoglobulins, and other potentially harm-ful components of the humoral part of the immune systemwith effective size hypothesized as 5–15 nm.[75] This valuewas confirmed by using microfabrication technology to cre-ate precisely defined uniform pores. A pore size of 18 nmis recommended to prevent passage of immunoglobulins.[22]
Nevertheless, this pore size does not exclude the proteinsreleased by encapsulated cells, which may stimulate animmune response and lead to membrane overgrowth byfibroses.[34] Decreasing the pore size to exclude these pro-teins would lead to impermeation for nutrients, and it is, atfirst glance, not an appropriate way to trigger the optimummembrane pore size on the nanometre scale. Fortunately, andas a part of the learning curve (Fig. 2), numerous exper-imental results using semipermeable membranes with theMWCO values from a few nanometres[73] to a few tens of
nanometres[76] have shown that the semipermeable mem-branes provided sufficient long-term immunoprotection assoon as the immune cells were excluded. Immunoglob-ulins, and even complement proteins, may permeate themembrane.[61]
Thus far, this information rests on the MWCO valuesfrom in vitro permeability measurements. The operatingMWCO under in vivo conditions can be significantly mod-ified, mainly if the surface of membrane is overgrownby collagen layers and/or vascularized, which could bethe case with either TheraCyte planar membranes[21] oragarose microcapsules.[61] Our recent data may confirm thishypothesis.[77] Microcapsules made of sodium alginate, cel-lulose sulfate, and poly(methylene-co-guanidine) explantedfrom the peritoneal cavity of Wistar rats after five monthsshowed that MWCO decreased from 40 to ∼10 kDa, deter-mined by dextran standards. The reason for this result couldbe a minor collagen layer a few micrometres thick detectedon the capsular surface by immunohistochemical analysis. Itis assumed that the chemical composition of the membranedid not change and cause decrease in pore size.
Although the previous paragraph suggests that permeabil-ity does not need to be strictly controlled in the nanometrerange, it is required, for consistency reasons, to characterizethe semipermeable membranes from the point of view of per-meability by at least defining the MWCO value. As outlinedby Schuldt and Hunkeler,[78] there are many ways to assess thecapsule permeability to obtain MWCO values and the poresize distribution.[72] To date, there still (and unnecessarily)seems to be no consistency among groups, despite round-robin studies and common publications. For example, a citedMWCO value is 50 kDa.The value of molecular weight statedin this way is, however, misleading. It is recommended tostate the macromolecular dimension instead of the molecularweight, since, for the same molecular weight, the size of pro-teins (globular conformation) is significantly lower than thesize of polysaccharides (statistical coil).[72] A simple remedyfor this inconsistency is to state the MWCO with the soluteused to measure this value.
Mechanical and Chemical Stability
The physical and chemical properties of a semiperme-able membrane for encapsulation and immunoprotectionof islets of Langerhans include mechanical and chemicalstability. If either of these two parameters is inadequate,long-term immunoprotection cannot be obtained. Once themembrane is not chemically resistant to the environment,not only mechanical resistance but other properties (e.g. per-meability followed by insufficient immunoprotection, andbiocompatibility by exposure of non-biocompatible mem-brane components) change in an uncontrolled way, whichlikely leads to transplant failure. Thus, swelling is an openissue which needs to be solved in case of the microcapsulesmade of sodium alginate and poly(l-lysine),[36,79,80] andalginate beads,[79,80] and depends on alginate chemical struc-ture and molecular weight parameters. Excessive fragilitywas a drawback of the polyelectrolyte complex-based micro-capsule made of sodium alginate and poly(l-lysine). Various
514 I. Lacík
approaches to overcome the fragility included (a) multiplecoating,[81] (b) decreasing the size,[82] (c) applying a silicatelayer,[83,84] (d) strengthening the core of this microcapsuleby using gelling ions of stronger affinity to alginate than cal-cium (e.g. barium),[85] (e) tailoring the gelling reaction toincrease the polymer concentration at the capsular surface,[85]
and (f) applying covalent crosslinking between alginate andpoly(l-lysine) modified by UV-sensitive groups.[86]
The magnitude of mechanical stability, set under in vitroconditions, has to take into account the compression andshear stresses imposed by tissue at the locus of transplanta-tion. Our prior[87,88] experiences and other recent results[77]
with sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) microcapsules indicate that the rupture loadshould be in the range of a few to tens of grams for transplan-tation to the intraperitoneal cavity. In current experiments,[77]
the rupture load of this microcapsule before transplanta-tion, and of capsules retrieved after several months fromperitoneal cavities of Wistar rats, did not differ greatly andwas in the range of approximately 10 g per capsule formicrocapsules of 800 µm diameter. The rupture load wasdetermined by a Texture Analyzer from Stable MicrosystemsInc.These data quantify the mechanical stability under in vivoconditions, indirectly quantify the chemical stability and non-biodegradability, and prove that this microcapsule resists thein vivo conditions.
