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Bioactive cell-hydrogel microcapsules for cell-based drug delivery
Journal of Controlled Release 135 (2009) 203–210
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l
Bioactive cell-hydrogel microcapsules for cell-based drug delivery Gorka Orive a, María De Castro a, Hyun-Joon Kong b, Rosa Ma Hernández a, Sara Ponce a, David J. Mooney b, José Luis Pedraz a,⁎ a b
Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria-Gasteiz, Spain School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA
a r t i c l e
i n f o
Article history: Received 6 October 2008 Accepted 12 January 2009 Available online 21 January 2009 Keywords: Biomimetic capsules RGD Alginates Cell encapsulation Drug delivery Cell therapy
a b s t r a c t Improvement of long-term drug release and design of mechanically more stable encapsulation devices are still major challenges in the field of cell encapsulation. This may be in part due to the weak in vivo stability of calcium-alginate beads and to the use of inactive biomaterials and inert scaffolds that do not mimic the physiological situation of the normal cell milieu. We hypothesized that designing biomimetic cell-hydrogel capsules might promote the in vivo long-term functionality of the enclosed drug-secreting cells and improve the mechanical stability of the capsules. Biomimetic capsules were fabricated by coupling the adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains and by using an alginate-mixture providing a bimodal molecular weight distribution. The biomimetic capsules provide cell adhesion for the enclosed cells, potentially also leading to mechanical stabilization of the cell-polymer system. Strikingly, the novel cell-hydrogel system significantly prolonged the in vivo long-term functionality and drug release, providing a sustained erythropoietin delivery during 300 days without immunosuppressive protocols. Additionally, controlling the cell-dose within the biomimetic capsules enables a controlled in vitro and in vivo drug delivery. Biomimetic cell-hydrogel capsules provide a unique microenvironment for the in vivo long-term de novo delivery of drugs from immobilized cells. © 2009 Published by Elsevier B.V.
1. Introduction The technology of cell microencapsulation is based on the immobilization of therapeutically active cells within a polymer matrix, often alginate, surrounded by a semipermeable membrane. Such encapsulated cells are protected against immune cell- and antibody-mediated rejection and have the potential to produce an array of therapeutically active substances [1–6]. These “living” drug delivery systems are especially interesting for controlled and continuous expression of hormones [7] and growth factors, for the local and targeted delivery of drugs [8] and to improve the pharmacokinetics of easily degradable peptides and proteins, which often have short half-lives in vivo. One potential impact of this drug delivery approach is that administration of immunosuppressants and implementation of strict immunosuppressive protocols can be partially or fully eliminated and therefore the serious risks associated with these drugs can also be avoided. Although several attempts have been explored to improve the long-term functionality of these bio-systems [9–11], designing mechanically more stable encapsulation devices and providing a
⁎ Corresponding author. Tel.: +34 945 013091; fax: +34 945 013040. E-mail address: [email protected] (J.L. Pedraz). 0168-3659/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.jconrel.2009.01.005
more physiological microenvironment for the immobilized cells still represent major challenges in the field. Typical calcium-alginate beads are sensitive to non-gelling agents such as sodium and potassium, which in physiological conditions, results in osmotic swelling of the beads, inevitably leading to increased pore size and destabilization and rupture of the polymer scaffold. Addressing some of these limitations may have a major impact on the long-term functionality of the enclosed drug-secreting cells. However, one major risk when designing strategies aimed to increase the stability of the devices is that this enhancement may sacrifice other properties such as capsule permeability, intra-cellular microenvironment and cell functionality. Interestingly, until now, only inactive biomaterials and polymers have been used for microcapsule elaboration and all the reported approaches have considered the three dimensional capsules as inert scaffolds in which cells are physically entrapped and transported. In contrast, we investigated the possibility of designing biomimetic cellhydrogel capsules to promote the in vivo long-term functionality of the enclosed cells and improve the mechanical stability of the capsules. To address these issues, first, we fabricated biomimetic alginates by coupling the fibronectin-derived adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains. An alginate-mixture providing a bimodal molecular weight distribution was used for capsule preparation with the aim of increasing the elastic modulus of the gel while minimally raising the viscosity of the pre-gelled solution. Increasing the mechanical properties of the gels by simply raising the
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concentration of the high molecular weight alginates typically used for cell encapsulation would also significantly raise the viscosity of the pre-gel solution, and increase cell damage during mixing [12]. Because alginate itself does not support cell attachment, the decoration of the polymer with RGD will facilitate its interaction with the integrin receptors of the enclosed cells and hypothetically prolong the survival and functionality of the latter [13,14]. We suggest that biomimetic cell-hydrogel capsules can increase the stability of the cell-loaded capsules and provide an improved microenvironment for the enclosed cells. In the present paper, we report that appropriately designed cell-hydrogel scaffolds significantly increase the mechanical resistance of the capsules while minimizing damage during cell encapsulation. The novel biomimetic capsules provide cell adhesion for the enclosed cells and prolong their long-term functionality and drug release. In addition, controlling the cell-dose within the biomimetic capsules enables a controlled in vitro and in vivo drug delivery. 2. Materials and methods 2.1. Alginate preparation Low viscosity and high guluronic acid containing alginate (LVG) and medium viscosity and high guluronic acid containing alginate (MVG) were purchased from Protanal LF 20/40 (FMC Technologies). The molecular weights (Mw) of alginates were adjusted by irradiating them with a cobalt-60 source. The irradiation dose was varied from 2 to 5 Mrads. The final Mw of alginate molecules were analyzed with gel permeation chromatography (Viscotek). Following irradiation, alginate molecules were functionalized with synthetic oligopeptides containing a sequence of (glysine)4-arginineglysine-aspartic acid-tyrosine (G4RGDY, Commonwealth Biotechnology Inc., Richmond, VA, USA) by using previously described carbodiimide chemistry [15]. A molar ration of 0.0016 RGD peptide per sugar residue of the alginate chains was maintain constant. Briefly, alginate solutions were prepared with a 2-[N-morpholino]ethanesulfonic acid hydrate (MES, Sigma) buffer and sequentially mixed with N-hydroxysulfosuccinimide (sulfo-NHS, Pierce Chemical), and 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC, Sigma). Oligopeptides were added to the reaction, and the alginates were allowed to react for 24 h. The modified alginates were purified by extensive dialysis against distilled water which removed the molecules smaller than 3500 g/mol. Following filtration for sterilization and lyophilization, the modified alginate was re-constituted to the desired concentration. 2.2. Viscosity measurement The viscosity of the alginates solutions was determined by a rotational rheometer (AR 1000 TA Instruments) with a cone-plate measuring head. The parameters of the cone were: diameter 40 mm and clearance between cone and plate 50 µm. The rheometer is connected to a thermostated bath at 25 °C to maintain the temperature constant during the measurement process. During the measurements the top part of the sample was covered in order to prevent water evaporation. The frequency was varied from 0.05 to 1000 s− 1. 2.3. Cell culture C2C12 myoblasts derived from the skeletal leg muscle of an adult C3H mouse and genetically engineered to secrete murine EPO were kindly provided by the Institute des Neurosciences (Ecole Polytechnique Federale of Lausanne, Lausanne, Switzerland). Cells were grown in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 2 mM L-glutamine, 4.5 g/l glucose, and 1% antibiotic/antimycotic solution. Cells were cultured in T-flasks, maintained at 37 °C in a
