Curr Pharm Des. 2015;21(33):4917-35.
Alginates in Pharmaceutics and Biomedicine: Is the Future so Bright?
Stefano Giovagnoli1*, Giovanni Luca2, Paolo Blasi1, Francesca Mancuso2, Iva Arato2, Aurélie Schoubben1, Mario Calvitti2, Giulia Falabella2, Giuseppe Basta3, Maria Bodo2, Riccardo Calafiore3, Maurizio Ricci1
1Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
2Department of Experimental Medicine, University of Perugia, Perugia, Italy
3Department of Medicine, University of Perugia, Perugia, Italy
Alginate represents one of the most appealing biopolymers for biomedical and biopharmaceutical applications. Alginate as a biomaterial for clinical use has been established, although not free from issues. Alginates as delivery systems have a central role in cell microencapsulation for xenotransplantation and delivery of biopharmaceuticals. Based on our experience, alginate-based delivery strategies can indeed impact patient’s quality of life especially for pathologies such a Type I diabetes mellitus.
Alginates are linear polysaccharides, made of β-(1-4) linked D-mannuronic acid (M) and α-(1-4)-linked L-guluronic acid (G) residues, produced by brown algae. Alginates consist of blocks of similar and strictly alternating residues (i.e. MMMM, GGGG and GMGM). Each polymer block has different conformational preferences and behaviors. Alginate hydrogels can be produced with bivalent cations (e.g., Ca2+, Ba2+, Fe2+) and trivalent cations (e.g., Fe3+, La3+) or polyelectrolytes, such as poly-l-ornithine (PLO) or chitosan. Thanks to a well established biocompatibility, alginate represents one of the most widely accepted material for clinical use. Therefore, these biopolymers present many of the intrinsic properties required for the preparation of micrometric and nanometric advanced delivery systems and biohybrid artificial organs.
Figure 1- Double coated alginate-PLO-alginate microcapsule structure and immunoisolation features. The microcapsule is obtained by crosslinking with calcium while the PLO layer serves to modulate alginate membrane cut-off.
Alginate technology and our contribution
The capacity of alginate to form highly biocompatible hydrogels, with calcium and other divalent and trivalent cations, has been exploited as base material for cell encapsulation and immobilization. Cell microencapsulation technology provides to the entrapped cells a functional barrier and biocompatible environment that grant access to oxygen, nutrients, and hormones, while preventing the entrance to toxic metabolites. The obtained microcapsules can impede the invasion of host immune responders, such as cells, immunoglobulins, antibodies, and complement, which, with a molecular weight of 160–900 kDa, cannot diffuse through the capsule membrane (Fig. 1). Immunoisolation can grant successful transplantation of cells in the absence of the usually required general immunosuppression (1) and transplantation of non-human cells (2,3). In this regard, porcine neonatal pancreatic cell clusters (NPCCs) have become a potential valuable substitute of human islets in diabetes treatment (4). Nevertheless, NPCCs show necessary delay time of maturation/differentiation post-transplantation prior to providing the required insulin secretion levels. To overcome this, we proposed a new protocol exploiting the sustained release of antioxidants, i.e. vitamins, superoxide dismutase and catalase (Fig. 2A), that improved dramatically β-cell maturation, with enhanced response to glucose stimulation (5,6). Alternatively, we also proved that Sertoli cells (SC), isolated from <3 day piglets, co-cultured with NPCCs and microencapsulated in alginate/PLO (Fig. 2B), can remarkably increase in vitro the β-cell mass and insulin secretion and in vivo a significantly longer remission of hyperglycemia (7).
Being SC unique immunomodulatory cells, we proposed administration of SC alone in alginate microcapsules to treat diabetic NOD mice (8). Such procedure led to diabetes prevention and reversion in the respective 88 and 81% of treated animals with no need for additional cell or insulin therapy. We demonstrated that such an approach can be a successfully employed for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans (9) as well as the prevention of skin tissue allograft rejection (10).
The remarkable properties of alginate allowed of clinical settings for human islet microencapsulated in our clinical-grade alginate (11) ALG-PLO-ALG microcapsules. In this study, four type 1 diabetes mellitus patients were transplanted intraperitoneally, under local anesthesia and ultrasound echography guidance except one case receiving three subsequent grafts the last of which was delivered under general anesthesia by laparoscopic surgery (12).
In all cases we observed no adverse reactions to the grafting procedure, nor evidence of immune sensitization. In terms of metabolic outcome, all patients showed decline of exogenous daily insulin consumption that resulted nearly halved, except one case that temporarily suspended the insulin injections. Remarkably, no anti-MHC class I–II or anti-GAD65 antibodies or islet cell antibodies were detected in any of the transplanted patients throughout the 5 years of post-transplantation follow-up (13,14). In this study, safety of the procedure and partial but evident graft function was clearly proven (15).
Following a collaboration with the University of Perugia on preclinical trials in rodents or primates, the Living Cell Technologies started a pilot phase I/II clinical trial on NPCCs microencapsulated in alginate-based microspheres (DIABECELL®) in type 1 diabetes mellitus patients (16). The product, now in Phase II, showed no marked adverse events at 18–96 weeks after transplantation.