Size and Shape
In principle, each encapsulation device shown in Fig. 3 ischaracterized by its size and shape and, vice versa, size andshape are determined by encapsulation protocol and materialused. The control over size and shape increases the flexibil-ity for the given material in adjusting other physicochemicalproperties (permeability, mechanical stability, and membranethickness), as well as the site of transplantation. In case ofmicrocapsules, a typical example is work from Calafiore’sgroup, which investigated the islet microencapsulation inthe semipermeable membrane based on sodium alginate andpoly(l-ornithine) forming conformal coating,[89] microcap-sules of medium size (∼400 µm), and microcapsules ofconventional size (800 µm).[90]
Process-Related Properties
Reproducibility
Reproducibility and consistency represent serious prob-lems in islet encapsulation field. Sources for the lack of inter-and intra-laboratory reproducibility were addressed in therecent review by Orive et al.[29] The main reasons are seen invery poorly standardized materials, and because informationon islets and encapsulation protocols is in-house and/or pro-prietary information. Consequently, it is difficult to reproducethe results as they depend on several steps from preparationof solutions, islet isolation and handling, and encapsulationprocess up to conditions of culturing and transplantation. Theproblem of reproducibility among different groups has to betackled and this is one of the tasks in the newly funded activ-ities within the EU-COST Action 865.[45] Another issue is
the repeatability in formation of a semipermeable membraneby one group in order to convincingly account only for thechanges in biological aspects (i.e. islets and recipient), andnot from material and encapsulation process aspects.
Uniformity
This term is particularly valid for microcapsules. Allmicrocapsules formed must have identical properties andthe same preparation history. The clinical applications willrequire transplantation of approximately 105–106 microcap-sules, which all must have uniform properties within theoptimum window. In case the capsules are non-uniform, then,in the worst case, a substantial fraction may appear as fail-ing and likely the entire transplant will be considered asa failure, potentially leading to discarding such a system.Hypothetically, non-uniformity may be one reason for eitherthe overgrowth or retrieval of only a fraction of microcap-sules often reported by several groups. An important step tothe continuous formation of uniform microcapsules was thedevelopment of the multi-loop reactor with a simple designby Anilkumar et al.[91] This reactor is highly suitable for pro-duction of microcapsules formed in a relatively short reactiontime in the range from a few tens of seconds to a few minutes.A more complicated apparatus for continuous production ofpolyelectrolyte microcapsules was described by Ceausogluand Hunkeler,[92] with the same aim to improve the controlover the capsule formation process.
Capacity
In order to eventually develop the technology to producesufficient numbers of encapsulation devices, capacity needsto be considered to move from the laboratory to clinics.This does not seem to be a problem in the case of pla-nar membranes[52,55] when they prove their readiness forclinical trials. For microcapsules, the continuous produc-tion of capsules under sterile conditions, using multi-nozzlesystems, has been developed in various laboratories,[93–95]
as well as commercially[96] claiming capacity sufficient fortransplantation to humans.
Availability
This property is important because once a device is devel-oped it should be available to all diabetic patients who electto use it under medical recommendation.
Microcapsules by Electrostatic Interactions
Microcapsules made by exploiting electrostatic interactionscurrently represent the most promising way to form semiper-meable membranes for encapsulation and transplantation ofislets of Langerhans. Such microcapsules are formed eitherby polyelectrolyte complexation between two oppositelycharged polyelectrolytes or by interaction of polyelectrolytewith a suitable, usually multivalent, ion with the abilityto gel a given polyelectrolyte, as schematically shown inFig. 4. It is not problematic to form a polymer networkbased on ionic interactions, since oppositely charged poly-electrolytes as well as polyelectrolyte and gelling ions will
Polymer Chemistry in Diabetes Treatment 515
� �
Polyanion
Polycation
POLYMER NETWORK
Polyelectrolyte
Gelling ion
Polyelectrolyte complexation Ionotropic gelation
(a) (b)
Fig. 4. Schematic representation of microcapsule formation byelectrostatic interactions, by either polyelectrolyte complexation orionotropic gelation. Interactions and conditions are selected so the poly-mer network is formed as a semipermeable membrane as a microcapsulewith distinct membrane made of (a) sodium alginate, cellulose sul-fate, and poly(methylene-co-guanidine), or (b) gelled calcium alginatebead.[77] Scale bars: 0.5 mm.
always interact. A more difficult task is to intelligently directthese interactions to form microcapsules, which possess theproperties summarized earlier. Since the electrostatic interac-tions are of physical origin,[97] it may not be possible to usemicrocapsules under in vivo conditions, where the electro-static interactions can be screened (and complex dissolved)by the action of ions and/or proteins. However, the com-plex is formed by the concerted action of electrostatic andother interactions (hydrogen bonded, hydrophobic, and polar)and chain entanglement supporting the primary electrostaticinteractions and network stability under vivo conditions. Thiseffect is documented by numerous examples of microcap-sules made by means of electrostatic interactions,[40] andstudies into stability of polyelectrolyte complexes on varyingionic strength.[98]
For educational purposes, and with the intent to avoidpropagation of errors, the author noticed publications incor-rectly stated that the polyelectrolyte complexation is the‘polymerization reaction’, or that in case of polyelectrolytecomplexation the ‘covalent bond’ is formed. Polymer chem-istry principles should be used in the design of polymericsemipermeable membranes to avoid mistakes in terminologyand in the understanding of the role of polymer chemistry.