humidified 5% CO2/95% air atmosphere standard incubator, and were passed every 2–3 days. All the components of the culture medium were purchased from Gibco BRL (Invitrogen S.A., Spain). Before cell encapsulation, EPO secretion from 106 cells/24 h was determined using a sandwich enzyme-linked immunoabsorbent assay (ELISA) kit for human EPO (R&D Systems, Minneapolis, MN, USA). Cross-reaction of the kit allowed detection of murine EPO in culture supernatants. 2.4. Cell encapsulation Genetically engineered C2C12 myoblasts were immobilized within RGD and non-RGD microcapsules using an electrostatic droplet generator. Briefly, cells (5 × 106 cells/ml) were suspended in RGDalginate mixture solution (2.75% w/v) (50:50 LVG-RGD and MVGRGD alginate) and non-RGD alginate mixture solution (1.4% w/v) (50:50 LVG and MVG alginate). In the dose–response study, different cell concentrations (5, 3.75, 2.5 or 1.25 × 106 cells/ml) were suspended in the RGD-alginate mixture solution (2.75% w/v) (50:50 LVG-RGD and MVG-RGD alginate). The cell suspension was extruded into a 55 mM calcium chloride solution and the resulting alginate beads were successively ionically linked with a 0.05% (w/v) poly-L-lysine solution for 5 min (PLL; hydrobromide Mw: 15,000–30,000 Da; Sigma, St. Louis Mo, USA) and alginate solution 0.1% (w/v) for 5 min. Microcapsules were prepared at room temperature, under aseptic conditions, and were cultured in complete medium. Morphology of microcapsules was assessed by an inverted optical microscopy (Nikon TMS) equipped with a camera (Sony CCD-Iris). 2.5. Mechanical stability studies: osmotic and compression resistance tests The osmotic resistance of RGD and non-RGD microcapsules was evaluated by a short-term stability study in which the swelling suffered by the microcapsules after 1% citrate solution (w/v) treatment was determined. Briefly, 100 µl of microcapsule suspension (50–100 microcapsules) was mixed with 900 µl of phosphate-buffered saline (PBS) and placed in a 24-well cell culture cluster. The cell cluster was put in a shaker at 500 rpm and 37 °C for 1 h. Afterwards, supernatants were eliminated and 800 µl of 1% citrate solution (w/v) was added. The cluster containing the microcapsules was maintained at static conditions at 37 °C for 24 h. The following day the diameter of 20 microcapsules of each type was measured. The washing and shaking step with PBS and the static conditions were repeated during the following days until a 6-day period was completed. Results are expressed as (Df/Di) where Di is the initial diameter of the capsule and Df is the final diameter after the treatment. The compression resistance of capsules was determined as the main force (g) needed to generate 70% uniaxial compression on a Texture Analyser (TA-XT2i, Stable Microsystems, Surrey, UK). The force exerted by the probe on the capsule was recorded as a function of the compression distance leading to a force versus strain relation. Thirty capsules per batch were analyzed in order to obtain statistically relevant data. 2.6. Animal protocols All the procedures involving animals were in accordance with the relevant Spanish legislation and the European Community Council Directive on “Protection of Animals used in Experimental and Other Scientific Purposes” of 24 November 1986 (86/609/EEC). 2.7. Microcapsule implantation Adult female Balb/c mice (Harlan Interfauna, Spain) were used as allogeneic recipients. Before implantation, microcapsules were
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washed several times in Hank's balanced salt solution (HBSS). Animals were anesthetized by isoflurane inhalation and a total volume of 0.2 ml of cell-loaded RGD and non-RGD microcapsules were implanted in the subcutaneous space of mice. In the second set of experiments 0.2 ml of RGD microcapsules containing 5, 3.75, 2.5 and 1.25 × 106 cells/ml suspended in HBSS were also implanted subcutaneously. An 18-gauge catheter was used for capsule implantation (Nipro Europe N.V., Belgium). Control animals received only HBSS by the same route. Upon recovery, animals had access to food and water ad libitum. No immunosuppressant protocols were applied to the animals during the study. At specific time points blood was collected by retroorbital puncture on anesthetized animals using heparinized capillaries (Deltalab, Spain). The hematocrit was measured by a standard microhematocrit method. Results are expressed as mean ± standard deviation. EPO production from RGD and non-RGD cell loaded microcapsules and from explanted capsules was measured using the sandwich ELISA for human EPO. Results are expressed as mean ± standard deviation. The cellular activity of immobilized cells was evaluated using the tetrazolium assay (MTT) (Sigma, St. Louis, MO, USA) as described elsewhere [16]. 2.8. Histological analysis At day 330 after transplantation, animals were sacrificed and microcapsules were retrieved and fixed in a 4% paraformaldehyde solution in 0.1 M sodium phosphate, pH 7.2. Thereafter, serial horizontal cryostat sections (14 µm) were processed for hematoxylin-eosin staining. 2.9. Statistical analysis Data are presented as mean ± standard deviation. Differences between control and experimental groups were analyzed for statistical significance using a Student t test according to the results of the Levene test of homogeneity of variances. A P-value b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Biomimetic cell-hydrogel capsules are mechanically and chemically more resistant We first fabricated biomimetic alginates by coupling the fibronectin-derived adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains. The molar ratio between RGD peptides and high MW alginate chains was kept constant at 0.016:1. Combining both polymers to form binary hydrogels realizes the advantages of both low and high MW alginates. The former contributes to the stiffness of the hydrogel while the latter maintains a high strain at failure [14]. With this approach, we increased considerably the elastic modulus of the gel while only minimally raising the viscosity of the pre-gelled solution, an important requirement for cell manipulation and immobilization as it dictates the magnitude of shear forces experienced by the immobilized cells. We elaborated two different binary gels: the RGD alginate mixture solution and the non-RGD alginate mixture solution. To confirm the solutions were comparable, from a viscosity point of view, the viscosities were quantified (Fig. 1A). The viscosity of binary alginate solutions was always lower than the viscosity that would result from using the same polymer concentration with only high molecular weight polymer, and low and similar overall viscosities were found with both binary gel types. Alginate has no capacity to support cell interaction and attachment due to the lack of mammalian receptors recognizing its surface chemistry [14,17]. RGD modification provides a specific mechanism for
Fig. 1. Comparison of RGD and non-RGD alginate solutions and microcapsules. (A) viscosity measurement of RGD and non-RGD mixture solutions. (B) Schematic description of the biomimetic cell-hydrogel capsules compared to non-RGD capsules. RGD modification provides a specific mechanism for cell adhesion. (C) and (D) RGD-modified scaffolds provide mechanical stabilization of the cell-polymer system. The swelling of biomimetic scaffolds was significantly lower than non-RGD devices (Pb0.05) whereas the resistance of the capsules against compression was significantly higher (Pb0.01).