Importance of findings
Our studies clearly prove the great benefits that the alginate technology can provide to the treatment of problematic diseases, such as diabetes. Although not free from issues, the use of alginate for cell microencapsulation is a worthwhile approach that can be exploited to develop cell-based therapies for a series of pathologic conditions, unsuccessfully and incompliantly treated by using conventional methods.
Innovation of alginate materials and techniques will push on the alginate technology application alongside the comprehension of the problems that prevent the progress towards therapy and market.
We believe that such approaches could dramatically improve patient’s life span and quality and, in some cases, open new perspectives for the development of a possible cure.
Nevertheless, prior to reaching such goals, the lack of a reliable microencapsulation systems, the large variability of alginate raw materials and manufacturing processes, and the partial unreliability of biocompatibility testing in animal models should be addressed. Essential to this purpose is the join venture among experts from different areas, involving regulatory agencies and pharmaceutical and biotech companies.
Figure 2- Double coated alginate-PLO-alginate microcapsule loaded with A) biodegradable microspheres releasing SOD and ketoprofen; the microspheres were engineered to release ketoprofen and SOD over short (1 week) and long-term (>1 month) timeframe; and B) with Sertoli cells. Both strategies were useful to improve NPCCs maturation.
This work has been supported by a grant from Mr. Gary Harlem (Altucell Inc. 3 Astor Court, Dix Hills, New York, NY).
- P. de Vos, M.M. Faas, B. Strand, R. Calafiore, Alginate-based microcapsules for immunoisolation of pancreatic islets, Biomaterials 27 (2006) 5603–5617.
- Omer A, Duvivier-Kali VF, Trivedi N, Wilmot K, Bonner-Weir S, Weir GC. Survival and maturation of microencapsulated porcineneonatal pancreatic cell clusters transplanted into immunocompetent diabetic mice. Diabetes 2003; 52:69–75.
- R. Calafiore, G. Basta, G. Luca, M. Calvitti, G. Calabrese, L. Racanicchi, G. Macchiarulo, F. Mancuso, L. Guido, P. Brunetti.Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals. Biotechnol Appl Biochem. 2004 Apr;39(Pt 2):159-64.
- Omer A, Duvivier-Kali VF, Trivedi N, Wilmot K, Bonner-Weir S, Weir GC. Survival and maturation of microencapsulated porcine neonatal pancreatic cell clusters transplanted into immunocompetent diabetic mice. Diabetes 2003; 52: 69-75.
- Luca G, Basta G, Calafiore R, Rossi C, Giovagnoli S, Esposito E, Nastruzzi C. Multifunctional microcapsules for pancreatic islet cell entrapment: design, preparation and in vitro characterization. Biomaterials 2003; 24: 3101–14.
- Giovagnoli S, Luca G, Casaburi I, Blasi P, Macchiarulo G, Ricci M, Calvitti M, Basta G, Calafiore R, Rossi C. Long-term delivery of superoxide dismutase and catalase entrapped in poly(lactide-co-glycolide) microspheres: In vitro effects on isolated neonatal porcine pancreatic cell clusters. J Control Rel 2005; 107: 65–77.
- Luca G, Nastruzzi C, Calvitti M et al. Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo post-transplant results in NOD mice. Cell Transplantation 2005; 14: 249-261.
- Fallarino F, Luca G, Calvitti M, Mancuso F, Nastruzzi C, Fioretti MC et al.Therapy of experimental type 1 diabetes by isolated Sertoli cell xenografts alone. J Exp Med 2009;206: 2511-2526.
- Luca G, Calvitti M, Mancuso F, Falabella G, Arato I, Bellucci C et al. Reversal of experimental Laron syndrome by graft of microencapsulated Sertoli Cells.J Control Release 2013; 165: 75-81.
- Bistoni G, Calvitti M, Mancuso F, Arato I, Falabella G, Cucchia R et al. Prolongation of skin allograft survival in rats by the transplantation of microencapsulated xenogeneic neonatal porcine Sertoli cells.Biomaterials. 2012;33:5333-5340.
- Basta G, Calafiore R. Process for the ultrapurification of alginates. US2010/0298262A1 Nov 25, 2010.
- R. Calafiore, G. Basta, G. Luca, A. Lemmi, M.P. Montanucci, G. Calabrese, L. Racanicchi, F. Mancuso, P. Brunetti, Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases, Diabetes Care 29 (2006) 137–138.
- R. Calafiore, G. Basta, G. Luca, A. Lemmi, M.P. Montanucci, G. Calabrese, L. Racanicchi, F. Mancuso, P. Brunetti, Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases, Diabetes Care. 29 (2006) 137-138.
- G. Basta, P. Montanucci, G. Luca, C. Boselli, G. Noya, B. Barbaro, M. Qi, K.P. Kinzer, J. Oberholzer, R. Calafiore, Long-term metabolic and immunological follow-up of nonimmunosuppressed patients with type 1 diabetes treated with microencapsulated islet allografts: four cases, Diabetes Care. 34 (2011) 2406-2409
- Calafiore R, Basta G. Clinical application of microencapsulated islets: actual prospectives on progress and challenges. Adv Drug Deliv Rev. 2014 Apr;67-68:84-92
- R.B. Elliott, Communication to 23th World Congress of IPITA. Prague, Czech Republic, 2011.