Islet microencapsulation by means of electrostatic inter-actions is popular because it is (a) fast and simple, (b) carriedout under physiological conditions, and therefore, is safefor encapsulated islets, (c) robust and can be used with avariety of polyelectrolytes, which together with selecting theprocess conditions, makes it possible to optimize the capsuleproperties, and (d) reproducible and scalable. Even thoughmicroencapsulation is generally denoted as a simple process,the simplicity has to be connected with control and under-standing of all steps leading to successful product capableof long-term immunoprotection of encapsulated islets and
diabetes reversal. In the next sections, the logistics of encap-sulation by electrostatic interactions is described in detail,indicating potential reasons for inconsistency and highlightspitfalls of this encapsulation process.
Encapsulation Protocol
The encapsulation protocol consists of several steps com-monly involving a large group of people from different fieldsof expertise. It is of utmost importance to define, and closelyfollow, each step in the encapsulation protocol in order toreach consistent and optimized results. Recommendationsfor capsule formation by polyelectrolyte complexation areprovided elsewhere.[40]
Fig. 5 shows the three-phase encapsulation protocolof (a) pre-encapsulation, (b) encapsulation, and (c) post-encapsulation. Each will be discussed separately.
Pre-Encapsulation Phase
Overall Goals. This phase is viewed as the mostchallenging one, as the decision on final design made withrespect to (a) site of transplantation, (b) materials to be used,and (c) process of encapsulation. The usual experience is thatmicrocapsules are made from selected polyelectrolytes byan optimized process of encapsulation, then they are testedin various animal models. Depending on recipient (large orsmall animal) and site of transplantation (for example, peri-toneal cavity or liver), the process has to account for therequired number of capsules and their size, respectively. Thisin turn relates to the properties of polyelectrolytes, whichby their characteristics determine the process of encapsula-tion and the range of achievable properties of microcapsules.Therefore, optimization of the entire encapsulation processfollows the link of material ↔ process ↔ capsule properties,which closely depend on each other. Any modification tothis link (polymer properties, process conditions, or targetedmicrocapsule properties such as size) may reset the wholedesign process to the beginning, which justifies attention tothe pre-encapsulation phase.
Selection of Polyelectrolytes. This is the most crucialstep. In the past, intensive polymer screening was carriedout. The suitability was sometimes related to simple testsof microcapsule and complex formation by using mostlycommercial polyelectrolytes of natural and synthetic ori-gin, without rigorously taking into account the effects ofmolecular weight, charge type, pH, and ionic strength.[69]
Experiments were generally based on the trial-and-errorapproach. Most of the materials used today have beenidentified in recent papers.[30,34,40,48] They include polysac-charides, predominantly sodium alginate, sodium cellulosesulfate, carboxymethylcellulose, and chitosan, and syntheticpolymers poly(l-lysine), poly(l-ornithine), poly(methylene-co-guanidine), polyacrylic acid, and poly(dimethyl diallylammonium chloride).
In order to ensure repeatability and standardization ofencapsulation protocols, polymers have to be characterizedas completely as possible taking into account that poly-electrolytes are polymers. Thus, polyelectrolytes used for
516 I. Lacík
3. Post-encapsulation phase
1. Pre-encapsulation phase
2. Encapsulation phase
IsletsSolution of
polyelectrolyte 1
Dispersion of islets in solution of polyelectrolyte 1
Droplet formation
Solution ofpolyelectrolyte 2 or gelling ionMembrane formation
Fig. 5. Encapsulation protocol for islet encapsulation in microcap-sules based on electrostatic interactions.
capsule formation should be characterized in terms of molec-ular weight, molecular weight distribution, and chemicalmicrostructure.Additional important issues include standard-ization and purity.[29] The former is connected with long-term reproducibility in encapsulation and transplantationdata, where the latter has been identified as the success-determining property in case of polysaccharides relatedto microcapsule biocompatibility. Much attention has beenlately devoted to sodium alginate, which historically is themain polysaccharide used for islet encapsulation, and whichpurification from endotoxins, proteins, polyphenols, andother impurities is well-established for laboratory[35,38,99–101]
and commercially available samples.[102,103]
Encapsulation Phase
Preparation of Solutions. Solutions of polyelectrolyte 1(Fig. 5) are almost solely made of polysaccharides. It seemsthat even the preparation of its solution requires a stan-dardized protocol. Some groups, in order to compare var-ious sodium alginates, adjust the polymer concentrationto constant viscosity in the islet encapsulation studies[104]
as well as in advanced studies into surface morphologyand mechanical properties of beads and capsules.[105] Thisapproach is sensible in terms of comparable conditions fordroplet production, but the network density, stability, perme-ability, and other properties corresponding to the network
density given by actual polymer concentration, may providean inadequate comparison. Other groups use specific condi-tions for the preparation of solutions such as storing of sterilealginate solution for three months at 4◦C, although withoutexplaining the reason behind this procedure.[106]
Islet Dispersion. After gentle mixing of polyelectrolyte 1solution with islets, a homogenous dispersion is achieved.The islet density, i.e. number of islets per milliliter of solution,should reflect the theoretical load of islets in capsules, usu-ally counting about one to four islets per microcapsule, basedon the total number of islets and targeted capsule size. Beforemaking the islet dispersion, islets have to be thoroughlywashed from serum proteins in buffer solution, because pro-teins interact with polyelectrolytes and may interfere with thecomplexation reaction.