cell adhesion (Fig. 1B), as integrin receptors on the cell surface have the ability to bind and crosslink multiple chains of the RGD-modified alginate scaffold, potentially also leading to mechanical stabilization of the cell-polymer system. To assess the adhesive and biomimetic effects of RGD alginates, genetically engineered mouse skeletal C2C12 myoblasts secreting erythropoietin (EPO) were selected as a model cell system, and mixed with the binary polymer solutions. It has been observed that RGD concentration and alginate composition can control myoblast phenotype [18]. Myoblasts are relatively easy to manipulate and once encapsulated and implanted into the host, they irreversibly exit the cell cycle and fuse into multinucleated myofibrils. The latter is a potential advantage to control the therapeutic dose and to avoid potential biosafety risks. EPO was selected as a model drug due to its emerging and therapeutic effects and because it is easy to monitor its
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expression and bioactivity in vivo by following the hematocrit level (Hct). Moreover, because it exhibits poor pharmacokinetics, we expect that cell encapsulation technology would avoid the repeated EPO injections currently practiced. Three dimensional (3D) microcapsules were fabricated with RGD and non-RGD alginate solutions (Fig. 1B). To ensure an optimal biocompatibility, long-term functionality and a suitable zero-order kinetic release of EPO, small diameter (450 μm) and uniform microcapsules were produced [19–21]. We hypothesized that together with the bimodal molecular weight distribution of alginate mixture, the cell–ligand interactions may be helpful to increase the mechanical properties of the matrices as cells themselves will take part in the matrix network formation. To evaluate and compare the mechanical integrity of the RGD and non-RGD alginate microcapsules, we studied the swelling behavior and the mechanical resistance of the capsules against compression. Although developing reproducible and efficacious methods aimed to measure microcapsule's mechanical resistance is also another important challenge, the osmotic swelling study and the compression resistance study have been widely accepted by the scientific community involved in the field. The swelling of RGD alginate microcapsules (1.12± 0.01) was significantly lower than
non-RGD devices (1.16 ±0.01) (n =20, P b 0.05) (Fig. 1C), whereas the resistance of the capsules against compression was significantly higher (49.6 ±6.7 g/microcapsule versus 39.1±4.7 g/microcapsule; n= 20, P b 0.01) (Fig. 1D). In addition, the binary gel system in either case significantly improved the mechanical resistance reported for classical single alginate-poly-L-lysine capsules, which is close to 16 g/microcapsule [22]. These results support the idea that a bimodal molecular weight distribution of alginate is an effective tool to increase the strength of the gels. In addition, the interaction of myoblasts with the adhesion ligands provides additional mechanical integrity to the scaffolds by acting as cross-linkers. 3.2. Biomimetic cell-hydrogel capsules prolong the in vivo long-term functionality of the enclosed cells To characterize the viability and functionality of the myoblasts within the matrices, we studied the metabolic cell activity and EPO release during 3 weeks in vitro. These short-term studies did not show major differences either in cell viability or EPO release between RGD and non-RGD microcapsules (data not shown). After 21 days in
Fig. 2. In vivo functionality of EPO-secreting cells immobilized in the biomimetic cell-hydrogel capsules. (A) Long-term hematocrit levels of mice subcutaneously implanted with biomimetic RGD capsules and non-RGD capsules compared to control animals. Cell-hydrogel RGD capsules exhibit a more sustained functionality, maintaining the hematocrit levels close to 85% during 10 months after only one administration (Pb0.01). (B) Photomicrographs of the explanted of the biomimetic RGD and the non-RGD capsules 330 days postimplantation. (C) Explanted biomimetic cell-hydrogel capsules showed significantly higher EPO release than non-RGD devices (Pb0.05). (D) Photomicrographs of the explanted devices showed a minimal inflammatory reaction based on a thin fibroblastic layer surrounding the cell-loaded capsules.
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culture, EPO secretion from 100 cell-loaded RGD-microcapsules was measured as 71 ± 9 mIU EPO/24 h whereas the production in nonRGD matrices was 72 ± 5 mIU EPO/24 h. To further evaluate the potential impact of RGD bioactivated matrices on the survival and long-term functionality of the encapsulated cells, we implanted 0.2 ml of RGD and non-RGD cell-loaded
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microcapsules (5 × 106 cells/ml alginate) in the subcutaneous tissue of immunocompetent Balb/c mice (n = 5) without implementation of immunosuppressive protocols. A subcutaneous site was selected as previous reports testing this administration route resulted in poor implant viability and inconsistency in the hematocrit response to EPO secretion, even in syngeneic recipients [23]. The subcutaneous
Fig. 3. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vitro and in vivo. (A) Photomicrographs of the biomimetic cell-hydrogel capsules loaded with different cell densities. (B) and (C) In vitro metabolic activity of the immobilized cells and EPO secretion were correlated with immobilized cell dose. (D) Hematocrit levels of all recipients subcutaneously implanted with RGD microcapsules, independently of the dose, were significantly higher than control mice for all the time-periods (*, P b 0.05). The higher cell-dose provided significantly higher hematocrit levels than the lower dose at different time-points (†, P b 0.05).
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administration of EPO versus the continuous bolus administration of the molecule should be taken into account for future potential clinical applications [23]. In fact, patients receiving subcutaneous constant release of EPO require lower doses and less frequent injections than patients treated with intravenous injections, suggesting the former is a more effective treatment [16,24]. Results indicated that hematocrit levels of all implanted recipients (RGD and non-RGD microcapsules) were significantly increased (P b 0.01), as compared to the control mice which showed a constant baseline during the study. Mice implanted with RGD-alginate devices increased their hematocrit until day 28 (88%) and then they maintained an asymptotic level between 80 and 90% until the end of the study. Recipients with non-RGD microcapsules showed a similar behavior until day 120. Thereafter, a progressive decline in the Hct of the animals was detected. Moreover, in the last 100 days of the study the hematocrit level of the recipients with RGD alginate matrices was significantly higher than non-RGD devices (P b 0.01) (Fig. 2A). According to these results, biomimetic cell-hydrogel capsules exhibit a more sustained functionality, maintaining the hematocrit levels close to 85% during 10 months after only one administration. To our knowledge, this is the first report describing such a sustained EPO release from encapsulated cells implanted subcutaneously and without immunosuppressive protocols. In previous studies using microcapsules elaborated with low MW alginate, we reported sustained Hcl
during 100 and 130 days respectively [8,24]. Our new data represents a three-fold increase in the efficacy of the drug delivery system. We explanted the cell-loaded microcapsules from the recipients 330 after the implantation. At explantation, both RGD and non-RGD capsules appeared together forming an irregular and neovascularized structure which was adhered to the subcutaneous tissue. Microcapsules were easily separated from the aggregates and they still maintained a defined semipermeable membrane (Fig. 2B). However, from a morphological point of view RGD capsules presented a more spherical shape and homogeneous surface. To asses the functionality of the explanted grafts, we measured the EPO released from the RGD and non-RGD devices (Fig. 2C). Explanted biomimetic cell-hydrogel capsules (100 capsules) released 51.8 ± 38 mIU EPO/24 h whereas non-RGD devices secreted 4.9 ± 3.9 mIU EPO/24 h (P b 0.05). These results suggest that biomimetic design of the capsules not only provides a physically and chemically favorable environment, but also an improved biological space by presenting signals that specifically binds to cell receptors. The histological analysis of the capsules revealed a minimal inflammatory reaction based on a thin fibroblastic layer surrounding both RGD and non-RGD capsules (Fig. 2D). A similar response has been reported previously using conventional alginate-poly-L-lysine capsules [9,25,26]. Although commercially purified alginates were chosen for these experiments, and polymer decoration was done in sterile
Fig. 4. Morphological and histological characterization of the explanted devices. (A) The biomimetic capsules loaded with different cell-densities were carefully retrieved 200 days after implantation. Capsules appeared as irregular aggregates surrounded by vascular networks. (B) The histological analysis of the RGD matrices showed minor inflammatory response and the presence of capillaries within and surrounding the capsule aggregates.
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conditions, a thin pericapsular layer was present around the capsule aggregates. It has been reported that the implantation procedure by itself attracts and activates immune cells and stimulates cytokine release. Assuming this, even an ideal, absolutely non-immunogenic microcapsule might not prevent such events to occur [26]. In this report, the weak immune-reaction detected did not alter the functionality of the biomimetic cell-hydrogel capsules nor limit capsule graft survival and function. However, further evaluations might be needed to determine if reducing this slight response may further improve the long-term viability of the enclosed cells. Interestingly, EPO directly addresses cellular inflammation by inhibiting several pro-inflammatory cytokines including IL-6, TNF-α and MCP-1 which exert deleterious effects and trigger graft failure [27,28]. The presence of a capillary network surrounding the capsule aggregates may also contribute to reducing the distance between the enclosed cells and the vasculature. The latter may ensure a better nutrient and oxygen supply, and thus improve the long-term functionality of the devices. In fact, effective diffusion of oxygen and nutrients from capillaries to neighbouring immobilized cells can occur over a maximum distance of 200 μm [29,30].
