Francesca Pierigè

Erythrocyte-based drug delivery

The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimising inevitable adverse side effects, is an extremely fascinating perspective. The behaviour of erythrocytes as a delivery system for several classes of molecules (i.e., proteins, including enzymes and peptides, therapeutic agents in the form of nucleotide analogues, glucocorticoid analogues) has been studied extensively as they possess several properties, which make them unique and useful carriers. Furthermore, the possibility of using carrier erythrocytes for selective drug targeting to differentiated macrophages increases the opportunities to treat intracellular pathogens and to develop new drugs. Finally, the availability of an apparatus that permits the encapsulation of drugs into autologous erythrocytes has made this technology available in many clinical settings and co-mpetitive with other drug delivery systems.

Macrophage depletion induced by clodronate-loaded erythrocytes.

Given the important role of macrophages in various disorders, the transient and organ specific suppression of their functions may benefit some patients. Until now, liposome-encapsulated bisphosphonate clodronate has been extensively proposed to this end. In this paper, we demonstrate that erythrocytes loaded with clodronate can also be effective in macrophage depletion. Here, clodronate was encapsulated in erythrocytes through hypotonic dialysis, isotonic resealing and reannealing to final concentrations of 4.1±0.4 and 10.1±0.8 μmol/ml of human and murine erythrocytes, respectively. The ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in vitro and in vivo. In vitro studies on human macrophages showed that a single administration of engineered erythrocytes was able to reduce cell adherence capacity in a time-dependent manner, reaching 50±4% reduction, 13 days post treatment. The administration of loaded erythrocytes to cultures of murine peritoneal macrophages was able to reduce macrophage adhesion 67±3%, 48 h post treatment. In vivo, the ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in Swiss and C57BL/6 mice. Swiss mice received 125 μg of clodronate through eryhtrocytes and 6 days post treatment 69±7% reduction in the number of adherent peritoneal macrophages and 75±5% reduction in number of spleen macrophages were observed. C57BL/6 mice received 220 μg clodronate by RBC and 3 and 8 days post treatment 65±7% reduction in the number of spleen macrophages and the complete depletion of liver macrophages were obtained. In summary, our results indicate that clodronate selectively targeted to the phagocytic cells by a single administration of engineered erythrocytes is able to deplete macrophages, even if not completely. The transient suppression of macrophage functions through clodronate-loaded erythrocytes can be used in many biomedical phenomena and research applications.

Immunophilin-loaded erythrocytes as a new delivery strategy for immunosuppressive drugs

Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5±1.3, 7.5±3.1 and 15.5±0.4nmol FKBP12 were able to bind 3.5±1.5, 6.0±1.9 and 11.4±2.9μg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0±0.6, 5.0±0.8 and 15.9±2.4nmol of cyclophilin A could bind 8.9±3.4, 12.2±3.5 and 17.0±3.2μg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0±0.3μg FK506 and 3.2±0.3μg CsA). Moreover, FK506 released from FKBP12-RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.

Erythrocyte-mediated delivery of phenylalanine ammonia lyase for the treatment of phenylketonuria in BTBR-Pahenu2 mice

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by defects in the phenylalanine hydroxylase gene. Preclinical and clinical investigations suggest that phenylalanine ammonia lyase (PAL) could be an effective alternative for the treatment of PKU. The aim of this study is to investigate if erythrocytes loaded with PAL may act as a safe delivery system able to overcome bioavailability issues and to provide, in vivo, a therapeutically relevant concentration of enzyme. Murine erythrocytes were loaded with recombinant PAL from Anabaena variabilis (rAvPAL) and their ability to perform as bioreactors was assessed in vivo in adult BTBR-Pah(enu2) mice, the genetic murine model of PKU. Three groups of mice were treated with a single i.v. injection of rAvPAL-RBCs at three different doses to select the most appropriate one for assessment of efficacy. Repeated administrations at 9-10 day-intervals of the selected dose for 10 weeks showed that the therapeutic effect was persistent and not affected by the generation of antibodies induced by the recombinant enzyme. This therapeutic approach deserves further in vivo evaluation either as a potential option for the treatment of PKU patients or as a possible model for the substitutive enzymatic treatment of other inherited metabolic disorders.

Prolonged islet allograft survival in diabetic mice upon macrophage depletion by clodronate-loaded erythrocytes

Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4+/-0.89 and 20+/-2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25+/-1.9 days in the group treated with free-clodronate and 35+/-6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.