Droplet Formation. Droplets of islet dispersed in poly-electrolyte 1 solution are usually formed by using nozzles.Nozzles operate on a variety of principles such as vibrat-ing nozzle, laminar jet break-up by cutting, coaxial gas flowextrusion (air-stripping), and electrostatic droplet generation.Changing the polymer molar mass can sufficiently alter theviscosity that a different nozzle must be employed.The vibrat-ing nozzle[96,107] and break-up of laminar jet by cutting[108]
are commercially available instruments, while air strippingand the electrostatic droplet generator[109] are in-house appa-ratuses. Nozzle selection is based on the required dropletsize, capacity, and viscosity of polyelectrolyte solution. Thesolution viscosity determines flow rate and droplet size, andeach nozzle possesses an optimum operational range.[110,111]
Since the viscosity of polyelectrolyte solution is directlyrelated to the molecular weight of polyelectrolyte and solu-tion composition (polyelectrolyte concentration and ionicstrength), which affect the properties and performance of amicrocapsule,[40] selection of an optimum nozzle is a veryimportant part of the encapsulation protocol. Scale-up ofdroplet production is the next step in the nozzle optimizationprocess.[93–96] Sometimes authors give information on dis-tance between nozzle and receiving (gelling) solution wherethe membrane is formed. This corresponds with optimiz-ing the capsule shape, which may be distorted because thisdistance may be insufficient to allow the spherical shapeto form.
Membrane Formation. Several points must be discussedregarding the step of membrane formation in Fig. 5, as it iscentral to the entire encapsulation process.
Polyelectrolytes. The recommended properties of poly-electrolytes used for microcapsule formation are stated inTable 3. Since islets are exposed to polyelectrolyte 1 solu-tion for the time from making the dispersion until they areextruded as droplets to the receiving solution (up to tens ofminutes), the solution of polyelectrolyte 1 should be harmlessto islets. Based on cytotoxicity studies,[69–71] polyanions aregenerally much less harmful than polycations; therefore, onlypolyanions are used as a polyelectrolyte 1. The molecularmass of polyanions should be in the range of a few hundredthousands of Daltons to ensure that droplet shape recoversafter leaving the nozzle as well as after penetrating into thereceiving solution at concentrations to a few weight percent,
Polymer Chemistry in Diabetes Treatment 517
Table 3. Recommended properties of polyelectrolytes 1 and 2 usedfor capsule formationA
Polyelectrolyte 1 Polyelectrolyte 2
Polyanion (or combination) Polycation (often oligocation)Polysaccharide Synthetic polymerMolecular weight >105 Da Molecular weight 103–104 DaIonotropic gelation FlexibleBalanced weak and strong charges High charge density
ASee Fig. 5.
as described in our previous work.[112] Therefore, the molarmass of the cell-contacting polymer is affected by rheologi-cal parameters and the need for viscoelasticity. The sphericalshape of the final microcapsule is affected by the rheologyof the polyelectrolyte 1 solution and its interaction with thereceiving solution. This shape is preferable for reasons ofbiocompatibility, mechanical resistance, suitable diffusioncharacteristics (high surface to volume ratio), and becauseit informs on the control over the capsule formation process.The shape is advantageous when the polyanion is capableof ionotropic gelation with a gelling cation (component ofreceiving solution), which stabilizes the spherical shape ofdroplet in the receiving bath, and hence forms a supportfor membrane formation between polyanion and polycation.Thus, polyanions are often selected from the ionotropicallygelled polysaccharides. In the cell encapsulation field, gellingof sodium alginate by either Ca2+ or Ba2+ represents a typicalexample.[37]
Alternatively, polyelectrolyte 2 is a polycation usually ofsynthetic origin, low molecular weight (oligocation), highcharge density, and flexibility to effectively interact with theanionic charge of the droplet to form a membrane. Althoughpolycations are generally more toxic than polyanions, it isnot expected that they are harmful to the islets, which areexposed to polycations usually only for a very short time (tensof seconds to a few minutes), and the membrane formed of thepolyelectrolyte complex is generally not toxic to islets.[69,70]
Interactions between polyelectrolytes must be balancedbetween consideration of the hydrogel character of the micro-capsule (low concentration of a polymer swollen in buffersolution), and avoidance of formation of a coacervate (com-plex which is phase-separated from aqueous solution bystrong interactions) where control over the membrane prop-erties is lost. The balance of interactions may be improved bymixing polyelectrolytes of the same charge.An example is thecombination sodium alginate with cellulose sulfate polyan-ions, which is used to form a membrane with poly(methylene-co-guanidine).[87] Cellulose sulfate that strongly interactswith poly(methylene-co-guanidine) is diluted with weakerinteracting sodium alginate, which is capable of gellingwith Ca2+ present in the poly(methylene-co-guanidine)solution.
Reasons against forming a capsular membrane ofdesired properties in the membrane formation stepinclude:[87,112]
• Low viscosity of polyanion solution and/or fast gelling,which result in a microcapsule of deformed shape.
• Too intense interactions, resulting in shrinking and loss ofthe hydrogel character of the membrane.
• Too weak interactions (caused by low concentration ofactive sites or high ionic strength), resulting in osmoticpressure-related defects.