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All the implanted biomimetic capsules were carefully retrieved 200 days after implantation. Microcapsules again formed irregular aggregates surrounded by vascular networks (Fig. 4A). The vascularization of the aggregates seemed to be dose-dependent, with minor or no capillary formation in the low dose RGD-capsule aggregates (1.25 × 106). EPO has been shown to possess pro-angiogenic properties as it enhances matrix metalloproteinase 2 production, Janus Kinase-2 phosphorylation and stimulates the proliferation and migration of endothelial cells and bone-marrow derived endothelial progenitor cells [33,34]. Although the present results suggest that a minimum EPO dose might be necessary to stimulate capillary formation around the capsule aggregates, no clear relations were found between the vessel outgrowth around the capsules and the long-term functionality of the devices loaded with the higher cell doses. The histological analysis of the RGD matrices showed minor inflammatory response and the presence of capillaries within and surrounding the capsule aggregates (Fig. 4 B). After isolating the matrices from the aggregates, we observed that all RGD capsules remained viable and released detectable amounts of EPO (data not shown). 4. Conclusions
3.3. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vitro In a second set of experiments, we made a dose–response study to evaluate the potential of biomimetic cell-hydrogel capsules to provide controlled drug delivery. We first, fabricated and loaded RGD-matrices with different cell densities (5, 3.75, 2.5 and 1.25 × 106 cells/ml alginate solution). Microscopic evaluation showed an increasing cell density within the biomimetic scaffolds as well as the uniformity and spherical shape of all types of devices (Fig. 3A). The in vitro metabolic activity of the immobilized cells was correlated with the cell dose, as was the EPO production (Fig. 3 B and C). In addition, both the mitochondrial activity of the cells and drug release were maintained constant during 3 weeks in vitro. According to this, it is possible to fabricate tailored cell-hydrogel capsules which release continuous drug doses. 3.4. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vivo To assess the therapeutic potential of this approach, we implanted subcutaneously 0.2 ml of the different cell-loaded biomimetic cell-hydrogel capsules in Balb/c mice. Results indicated that hematocrit levels of all recipients implanted with RGD microcapsules, independently of the dose, were significantly higher than control mice for all the time-periods (P b 0.05) (Fig. 3 D). The hematocrit level of all mice implanted with RGD-microcapsules rose from a baseline value (46 ± 7%) to a peak value at day 28. At that point, the levels were 87 ± 3%; 88 ± 4%; 86.4 ± 1.5%; and 81 ± 8% for the cell-densities of 5, 3.75, 2.5 and 1.25 × 106 respectively. A dose response effect in the hematocrit levels was observed in the initial 14 days. From day 28 till day 60, the Hct obtained with all the formulations was not significantly different, indicating that a maximum hematopoietic effect was obtained with all the formulations. In the duration of the time period, mice receiving the higher dose showed significantly higher Hct than recipients receiving the lower dose (P b 0.05). Additionally, the hematocrit levels of mice implanted with the lowest cell-dose scaffold decreased progressively whereas animals receiving the other 3 formulations maintained the Hct levels constant until the end of the study. These results may be explained considering the pharmacodynamic behaviour of EPO, which is characterized by high therapeutic effects obtained with relatively low EPO doses. Although lower EPO levels will be recommended for the treatment of anaemia, higher doses might be required for neuroprotection [31] and the treatment of schizophrenia [32].
Biomimetic cell-hydrogel capsules are mechanically and chemically more resistant and prolong drug release and long-term efficacy of immobilized cells. The overall results indicate that biomimetic cellhydrogel capsules can alter the pharmacokinetics and dose regimen of EPO, eliminating the need for repeated parenteral administrations. Unlike other approaches encapsulating the peptide alone, this cellbased strategy permits the continuous de novo secretion of EPO, avoiding drug instability. Biomimetic design of cell-loaded capsules can improve cell performance as cells are immobilized in a functional scaffold which mimics the natural microenvironment of the cells. This approach may be broadly applicable to any therapeutic approach in which continuous and controlled drug or growth factor delivery is necessary, including deficiencies of a gene product, central nervous system diseases and pathologies demanding chronic drug treatments. Acknowledgement This project was partially supported by the Ministry of Education and Science (BIO2005-02659). EPO-secreting myoblasts were provided by Dr. Patrick Aebischer and the Institute des Neurosciences of Lausanne (EPFL), Lausanne, Switzerland. We also thank the Department of Histology and Pathological Anatomy of the University of Navarra (Pamplona, Spain) for technical assistance with histological analyses. References [1] F. Lim, A.M. Sun, Microencapsulated islets as bioartificial endocrine pancreas, Science 210 (1980) 908–910. [2] R.P. Lanza, J.L. Hayes, W.L. Chick, Encapsulated cell technology, Nat. Biotechnol. 14 (1996) 1107–1111. [3] G. Orive, R.M. Hernández, A.R. Gascón, et al., Cell encapsulation: highlights and promise, Nat. Med. 9 (2003) 104–107. [4] T.M.S. Chang, Therapeutic applications of polymeric artificial cells, Nat. Rev. Drug Discov. 4 (2005) 221–235. [5] G. Orive, R.M. Hernández, A.R. Gascón, M. Igartua, J.L. Pedraz, Cell microencapsulation technology for biomedical purposes: novel insights and challenges, Trends Pharm. Sci. 24 (2003) 207–210. [6] A. Townsend-Nicholson, S.N. Jayasinghe, Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds, Biomacromolecules 7 (2006) 3364–3369. [7] P. De Vos, M.M. Faas, B. Strand, R. Calafiore, Alginate-based microcapsules for immunoisolation of pancreatic islets, Biomaterials 27 (2006) 5603–5617. [8] M. Lörh, A. Hoffmeyer, J. Kroger, et al., Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma, Lancet 357 (2001) 1591–1592. [9] G. Orive, M. De Castro, S. Ponce, et al., Long-term expression of erythropoietin from myoblasts immobilized in biocompatible and neovascularized microcapsules, Mol. Ther. 12 (2005) 283–289.
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[10] A.M. Rokstad, I. Donati, M. Borgogna, et al., Cell-compatible covalently reinforced beads obtained from a chemoenzymatically engineered alginate, Biomaterials 27 (2006) 4726–4737. [11] R. Calafiore, G. Basta, G. Luca, et al., Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases, Diabetes Care 29 (2006) 137–138. [12] H.J. Kong, M.K. Smith, D.J. Mooney, Designing alginate hydrogels to maintain viability of immobilized cells, Biomaterials 24 (2003) 4023–4029. [13] E. Alsberg, K.W. Anderson, A. Albeiruti, J.A. Rowley, D.J. Mooney, Engineering growing tissues, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 12025–12030. [14] A.D. Augst, J.J. Kong, D.J. Mooney, Alginate hydrogels as biomaterials, Macromol. Biosci. 6 (2006) 623–633. [15] J.A. Rowley, D.J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials, Biomaterials 20 (1999) 45–53. [16] M. Sohmiya, T. Kakiba, Y. Kato, Therapeutic use of continuous subcutaneous infusion of recombinant human erythropoietin in malnourished predialysis anemic patients with diabetic nephropathy, Eur. J. Endocrinol. 139 (1998) 367–370. [17] A.B. Lansdown, M.J. Payne, An evaluation of the local reaction and biodegradation of calcium sodium alginate (Kaltostat) following subcutaneous implantation in the rat, J. R. Coll. Surg. Edinb. 39 (1994) 284–288. [18] J.A. Rowley, D.J. Mooney, Alginate type and RGD density control myoblast phenotype, J. Biomed. Mat. Res. 60 (2006) 217–223. [19] R. Robitaille, J.F. Pariseau, F.A. Leblond, M. Lamoureux, Y. Lepage, J.P. Hallé, Studies on small (b350 microm) alginate-poly-L-lysine microcapsules. III. Biocompatibility of smaller versus standard microcapsules, J. Biomed. Mater. Res. 44 (1999) 116–120. [20] Z.P. Lum, M. Krestow, I.T. Tai, I. Vacek, A.M. Sun, Xenografts of rat islets into diabetic mice. An evaluation of new smaller capsules, Transplantation 53 (1992) 1180–1183. [21] D. Chicheportiche, G. Reach, In vitro kinetics of insulin release by microencapsulated rat islets: effect of the size of the microcapsules, Diabetologia 31 (1998) 54–57. [22] L. Grandoso, S. Ponce, I. Manuel, et al., Long-term survival of encapsulated GDNF secreting cells implanted within the striatum of parkinsonized rats, Int. J. Pharm. 343 (2007) 69–78.