Inhibition of HIV-1 Replication in Macrophages by Red Blood Cell-Mediated Delivery of a Heterodinucleotide of Lamivudine and Tenofovir

Homo- and heterodimers of nucleoside/nucleotide analogues as reverse transcriptase inhibitors are effective on HIV-1-infected human monocyte-derived macrophages (M/M) compared to the single drugs or their combination. Since the combined treatment of lamivudine (3TC) and tenofovir ((R)PMPA) has an antiretroviral efficacy and a synergic effect respect to separate drugs, the heterodinucleotide 3TCpPMPA was synthesized. A single administration of the dimer as free drug or 3TCpPMPA-loaded RBC selectively targeted to M/M was able to almost completely protect macrophages from “de novo” infection.

Engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution

Pharmacokinetics, biodistribution, and biological activity are key parameters that determine the success or failure of therapeutics. Many developments intended to improve their in vivo performance, aim at modulating concentration, biodistribution, and targeting to tissues, cells or subcellular compartments. Erythrocyte-based drug delivery systems are especially efficient in maintaining active drugs in circulation, in releasing them for several weeks or in targeting drugs to selected cells. Erythrocytes can also be easily processed to entrap the desired pharmaceutical ingredients before re-infusion into the same or matched donors. These carriers are totally biocompatible, have a large capacity and could accommodate traditional chemical entities (glucocorticoids, immunossuppresants, etc.), biologics (proteins) and/or contrasting agents (dyes, nanoparticles). Carrier erythrocytes have been evaluated in thousands of infusions in humans proving treatment safety and efficacy, hence gaining interest in the management of complex pathologies (particularly in chronic treatments and when side-effects become serious issues) and in new diagnostic approaches.

Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment

Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

Reengineering red blood cells for cellular therapeutics and diagnostics

Recently optimized technologies that permit the reversible opening of nanopores across the red blood cell membrane, give the extraordinary opportunity for reengineering human erythrocytes to be used in different biomedical applications, both for therapeutic and diagnostic purposes. Engineered erythrocytes have been exploited as a system for the controlled release of drugs in circulation upon encapsulation of prodrugs or small molecules; as bioreactors when they are endowed of recombinant enzymes able to catalyze the conversion of toxic metabolite into inert products; as drug targeting system for the delivery of compounds to the reticuloendothelial system inducing proper senescent signals on the drug-loaded erythrocyte membrane; as carrier of contrasting agents for diagnostic procedures. Preclinical development of these different applications has taken advantage from the use of proper animal models whose erythrocytes can be reengineered as the human ones or the encapsulation procedures can be adapted on the basis of their specific erythrocyte biological features. Successful results, obtained both in vitro and in preclinical studies, have prompted several clinicians to start pilot clinical investigations in different conditions and some new companies to start the industrialization of selected loading technologies and to initiate clinical development programs. This short review summarizes the key features that, to the best of our knowledge, have been crucial to advance the products toward regulatory clinical approval making reengineering of erythrocytes a modality to treat patients with limited or absent therapeutic options. WIREs Nanomed Nanobiotechnol 2017, 9:e1454. doi: 10.1002/wnan.1454 For further resources related to this article, please visit the WIREs website.

A new therapy prevents intellectual disability in mouse with phenylketonuria

Untreated phenylketonuria (PKU) results in severe neurodevelopmental disorders, which can be partially prevented by an early and rigorous limitation of phenylalanine (Phe) intake. Enzyme substitution therapy with recombinant Anabaena variabilis Phe Ammonia Lyase (rAvPAL) proved to be effective in reducing blood Phe levels in preclinical and clinical studies of adults with PKU. Aims of present study were: a) to gather proofs of clinical efficacy of rAvPAL treatment in preventing neurological impairment in an early treated murine model of PKU; b) to test the advantages of an alternative delivering system for rAvPAL such as autologous erythrocytes. BTBR-Pahenu2-/- mice were treated from 15 to 64 post-natal days with weekly infusions of erythrocytes loaded with rAvPAL. Behavioral, neurochemical, and brain histological markers denoting untreated PKU were examined in early treated adult mice in comparison with untreated and wild type animals. rAvPAL therapy normalized blood and brain Phe; prevented cognitive developmental failure, brain depletion of serotonin, dendritic spine abnormalities, and myelin basic protein reduction. No adverse events or inactivating immune reaction were observed. In conclusion present study testifies the clinical efficacy of rAvPAL treatment in a preclinical model of PKU and the advantages of erythrocytes as carrier of the enzyme in term of frequency of the administrations and prevention of immunological reactions.