• High density of receiving solution, which obstructs dropletpenetration and results in membrane defects.
• Weak or unstable interactions result in released polymerchains from individual capsules, and these polymers tangleto cause capsule stickiness.
Therefore, when using a combination of polymers andencapsulation process, the optimization process needs to testfactors such as molecular weight and molecular weight dis-tribution, concentration, viscosity, and reaction time. Thereis enormous flexibility in adjusting the microcapsule prop-erties with the possibility to decouple some properties, e.g.mechanical stability and permeability,[73,113] and to optimizethe process of encapsulation to improve smoothness.[112]
Combination of Polyelectrolyte Complexation withIonotropic Gelation. The presence of gelling ions in thereceiving solution for ionotropic gelation of polyaniondroplets is an advantage. As shown in Fig. 6, membrane for-mation in the presence of the cation capable of the ionotropicgelation of polyanion droplets can proceed by either (a) atwo-step process, where in the first step gelled beads areformed then are coated in the second step by polycation form-ing the membrane, or (b) membrane formation through aone-step process upon mutual action of gelling cation andpolycation both present in receiving solution. The two-stepprocess is typically represented by the encapsulation methodpioneered by Lim and Sun[28] for alginate–poly(l-lysine)microcapsules, and is used by most groups working in isletencapsulation. A typical microcapsule prepared by the one-step process is made of sodium alginate, cellulose sulfate,and poly(methylene-co-guanidine).[87]
A potential pitfall of the two-step process is noted in theintermediate step of washing the beads before coating withthe polycation solution (Fig. 6). It has been shown by confo-cal laser scanning microscopy[114] that the gel of alginatewith calcium, even in the presence of a small amount ofbarium (forming a more stable gel with alginate than cal-cium), is not highly stable in buffer solution that exchangesgelling cations with non-gelling sodium cations. If beads arewashed and stored in buffer solution for different times, sig-nificant polydispersity in capsule properties and impaireduniformity can be obtained with possibly questionable trans-plantation results (see also ‘process-related properties’ aboveand discussion on capsule uniformity). Expansion of calciumalginate beads in saline solution and their homogenizationbefore deposition of poly(l-lysine) was proposed many yearsago.[115] Therefore, it is recommended that wherever possi-ble, either the membrane should be formed by the one-stepprocess, or the intermediate steps before the membrane isfinalized are controlled and documented for consistency andreproducibility.
Capsules made of sodium alginate, cellulose sulfate, andpoly(methylene-co-guanidine) can also be prepared by the
518 I. Lacík
Solution ofgelling cation
STEP 1
STEP 2
TWO-STEP PROCESS
Polyanion droplets
Beads of polyanion inionotropically gelled
Beads of polyanionwashed in buffer
Formation of membranein polycation solution
Buffer solution
Polycationsolution
STEP 1
Polyanion droplets
Formation of membrane
Solution of gellingcation and polycation
ONE-STEP PROCESS
Fig. 6. Comparison between two-step and one-step processes for microcapsule formation by ionic interactions involving ionotropic gelation andpolyelectrolyte complexation.
two-step process: The gelled beads from the mixed solu-tion of sodium alginate and cellulose sulfate are preparedin the first step, which are exposed to poly(methylene-co-guanidine) in the second step.[113,116–119] In spite of the samemembrane components, these two-step microcapsules exhibitdifferent properties in terms of chemical and mechanical sta-bility, permeability, and membrane composition compared tothe one-step microcapsules, including also observed instabil-ity during washing caused by leaching of cellulose sulfate notcomplexed by calcium cations during gelling.[77] In the one-step process, cellulose sulfate is fixed by interactions withpoly(methylene-co-guanidine), which is the simultaneousprocess to gelling of sodium alginate by calcium.[87] Thus, thesame microcapsules prepared by one- or two-step processesare different because of different gelling sequences and mech-anisms of membrane formation. This result is an example ofhow capsule properties can be adjusted by modification ofthe encapsulation process.
Dilemma of Collection and Reaction Times.[40] Fig. 6shows that for one- and two-step processes, the residencetime of the polyanion droplets in the collection bath variesbetween collecting the first and the last droplet. The beads (orcapsules) formed in the receiving bath cannot be processed(e.g. washed) immediately because their reaction time untilthis point spans from zero (last droplet) to total (first droplet),giving a total collection time. Therefore, it is a common prac-tice to continue the gelling reaction for an additional time,denoted as reaction time. For example,[120] alginate dropletscollected for 90 s and gelled for additional 90 s result in a
large distribution in reaction times (from 90 to 180 s). Unlessthe collection time is much shorter than the reaction time, thegel characteristics of the beads (capsules) within one batchare heterogeneous because the collection time contributesto the reaction time differently for each individual capsule.Thus, non-uniform microcapsules are formed in terms ofpermeability, mechanical resistance, chemical stability, andcomplex composition, and, hence, also in terms of biocom-patibility and overall performance after transplantation if theproperties are out of the optimum window discussed above.