[23] B.L. Schneider, F. Schwenter, W.F. Pralong, P. Aebischer, Prevention of the initial host immuno-inflammatory response determines the long-term survival of encapsulated myoblasts genetically engineered for erythropoietin delivery, Mol. Ther. 7 (2003) 506–514. [24] I.C. Macdougall, S.J. Gray, O. Elston, et al., Pharmacokinetics of novel erythropoiesis stimulating protein compared with epoetin alfa in dialysis patients, J. Am. Soc. Nephrol. 10 (1999) 2392–2395. [25] S. Ponce, G. Orive, R.M. Hernández, et al., In vivo evaluation of EPO-secreting cells immobilized in different alginate-PLL microcapsules, J. Control. Rel. 116 (2006) 28–34. [26] A. Murua, M. De Castro, G. Orive, R.M. Hernandez, J.L. Pedraz, In vitro characterization and in vivo functionality of erythropoietin-secreting cells immobilized in alginate-poly-L-lysine-alginate microcapsules, Biomacromolecules 8 (2007) 3302–3307. [27] R. Robitaille, J. Dusseault, N. Henley, K. Desbiens, N. Labrecque, J.P. Hallé, Inflammatory response to peritoneal implantation of alginate-poly-L-lysine microcapsules, Biomaterials 26 (2005) 4119–4127. [28] N.V. Olsen, Central nervous system frontiers for the use of erythropoietin, Clin. Infect. Dis. 37 (2003) S323–S330. [29] M.S. Martijin de Groot, T.A. Schuurs, H.G.D. Leuvenink, M.R.D. Van Schilfgaarde, Macrophage overgrowth affects neighboring nonovergrown encapsulated islets, J. Surg. Res. 115 (2003) 235–241. [30] G. Orive, S.K. Tam, J.L. Pedraz, J.P. Hallé, Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy, Biomaterials 27 (2006) 3691–3700. [31] M. Brines, A. Cerami, Emerging biological roles for erythropoietin in the nervous system, Nat. Rev. Neurosci. 6 (2005) 484–494. [32] H. Ehrenreich, D. Degner, J. Meller, et al., Erythropoietin: a candidate compound for neuroprotection in schizophrenia, Mol. Phychiatry 9 (2004) 42–54. [33] D. Ribatti, M. Presta, A. Vacca, et al., Human erythropoietin induces a proangiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo, Blood 93 (1999) 2627–2636. [34] K.J. Zwezdaryk, S.B. Coffelt, Y.G. Figueroa, et al., Erythropoietin, a hypoxiaregulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells, Exp. Hematol. 35 (2007) 640–652.
Contents lists available at ScienceDirect
Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l
Bioactive cell-hydrogel microcapsules for cell-based drug delivery Gorka Orive a, María De Castro a, Hyun-Joon Kong b, Rosa Ma Hernández a, Sara Ponce a, David J. Mooney b, José Luis Pedraz a,⁎ a b
Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria-Gasteiz, Spain School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA
a r t i c l e
i n f o
Article history: Received 6 October 2008 Accepted 12 January 2009 Available online 21 January 2009 Keywords: Biomimetic capsules RGD Alginates Cell encapsulation Drug delivery Cell therapy
a b s t r a c t Improvement of long-term drug release and design of mechanically more stable encapsulation devices are still major challenges in the field of cell encapsulation. This may be in part due to the weak in vivo stability of calcium-alginate beads and to the use of inactive biomaterials and inert scaffolds that do not mimic the physiological situation of the normal cell milieu. We hypothesized that designing biomimetic cell-hydrogel capsules might promote the in vivo long-term functionality of the enclosed drug-secreting cells and improve the mechanical stability of the capsules. Biomimetic capsules were fabricated by coupling the adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains and by using an alginate-mixture providing a bimodal molecular weight distribution. The biomimetic capsules provide cell adhesion for the enclosed cells, potentially also leading to mechanical stabilization of the cell-polymer system. Strikingly, the novel cell-hydrogel system significantly prolonged the in vivo long-term functionality and drug release, providing a sustained erythropoietin delivery during 300 days without immunosuppressive protocols. Additionally, controlling the cell-dose within the biomimetic capsules enables a controlled in vitro and in vivo drug delivery. Biomimetic cell-hydrogel capsules provide a unique microenvironment for the in vivo long-term de novo delivery of drugs from immobilized cells. © 2009 Published by Elsevier B.V.
1. Introduction The technology of cell microencapsulation is based on the immobilization of therapeutically active cells within a polymer matrix, often alginate, surrounded by a semipermeable membrane. Such encapsulated cells are protected against immune cell- and antibody-mediated rejection and have the potential to produce an array of therapeutically active substances [1–6]. These “living” drug delivery systems are especially interesting for controlled and continuous expression of hormones [7] and growth factors, for the local and targeted delivery of drugs [8] and to improve the pharmacokinetics of easily degradable peptides and proteins, which often have short half-lives in vivo. One potential impact of this drug delivery approach is that administration of immunosuppressants and implementation of strict immunosuppressive protocols can be partially or fully eliminated and therefore the serious risks associated with these drugs can also be avoided. Although several attempts have been explored to improve the long-term functionality of these bio-systems [9–11], designing mechanically more stable encapsulation devices and providing a
⁎ Corresponding author. Tel.: +34 945 013091; fax: +34 945 013040. E-mail address: [email protected] (J.L. Pedraz). 0168-3659/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.jconrel.2009.01.005
more physiological microenvironment for the immobilized cells still represent major challenges in the field. Typical calcium-alginate beads are sensitive to non-gelling agents such as sodium and potassium, which in physiological conditions, results in osmotic swelling of the beads, inevitably leading to increased pore size and destabilization and rupture of the polymer scaffold. Addressing some of these limitations may have a major impact on the long-term functionality of the enclosed drug-secreting cells. However, one major risk when designing strategies aimed to increase the stability of the devices is that this enhancement may sacrifice other properties such as capsule permeability, intra-cellular microenvironment and cell functionality. Interestingly, until now, only inactive biomaterials and polymers have been used for microcapsule elaboration and all the reported approaches have considered the three dimensional capsules as inert scaffolds in which cells are physically entrapped and transported. In contrast, we investigated the possibility of designing biomimetic cellhydrogel capsules to promote the in vivo long-term functionality of the enclosed cells and improve the mechanical stability of the capsules. To address these issues, first, we fabricated biomimetic alginates by coupling the fibronectin-derived adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains. An alginate-mixture providing a bimodal molecular weight distribution was used for capsule preparation with the aim of increasing the elastic modulus of the gel while minimally raising the viscosity of the pre-gelled solution. Increasing the mechanical properties of the gels by simply raising the
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concentration of the high molecular weight alginates typically used for cell encapsulation would also significantly raise the viscosity of the pre-gel solution, and increase cell damage during mixing [12]. Because alginate itself does not support cell attachment, the decoration of the polymer with RGD will facilitate its interaction with the integrin receptors of the enclosed cells and hypothetically prolong the survival and functionality of the latter [13,14]. We suggest that biomimetic cell-hydrogel capsules can increase the stability of the cell-loaded capsules and provide an improved microenvironment for the enclosed cells. In the present paper, we report that appropriately designed cell-hydrogel scaffolds significantly increase the mechanical resistance of the capsules while minimizing damage during cell encapsulation. The novel biomimetic capsules provide cell adhesion for the enclosed cells and prolong their long-term functionality and drug release. In addition, controlling the cell-dose within the biomimetic capsules enables a controlled in vitro and in vivo drug delivery. 2. Materials and methods 2.1. Alginate preparation Low viscosity and high guluronic acid containing alginate (LVG) and medium viscosity and high guluronic acid containing alginate (MVG) were purchased from Protanal LF 20/40 (FMC Technologies). The molecular weights (Mw) of alginates were adjusted by irradiating them with a cobalt-60 source. The irradiation dose was varied from 2 to 5 Mrads. The final Mw of alginate molecules were analyzed with gel permeation chromatography (Viscotek). Following irradiation, alginate molecules were functionalized with synthetic oligopeptides containing a sequence of (glysine)4-arginineglysine-aspartic acid-tyrosine (G4RGDY, Commonwealth Biotechnology Inc., Richmond, VA, USA) by using previously described carbodiimide chemistry [15]. A molar ration of 0.0016 RGD peptide per sugar residue of the alginate chains was maintain constant. Briefly, alginate solutions were prepared with a 2-[N-morpholino]ethanesulfonic acid hydrate (MES, Sigma) buffer and sequentially mixed with N-hydroxysulfosuccinimide (sulfo-NHS, Pierce Chemical), and 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC, Sigma). Oligopeptides were added to the reaction, and the alginates were allowed to react for 24 h. The modified alginates were purified by extensive dialysis against distilled water which removed the molecules smaller than 3500 g/mol. Following filtration for sterilization and lyophilization, the modified alginate was re-constituted to the desired concentration. 