Thus, it is important to understand how to ensure that col-lection time does not interfere with reaction time. Althoughthis discontinuous process involving collection and reactiontimes is almost only used for preparation of microcapsulesby electrostatic interactions, in the literature the informationon collection time is generally not provided (in the range ofa few percent). Further, information on reaction time is rel-atively scarce (in the range of a few tens of percent). Thisdiscrepancy can represent an important source for inconsis-tency of data among groups and frustrates the standardizationof process and product.
There are two ways to tackle this problem: (a) for the two-step process in Fig. 6, the ionotropic gelation is carried outto saturated gel density, which leads to uniform beads coatedafterwards by a polycation (this assumes no harmful effect offully gelled beads on islet viability), and (b) for the one-stepprocess in Fig. 6, collection time is considerably shorter thanthe reaction time. The latter possibility can be laborious formicrocapsules prepared at short reaction times (in the range
Polymer Chemistry in Diabetes Treatment 519
Air
Polycation
Capsule formation
Air-stripping nozzle
Polyanion(�cells)
Droplets
Multi-loop reactor Finalcapsules
Fig. 7. Encapsulation set-up used in our laboratory[77,122,123] involv-ing multiloop reactor[91] for continuous production of microcapsulesmade of polyanions sodium alginate and cellulose sulfate, and poly-cation poly(methylene-co-guanidine).
of tens of seconds to a few minutes), and is non-feasible forencapsulation of therapeutic amounts of islets in transplan-tation studies even using small animals. For this situation,a continuous multiloop reactor was designed by Anilkumaret al.,[91] shown in Fig. 7, which eliminates the interferencebetween collection time and reaction times by removal of col-lection time. Polyanion droplets are carried by the stream ofpolycation solution. Reaction time, identical for each capsule,is controlled by the number of loops and the flow rate of poly-cation solution. The reaction is quenched after the capsulesexit the reactor in buffer solution.
The discussion on membrane formation demonstrates it isimportant to consider the encapsulation process in order tocontrol the properties of final microcapsule. In addition to theabove common approach for microcapsule formation, whichinvolves nozzles for droplet formation (Figs 6 and 7), emul-sion strategies represent another option to produce dropletsand capsules. They may answer concerns regarding nozzleselection, production scale-up, and microcapsule uniformityin terms of the reaction time. For example, a semiper-meable membrane formed as a conformal coating on thesurface of islets by an alginate–poly(l-ornithine)–alginatelayer was proposed over ten years ago[89] and advancedrecently by colloid chemists.[121] This technology has shownits potential but still needs to further prove that the processparameters are understood, the process is reproducible, andthe formed semipermeable membrane provides long-termimmunoprotection of coated islets.
Post-Encapsulation Phase
Microcapsules with encapsulated islets are typically sub-sequently treated by applying coating solutions of anionicallycharged polymers predominantly to screen residual positivecharge, to increase the mechanical and chemical stability ofthe membrane, and to decrease the membrane’s MWCO. It
was common to dissolve the ionotropic gel by using chelat-ing agents. Currently, this step has been omitted anticipatingthat the membrane stability is disturbed by applying chelatingagents and that islets may be damaged. Nevertheless, there isno clear justification given for either of these negative featuresof using chelating agents.
It may be recommendable to let the microcapsule to equili-brate after islet encapsulation before transplantation for sometime in a culture medium. The polyelectrolyte complex has tobe equilibrated by lateral aggregation,[97] which may be con-nected with leaching of individual membrane componentswith a potential to initiate immune reaction towards capsulesat the site of transplantation.
The goal of this work, i.e. microencapsulation of islets ofLangerhans, is shown in Fig. 8. These microcapsules weremade of polyanions sodium alginate and cellulose sulfate,and polycation poly(methylene-co-guanidine)[77] followingthe process shown in Fig. 7.The capsules contain human isletsisolated at the Division of Transplantation of the Universityof Illinois at Chicago (UIC), and delivered to our labora-tory for viability and transplantation studies. Details of theseexperiments are beyond the scope of this review. These ongo-ing collaboration activities are a part of the activities withinthe proposed Milestone 5 in Fig. 2 testing the intercontinen-tal logistics with high-quality the islets originating from theislet-isolation center at UIC, which successfully performs theislet transplantation to diabetic patients.[124]
Microencapsulation: Focussing on the Final Solution?
The last column in Table 1 summarizes issues ‘To be solved’.These points have been recognized based on broad experiencefrom past years, and their elucidation represents the basisfor the rising curve in Fig. 2 towards Milestone 5. Most ofthe points in Table 1 have been discussed in the precedingsections. Here, final comments are made concerning howfar microencapsulation by electrostatic interactions might befrom the ‘final solution’.
Membrane Material
The most studied microcapsule is that made of sodiumalginate and poly(l-lysine) announced 26 years ago. Thereare still some discrepancies among groups regarding theoptimum chemical composition of alginate. The main mes-sage taken from recent studies is that it remains problematic,if not impossible, to screen the positive charges of poly(l-lysine),[100,101] which provoke the immune reaction.[125]
As a recent review indicates,[35] there will be continuousinterest in the alginate–poly(l-lysine) capsule, although anunambiguous way to resolve the problem of biocompati-bility has not been proposed. Excellent papers have beenwritten on this type of microcapsule in the last five yearsby groups including de Vos, Skjåk-Bræk, and Hallé. Inspite of this, in author’s opinion, there does not seem tobe a focussed strategy on envisioning the final solutionusing this type of microcapsule. There have been manyimprovements to this capsule over the years as well assuggestions to use other capsule compositions to replace
520 I. Lacík
0.1 mm
(a) (b)
0.5 mm
Fig. 8. Optical micrographs of human islets in microcapsules made of sodium alginate, cellulose sulfate, and polycation poly(methylene-co-guanidine), (a) population of microcapsules with islets, (b) detail of encapsulated islets.
alginate–poly(l-lysine). Unfortunately, none of the lattersystems attracted rigorous comparative studies using thesame experimental techniques and islet sources, with a sim-ilar thoroughness as in case of the alginate–poly(l-lysine)capsule.