2.2. Viscosity measurement The viscosity of the alginates solutions was determined by a rotational rheometer (AR 1000 TA Instruments) with a cone-plate measuring head. The parameters of the cone were: diameter 40 mm and clearance between cone and plate 50 µm. The rheometer is connected to a thermostated bath at 25 °C to maintain the temperature constant during the measurement process. During the measurements the top part of the sample was covered in order to prevent water evaporation. The frequency was varied from 0.05 to 1000 s− 1. 2.3. Cell culture C2C12 myoblasts derived from the skeletal leg muscle of an adult C3H mouse and genetically engineered to secrete murine EPO were kindly provided by the Institute des Neurosciences (Ecole Polytechnique Federale of Lausanne, Lausanne, Switzerland). Cells were grown in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 2 mM L-glutamine, 4.5 g/l glucose, and 1% antibiotic/antimycotic solution. Cells were cultured in T-flasks, maintained at 37 °C in a
humidified 5% CO2/95% air atmosphere standard incubator, and were passed every 2–3 days. All the components of the culture medium were purchased from Gibco BRL (Invitrogen S.A., Spain). Before cell encapsulation, EPO secretion from 106 cells/24 h was determined using a sandwich enzyme-linked immunoabsorbent assay (ELISA) kit for human EPO (R&D Systems, Minneapolis, MN, USA). Cross-reaction of the kit allowed detection of murine EPO in culture supernatants. 2.4. Cell encapsulation Genetically engineered C2C12 myoblasts were immobilized within RGD and non-RGD microcapsules using an electrostatic droplet generator. Briefly, cells (5 × 106 cells/ml) were suspended in RGDalginate mixture solution (2.75% w/v) (50:50 LVG-RGD and MVGRGD alginate) and non-RGD alginate mixture solution (1.4% w/v) (50:50 LVG and MVG alginate). In the dose–response study, different cell concentrations (5, 3.75, 2.5 or 1.25 × 106 cells/ml) were suspended in the RGD-alginate mixture solution (2.75% w/v) (50:50 LVG-RGD and MVG-RGD alginate). The cell suspension was extruded into a 55 mM calcium chloride solution and the resulting alginate beads were successively ionically linked with a 0.05% (w/v) poly-L-lysine solution for 5 min (PLL; hydrobromide Mw: 15,000–30,000 Da; Sigma, St. Louis Mo, USA) and alginate solution 0.1% (w/v) for 5 min. Microcapsules were prepared at room temperature, under aseptic conditions, and were cultured in complete medium. Morphology of microcapsules was assessed by an inverted optical microscopy (Nikon TMS) equipped with a camera (Sony CCD-Iris). 2.5. Mechanical stability studies: osmotic and compression resistance tests The osmotic resistance of RGD and non-RGD microcapsules was evaluated by a short-term stability study in which the swelling suffered by the microcapsules after 1% citrate solution (w/v) treatment was determined. Briefly, 100 µl of microcapsule suspension (50–100 microcapsules) was mixed with 900 µl of phosphate-buffered saline (PBS) and placed in a 24-well cell culture cluster. The cell cluster was put in a shaker at 500 rpm and 37 °C for 1 h. Afterwards, supernatants were eliminated and 800 µl of 1% citrate solution (w/v) was added. The cluster containing the microcapsules was maintained at static conditions at 37 °C for 24 h. The following day the diameter of 20 microcapsules of each type was measured. The washing and shaking step with PBS and the static conditions were repeated during the following days until a 6-day period was completed. Results are expressed as (Df/Di) where Di is the initial diameter of the capsule and Df is the final diameter after the treatment. The compression resistance of capsules was determined as the main force (g) needed to generate 70% uniaxial compression on a Texture Analyser (TA-XT2i, Stable Microsystems, Surrey, UK). The force exerted by the probe on the capsule was recorded as a function of the compression distance leading to a force versus strain relation. Thirty capsules per batch were analyzed in order to obtain statistically relevant data. 2.6. Animal protocols All the procedures involving animals were in accordance with the relevant Spanish legislation and the European Community Council Directive on “Protection of Animals used in Experimental and Other Scientific Purposes” of 24 November 1986 (86/609/EEC). 2.7. Microcapsule implantation Adult female Balb/c mice (Harlan Interfauna, Spain) were used as allogeneic recipients. Before implantation, microcapsules were
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washed several times in Hank's balanced salt solution (HBSS). Animals were anesthetized by isoflurane inhalation and a total volume of 0.2 ml of cell-loaded RGD and non-RGD microcapsules were implanted in the subcutaneous space of mice. In the second set of experiments 0.2 ml of RGD microcapsules containing 5, 3.75, 2.5 and 1.25 × 106 cells/ml suspended in HBSS were also implanted subcutaneously. An 18-gauge catheter was used for capsule implantation (Nipro Europe N.V., Belgium). Control animals received only HBSS by the same route. Upon recovery, animals had access to food and water ad libitum. No immunosuppressant protocols were applied to the animals during the study. At specific time points blood was collected by retroorbital puncture on anesthetized animals using heparinized capillaries (Deltalab, Spain). The hematocrit was measured by a standard microhematocrit method. Results are expressed as mean ± standard deviation. EPO production from RGD and non-RGD cell loaded microcapsules and from explanted capsules was measured using the sandwich ELISA for human EPO. Results are expressed as mean ± standard deviation. The cellular activity of immobilized cells was evaluated using the tetrazolium assay (MTT) (Sigma, St. Louis, MO, USA) as described elsewhere [16]. 2.8. Histological analysis At day 330 after transplantation, animals were sacrificed and microcapsules were retrieved and fixed in a 4% paraformaldehyde solution in 0.1 M sodium phosphate, pH 7.2. Thereafter, serial horizontal cryostat sections (14 µm) were processed for hematoxylin-eosin staining. 2.9. Statistical analysis Data are presented as mean ± standard deviation. Differences between control and experimental groups were analyzed for statistical significance using a Student t test according to the results of the Levene test of homogeneity of variances. A P-value b 0.05 was considered statistically significant. 3. Results and discussion 3.1. Biomimetic cell-hydrogel capsules are mechanically and chemically more resistant We first fabricated biomimetic alginates by coupling the fibronectin-derived adhesion peptide arginine glycine aspartic acid (RGD) to alginate polymer chains. The molar ratio between RGD peptides and high MW alginate chains was kept constant at 0.016:1. Combining both polymers to form binary hydrogels realizes the advantages of both low and high MW alginates. The former contributes to the stiffness of the hydrogel while the latter maintains a high strain at failure [14]. With this approach, we increased considerably the elastic modulus of the gel while only minimally raising the viscosity of the pre-gelled solution, an important requirement for cell manipulation and immobilization as it dictates the magnitude of shear forces experienced by the immobilized cells. We elaborated two different binary gels: the RGD alginate mixture solution and the non-RGD alginate mixture solution. To confirm the solutions were comparable, from a viscosity point of view, the viscosities were quantified (Fig. 1A). The viscosity of binary alginate solutions was always lower than the viscosity that would result from using the same polymer concentration with only high molecular weight polymer, and low and similar overall viscosities were found with both binary gel types. Alginate has no capacity to support cell interaction and attachment due to the lack of mammalian receptors recognizing its surface chemistry [14,17]. RGD modification provides a specific mechanism for
Fig. 1. Comparison of RGD and non-RGD alginate solutions and microcapsules. (A) viscosity measurement of RGD and non-RGD mixture solutions. (B) Schematic description of the biomimetic cell-hydrogel capsules compared to non-RGD capsules. RGD modification provides a specific mechanism for cell adhesion. (C) and (D) RGD-modified scaffolds provide mechanical stabilization of the cell-polymer system. The swelling of biomimetic scaffolds was significantly lower than non-RGD devices (Pb0.05) whereas the resistance of the capsules against compression was significantly higher (Pb0.01).
cell adhesion (Fig. 1B), as integrin receptors on the cell surface have the ability to bind and crosslink multiple chains of the RGD-modified alginate scaffold, potentially also leading to mechanical stabilization of the cell-polymer system. To assess the adhesive and biomimetic effects of RGD alginates, genetically engineered mouse skeletal C2C12 myoblasts secreting erythropoietin (EPO) were selected as a model cell system, and mixed with the binary polymer solutions. It has been observed that RGD concentration and alginate composition can control myoblast phenotype [18]. Myoblasts are relatively easy to manipulate and once encapsulated and implanted into the host, they irreversibly exit the cell cycle and fuse into multinucleated myofibrils. The latter is a potential advantage to control the therapeutic dose and to avoid potential biosafety risks. EPO was selected as a model drug due to its emerging and therapeutic effects and because it is easy to monitor its
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expression and bioactivity in vivo by following the hematocrit level (Hct). Moreover, because it exhibits poor pharmacokinetics, we expect that cell encapsulation technology would avoid the repeated EPO injections currently practiced. Three dimensional (3D) microcapsules were fabricated with RGD and non-RGD alginate solutions (Fig. 1B). To ensure an optimal biocompatibility, long-term functionality and a suitable zero-order kinetic release of EPO, small diameter (450 μm) and uniform microcapsules were produced [19–21]. We hypothesized that together with the bimodal molecular weight distribution of alginate mixture, the cell–ligand interactions may be helpful to increase the mechanical properties of the matrices as cells themselves will take part in the matrix network formation. To evaluate and compare the mechanical integrity of the RGD and non-RGD alginate microcapsules, we studied the swelling behavior and the mechanical resistance of the capsules against compression. Although developing reproducible and efficacious methods aimed to measure microcapsule's mechanical resistance is also another important challenge, the osmotic swelling study and the compression resistance study have been widely accepted by the scientific community involved in the field. The swelling of RGD alginate microcapsules (1.12± 0.01) was significantly lower than
non-RGD devices (1.16 ±0.01) (n =20, P b 0.05) (Fig. 1C), whereas the resistance of the capsules against compression was significantly higher (49.6 ±6.7 g/microcapsule versus 39.1±4.7 g/microcapsule; n= 20, P b 0.01) (Fig. 1D). In addition, the binary gel system in either case significantly improved the mechanical resistance reported for classical single alginate-poly-L-lysine capsules, which is close to 16 g/microcapsule [22]. These results support the idea that a bimodal molecular weight distribution of alginate is an effective tool to increase the strength of the gels. In addition, the interaction of myoblasts with the adhesion ligands provides additional mechanical integrity to the scaffolds by acting as cross-linkers. 3.2. Biomimetic cell-hydrogel capsules prolong the in vivo long-term functionality of the enclosed cells To characterize the viability and functionality of the myoblasts within the matrices, we studied the metabolic cell activity and EPO release during 3 weeks in vitro. These short-term studies did not show major differences either in cell viability or EPO release between RGD and non-RGD microcapsules (data not shown). After 21 days in
Fig. 2. In vivo functionality of EPO-secreting cells immobilized in the biomimetic cell-hydrogel capsules. (A) Long-term hematocrit levels of mice subcutaneously implanted with biomimetic RGD capsules and non-RGD capsules compared to control animals. Cell-hydrogel RGD capsules exhibit a more sustained functionality, maintaining the hematocrit levels close to 85% during 10 months after only one administration (Pb0.01). (B) Photomicrographs of the explanted of the biomimetic RGD and the non-RGD capsules 330 days postimplantation. (C) Explanted biomimetic cell-hydrogel capsules showed significantly higher EPO release than non-RGD devices (Pb0.05). (D) Photomicrographs of the explanted devices showed a minimal inflammatory reaction based on a thin fibroblastic layer surrounding the cell-loaded capsules.
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culture, EPO secretion from 100 cell-loaded RGD-microcapsules was measured as 71 ± 9 mIU EPO/24 h whereas the production in nonRGD matrices was 72 ± 5 mIU EPO/24 h. To further evaluate the potential impact of RGD bioactivated matrices on the survival and long-term functionality of the encapsulated cells, we implanted 0.2 ml of RGD and non-RGD cell-loaded
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microcapsules (5 × 106 cells/ml alginate) in the subcutaneous tissue of immunocompetent Balb/c mice (n = 5) without implementation of immunosuppressive protocols. A subcutaneous site was selected as previous reports testing this administration route resulted in poor implant viability and inconsistency in the hematocrit response to EPO secretion, even in syngeneic recipients [23]. The subcutaneous
Fig. 3. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vitro and in vivo. (A) Photomicrographs of the biomimetic cell-hydrogel capsules loaded with different cell densities. (B) and (C) In vitro metabolic activity of the immobilized cells and EPO secretion were correlated with immobilized cell dose. (D) Hematocrit levels of all recipients subcutaneously implanted with RGD microcapsules, independently of the dose, were significantly higher than control mice for all the time-periods (*, P b 0.05). The higher cell-dose provided significantly higher hematocrit levels than the lower dose at different time-points (†, P b 0.05).
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administration of EPO versus the continuous bolus administration of the molecule should be taken into account for future potential clinical applications [23]. In fact, patients receiving subcutaneous constant release of EPO require lower doses and less frequent injections than patients treated with intravenous injections, suggesting the former is a more effective treatment [16,24]. Results indicated that hematocrit levels of all implanted recipients (RGD and non-RGD microcapsules) were significantly increased (P b 0.01), as compared to the control mice which showed a constant baseline during the study. Mice implanted with RGD-alginate devices increased their hematocrit until day 28 (88%) and then they maintained an asymptotic level between 80 and 90% until the end of the study. Recipients with non-RGD microcapsules showed a similar behavior until day 120. Thereafter, a progressive decline in the Hct of the animals was detected. Moreover, in the last 100 days of the study the hematocrit level of the recipients with RGD alginate matrices was significantly higher than non-RGD devices (P b 0.01) (Fig. 2A). According to these results, biomimetic cell-hydrogel capsules exhibit a more sustained functionality, maintaining the hematocrit levels close to 85% during 10 months after only one administration. To our knowledge, this is the first report describing such a sustained EPO release from encapsulated cells implanted subcutaneously and without immunosuppressive protocols. In previous studies using microcapsules elaborated with low MW alginate, we reported sustained Hcl
during 100 and 130 days respectively [8,24]. Our new data represents a three-fold increase in the efficacy of the drug delivery system. We explanted the cell-loaded microcapsules from the recipients 330 after the implantation. At explantation, both RGD and non-RGD capsules appeared together forming an irregular and neovascularized structure which was adhered to the subcutaneous tissue. Microcapsules were easily separated from the aggregates and they still maintained a defined semipermeable membrane (Fig. 2B). However, from a morphological point of view RGD capsules presented a more spherical shape and homogeneous surface. To asses the functionality of the explanted grafts, we measured the EPO released from the RGD and non-RGD devices (Fig. 2C). Explanted biomimetic cell-hydrogel capsules (100 capsules) released 51.8 ± 38 mIU EPO/24 h whereas non-RGD devices secreted 4.9 ± 3.9 mIU EPO/24 h (P b 0.05). These results suggest that biomimetic design of the capsules not only provides a physically and chemically favorable environment, but also an improved biological space by presenting signals that specifically binds to cell receptors. The histological analysis of the capsules revealed a minimal inflammatory reaction based on a thin fibroblastic layer surrounding both RGD and non-RGD capsules (Fig. 2D). A similar response has been reported previously using conventional alginate-poly-L-lysine capsules [9,25,26]. Although commercially purified alginates were chosen for these experiments, and polymer decoration was done in sterile
Fig. 4. Morphological and histological characterization of the explanted devices. (A) The biomimetic capsules loaded with different cell-densities were carefully retrieved 200 days after implantation. Capsules appeared as irregular aggregates surrounded by vascular networks. (B) The histological analysis of the RGD matrices showed minor inflammatory response and the presence of capillaries within and surrounding the capsule aggregates.