Recently, the approach to completely exclude poly-cation from capsule formation[76,126] was revived and theislets were encapsulated in alginate beads stabilized onlyby barium as a gelling cation.[127,128] This approach may bepromising, as there have been developments in the chem-istry, purification, and standardization of alginates, novelgelling mechanisms have been recognized,[129,130] and thechemical compositions of epimerized alginates[79,80] can betailored by enzymes. These activities have a high potential toresult in biocompatible and stable beads exhibiting variousmicrostructures in respect to the alginate concentration pro-file across the microcapsule,[114] including homogeneouslycross-linked alginate with simultaneous internal and exter-nal gelation.[131] The encapsulation of islets of Langerhansin alginate beads, at least for allotransplantation, would be anexciting parallel and, importantly, immunosuppression-freeactivity to the Edmonton protocol.[13,17] It appears to be alsoadvantageous in terms of simplicity of the encapsulation pro-cess. The immediate future should tell whether this materialis going to shift islet microencapsulation closer to the finalstage.
It is not expected that the entire encapsulation communitywill work on allotransplantation in alginate beads. Explo-ration of other microencapsulation systems should continue,since the properties of microcapsules involving the mem-brane made of polyelectrolyte complexation should be easierto adjust than the properties of alginate beads. However, it istempting to propose that alginate beads may define a materialwith the desired optimum window properties.
Another group of materials are synthetic analogues tohistorically used gelling polysaccharides. Current syntheticpolymer and organic chemistries are able to design vari-ous polymers of different architectures and chemical com-positions by using controlled radical polymerization. Theadvantage of synthetic polymers is in their simpler char-acterization, purification, and standardization compared tocurrently used natural polymers. So far there are only a fewexamples of synthetic polymers synthesized with the ambi-tious goal to replace alginate.[132] Other modern polymericmaterials with a potential for islet encapsulation can be basedon the amphiphilic conetworks summarized by Erdodi andKennedy.[133] However, there seems a long way until thesematerials will be able to compete with the gelling polysaccha-rides with respect to properties and availability. The startingpoint for synthetic polymer chemistry could be in designingpolycations with the properties summarized in Table 3.
Microcapsule Size
In development of microencapsulation technology, therehas been an ongoing debate on the optimum capsulesize[36,90,134,135] from conformally coated islets to ∼1 mmin diameter. The trend was to make smaller capsules in therange 300–400 µm in order to lower the transplant volume,to decrease the diffusion distance of oxygen and nutrients,and specifically[134] to allow for transplantation to the liver.A reduction of the microcapsule size may, however, changethe entire conditions for capsule formation and capsule prop-erties because of the increased surface area for electrostaticinteractions.The reduction in size may result in islets protrud-ing through the membrane[36] and shrinkage of the capsularmembrane.[135] Hence, the encapsulation protocol has to beadjusted for targeted capsule size. The current trend is to
Polymer Chemistry in Diabetes Treatment 521
produce microcapsules between 400 and 700 µm, which is agood compromise between transplant volume, islet survival,islet performance, and production capacity using variousencapsulation set-ups.
Transplantation Site
Transplantation sites for microencapsulated islets consid-ered so far are peritoneal cavity, veins of liver and spleen,kidney capsule, and subcutaneous transplantation.Transplan-tation sites are selected for a close proximity of islets to bloodto ensure effective insulin release upon changed blood glu-cose concentration and islet survival. The latter condition isconnected not only with the need for nutrients but also thesupply of oxygen, which is equally important for long-termislet viability and insulin production.[36,136] Intense attentionwas devoted to transplantation of small microcapsules to theportal vein of liver of rats[137] and pigs.[138] These resultsshow the possibility for transplantation to the liver. However,because of strong immune reaction and, mainly, consideringthe safety of transplantation in the case of microcapsules, theliver does not seem to be suitable site for the transplantationof encapsulated islets.
Our internal literature survey (not published) shows thatthere are tens of successful cases of transplantation of encap-sulated islets into the peritoneal cavity of various recipients.It is notable that these successful cases were achieved inspite of the indirect contact of microcapsules containingislets with the blood stream. This leads to the conclusionthat questions raised about the efficient insulin release, andnutrient and oxygen supply should not be so critical whenusing the peritoneal cavity as the transplantation site. In somecases, microcapsules were transplanted to either the omen-tal pouch or subcutaneously, which may be advantageoussites because of localization of microcapsules that may bevascularized.[61,139] In summary, there seems to be no impor-tant reason why the peritoneal cavity should not stay as themain transplantation site.The peritoneal cavity offers the pos-sibility for retrieval of free-floated microcapsules, and it hasa high capacity if microcapsules are embedded in the tissue,as for example in the case of agarose microcapsules.[61]
For microcapsules, a discussion may be opened concern-ing whether their complete retrieval from peritoneal cavityis a condition for ultimate regulatory approval. There are noreports on acute inflammatory reaction of microcapsules aftertheir adherence to organs and the volume capacity of the peri-toneal cavity is high.Although capsules are designed for free-floating application, some surface chemistries may promotevascularization,[53,61,139] which could be even advantageousbecause it allows for lowering the number of transplantedislets.