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conditions, a thin pericapsular layer was present around the capsule aggregates. It has been reported that the implantation procedure by itself attracts and activates immune cells and stimulates cytokine release. Assuming this, even an ideal, absolutely non-immunogenic microcapsule might not prevent such events to occur [26]. In this report, the weak immune-reaction detected did not alter the functionality of the biomimetic cell-hydrogel capsules nor limit capsule graft survival and function. However, further evaluations might be needed to determine if reducing this slight response may further improve the long-term viability of the enclosed cells. Interestingly, EPO directly addresses cellular inflammation by inhibiting several pro-inflammatory cytokines including IL-6, TNF-α and MCP-1 which exert deleterious effects and trigger graft failure [27,28]. The presence of a capillary network surrounding the capsule aggregates may also contribute to reducing the distance between the enclosed cells and the vasculature. The latter may ensure a better nutrient and oxygen supply, and thus improve the long-term functionality of the devices. In fact, effective diffusion of oxygen and nutrients from capillaries to neighbouring immobilized cells can occur over a maximum distance of 200 μm [29,30].
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All the implanted biomimetic capsules were carefully retrieved 200 days after implantation. Microcapsules again formed irregular aggregates surrounded by vascular networks (Fig. 4A). The vascularization of the aggregates seemed to be dose-dependent, with minor or no capillary formation in the low dose RGD-capsule aggregates (1.25 × 106). EPO has been shown to possess pro-angiogenic properties as it enhances matrix metalloproteinase 2 production, Janus Kinase-2 phosphorylation and stimulates the proliferation and migration of endothelial cells and bone-marrow derived endothelial progenitor cells [33,34]. Although the present results suggest that a minimum EPO dose might be necessary to stimulate capillary formation around the capsule aggregates, no clear relations were found between the vessel outgrowth around the capsules and the long-term functionality of the devices loaded with the higher cell doses. The histological analysis of the RGD matrices showed minor inflammatory response and the presence of capillaries within and surrounding the capsule aggregates (Fig. 4 B). After isolating the matrices from the aggregates, we observed that all RGD capsules remained viable and released detectable amounts of EPO (data not shown). 4. Conclusions
3.3. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vitro In a second set of experiments, we made a dose–response study to evaluate the potential of biomimetic cell-hydrogel capsules to provide controlled drug delivery. We first, fabricated and loaded RGD-matrices with different cell densities (5, 3.75, 2.5 and 1.25 × 106 cells/ml alginate solution). Microscopic evaluation showed an increasing cell density within the biomimetic scaffolds as well as the uniformity and spherical shape of all types of devices (Fig. 3A). The in vitro metabolic activity of the immobilized cells was correlated with the cell dose, as was the EPO production (Fig. 3 B and C). In addition, both the mitochondrial activity of the cells and drug release were maintained constant during 3 weeks in vitro. According to this, it is possible to fabricate tailored cell-hydrogel capsules which release continuous drug doses. 3.4. Biomimetic cell-hydrogel capsules provide control over cell dose and drug delivery in vivo To assess the therapeutic potential of this approach, we implanted subcutaneously 0.2 ml of the different cell-loaded biomimetic cell-hydrogel capsules in Balb/c mice. Results indicated that hematocrit levels of all recipients implanted with RGD microcapsules, independently of the dose, were significantly higher than control mice for all the time-periods (P b 0.05) (Fig. 3 D). The hematocrit level of all mice implanted with RGD-microcapsules rose from a baseline value (46 ± 7%) to a peak value at day 28. At that point, the levels were 87 ± 3%; 88 ± 4%; 86.4 ± 1.5%; and 81 ± 8% for the cell-densities of 5, 3.75, 2.5 and 1.25 × 106 respectively. A dose response effect in the hematocrit levels was observed in the initial 14 days. From day 28 till day 60, the Hct obtained with all the formulations was not significantly different, indicating that a maximum hematopoietic effect was obtained with all the formulations. In the duration of the time period, mice receiving the higher dose showed significantly higher Hct than recipients receiving the lower dose (P b 0.05). Additionally, the hematocrit levels of mice implanted with the lowest cell-dose scaffold decreased progressively whereas animals receiving the other 3 formulations maintained the Hct levels constant until the end of the study. These results may be explained considering the pharmacodynamic behaviour of EPO, which is characterized by high therapeutic effects obtained with relatively low EPO doses. Although lower EPO levels will be recommended for the treatment of anaemia, higher doses might be required for neuroprotection [31] and the treatment of schizophrenia [32].
Biomimetic cell-hydrogel capsules are mechanically and chemically more resistant and prolong drug release and long-term efficacy of immobilized cells. The overall results indicate that biomimetic cellhydrogel capsules can alter the pharmacokinetics and dose regimen of EPO, eliminating the need for repeated parenteral administrations. Unlike other approaches encapsulating the peptide alone, this cellbased strategy permits the continuous de novo secretion of EPO, avoiding drug instability. Biomimetic design of cell-loaded capsules can improve cell performance as cells are immobilized in a functional scaffold which mimics the natural microenvironment of the cells. This approach may be broadly applicable to any therapeutic approach in which continuous and controlled drug or growth factor delivery is necessary, including deficiencies of a gene product, central nervous system diseases and pathologies demanding chronic drug treatments. Acknowledgement This project was partially supported by the Ministry of Education and Science (BIO2005-02659). EPO-secreting myoblasts were provided by Dr. Patrick Aebischer and the Institute des Neurosciences of Lausanne (EPFL), Lausanne, Switzerland. We also thank the Department of Histology and Pathological Anatomy of the University of Navarra (Pamplona, Spain) for technical assistance with histological analyses. References [1] F. Lim, A.M. Sun, Microencapsulated islets as bioartificial endocrine pancreas, Science 210 (1980) 908–910. [2] R.P. Lanza, J.L. Hayes, W.L. Chick, Encapsulated cell technology, Nat. Biotechnol. 14 (1996) 1107–1111. [3] G. Orive, R.M. Hernández, A.R. Gascón, et al., Cell encapsulation: highlights and promise, Nat. Med. 9 (2003) 104–107. [4] T.M.S. Chang, Therapeutic applications of polymeric artificial cells, Nat. Rev. Drug Discov. 4 (2005) 221–235. [5] G. Orive, R.M. Hernández, A.R. Gascón, M. Igartua, J.L. Pedraz, Cell microencapsulation technology for biomedical purposes: novel insights and challenges, Trends Pharm. Sci. 24 (2003) 207–210. [6] A. Townsend-Nicholson, S.N. Jayasinghe, Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds, Biomacromolecules 7 (2006) 3364–3369. [7] P. De Vos, M.M. Faas, B. Strand, R. Calafiore, Alginate-based microcapsules for immunoisolation of pancreatic islets, Biomaterials 27 (2006) 5603–5617. [8] M. Lörh, A. Hoffmeyer, J. Kroger, et al., Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma, Lancet 357 (2001) 1591–1592. [9] G. Orive, M. De Castro, S. Ponce, et al., Long-term expression of erythropoietin from myoblasts immobilized in biocompatible and neovascularized microcapsules, Mol. Ther. 12 (2005) 283–289.
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