Analytical Tools for Capsule Characterization
The last few years were marked by enormous improve-ments in analytical techniques for characterization of micro-capsules. Apart from the usual methods,[78] new techniquesinclude spectroscopy and microscopy techniques such asATR-FTIR,[100,140] ToF-SIMS and XPS,[100] AFM,[105,141]
and CLSM.[114,141]
These techniques have been useful in identifying thenew features, in particular, the capsular surface and mem-brane composition. It is anticipated that they will lead tothe comparative tests among various microcapsules. Moreimportantly, they could characterize explanted microcapsulesto supplement immunohistochemical analysis of the surfaceovergrowth. This information on changes in the polymericmembrane under in vivo conditions may help to identify rea-sons for observed transplant performance. The analysis ofexplanted capsules from this point of view, as for the micro-capsule made of sodium alginate and poly(l-ornithine),[142]
is so far rare and, in the future, it should belong to a standardprotocol of analysis of the transplanted microcapsules.
Islets
It is not useful to rigorously optimize the membrane mate-rial unless there are islets of standard and high quality. Inthe previous section, standardized polymers used for capsuleformation as well for inter-laboratory comparison as encap-sulation protocol and data consistency were advocated. Thesame must apply for islets. Even though the islet isolationprotocols are generally well established,[12] each group islikely to base the islet isolation protocol on their own expe-rience. Hence, the best description cannot be complete andprovide the recipe for reproducible islet isolation by others.Therefore, it is suggested that apart from the standard pro-tocols used for testing the islet viability (dithizone staining,viability fluorescence kits, static incubation, and perifusionstudies), standardizing the long-term viability of islets needsto be developed and implemented.
Current Activities Towards Clinical Transplantationof Encapsulated Islets
Three recently approved clinical trials demonstrate that thefield of diabetes treatment by encapsulated islets is devel-oping. Calafiore et al. obtained approval for clinical trans-plantation of human islets to ten diabetic patients usingsodium alginate and poly(l-ornithine) microcapsules.[143]
The progress report with the first two patients summarizesthat the transplanted islets survived for six months and oneyear, respectively, even though the exogenous insulin couldnot be withdrawn likely due to insufficient islet mass. Lastyear AmCyte Inc. announced approval for performing clini-cal trials with twelve patients at Toronto hospital.[144] Basedon this information, islets are planned to be encapsulated inmicrocapsules made of sodium alginate and poly(l-lysine),which are further immobilized in an alginate macrocapsule.This follows a device patented some years ago by Soon-Shiong et al.,[145] who performed the first clinical trial in1994.[33] Finally, another twelve-patient clinical trial wasannounced by Novocell, Inc.[62] for islets encapsulated in thepoly(ethylene glycol) membrane.
It is hoped that these clinical trials will lead to positiveresults and will answer questions on islet mass and requiredcapsule properties, and promote future clinical trials. It wouldbe highly encouraging to see that the transplantation of encap-sulated islets develops in parallel to the Edmonton protocol,
522 I. Lacík
for which the major challenges are long-term islet survival,cell supply, and safer immunosuppression.[146] The semiper-meable membrane has potential to overcome the problem ofusing immunosuppressive drugs and hopefully to allow usingislets from sources other than allotransplantation.[18–21]
Conclusions
The development of functional polymeric semipermeablemembranes for immunoprotection of transplanted islets ofLangerhans in diabetes treatment requires investigators andgroups alike to learn from the past. This experience includespositive experiences and mistakes. One of the main con-clusions of this review is the focus on consistency andstandardization regarding materials, processes and islets.These are seen as the inevitable conditions to avoid repeatingpast mistakes.
This review demonstrates that encapsulation of islets ina polymeric membrane is certainly a challenging task crit-ically testing the application of polymer chemistry to thistype of application, and abilities of polymer chemists. Theprinciples for successful design have been defined, and poly-mer chemistry, in connection with other disciplines, shouldfulfill them and provide material capable for long-termimmunoprotection of transplanted islets of Langerhans.
Acknowledgements
This work was supported by the Science and TechnologyAssistanceAgency under contract numbersAPVT-51-016002and APVV-51-033205, and the Centre of Excellence of theSlovak Academy of Sciences CEDEBIPO. I.L. is grateful toProfessor R. G. Gilbert, Director of KCPC at the Universityof Sydney, and Dr B. Morrison from BASF Australia for theairfare to attend the 16th annual meeting of the AustralasianSociety for Biomaterials and the 28th Australasian PolymerSymposium in New Zealand in February 2006. I.L. alsowishes to thank David Hunkeler, Director of Aqua+TechSpecialties, for comments during manuscript preparation andstimulating discussions over the past years.
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