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Advanced Drug Delivery Revi

Cell-based drug delivery☆

F. Pierigè, S. Serafini, L. Rossi, M. Magnani ⁎

Institute of Biological Chemistry “Giorgio Fornaini”, University of Urbino “Carlo Bo”, Urbino, Italy

Received 31 July 2007; accepted 14 August 2007Available online 5 October 2007

Abstract

Drug delivery has been greatly improved over the years by means of chemical and physical agents that increase bioavailability, improvepharmacokinetic and reduce toxicities. At the same time, cell based delivery systems have also been developed. These possesses a number ofadvantages including prolonged delivery times, targeting of drugs to specialized cell compartments and biocompatibility. Here we'll focus onerythrocyte-based drug delivery. These systems are especially efficient in releasing drugs in circulations for weeks, have a large capacity, can beeasily processed and could accommodate traditional and biologic drugs. These carriers have also been used for delivering antigens and/orcontrasting agents. Carrier erythrocytes have been evaluated in thousands of drug administration in humans proving safety and efficacy of thetreatments. Erythrocyte-based delivery of new and conventional drugs is thus experiencing increasing interests in drug delivery and in managingcomplex pathologies especially when side effects could become serious issues.© 2007 Elsevier B.V. All rights reserved.

Keywords: Transduced cells as drug delivery systems; Cell carriers of drugs; Red blood cells as drug delivery systems

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2862. Transduced cells as a drug delivery system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2873. Cell carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

3.1. Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2873.2. Red blood cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

3.2.1. Preparation of carrier RBCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2893.2.2. Active substances encapsulated into red blood cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2903.2.3. Coupled molecules to RBC membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2903.2.4. Encapsulation of drugs and contrasting agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

4. Clinical experiences with cell based drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2915. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on“Emerging Trends in Cell-Based Therapeutics”.⁎ Corresponding author. Istituto di Chimica Biologica “G. Fornaini”,

Università degli Studi di Urbino “Carlo Bo”, Via Saffi, 2-61029, Urbino,Italy. Tel.: +39 722 305211; fax: +39 722 305324.

E-mail address: mauro.magnani@uniurb.it (M. Magnani).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2007.08.029

1. Introduction

At present, there are 30 main drug delivery products on themarket. The total annual income for all of these is approximately

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US$33 billion with an annual growth of 15% (based on globalproduct revenue). The reasons for this increasing interest in drugdelivery is due to the increasing need of safe drugs, capable ofreaching the target andwithminimal side effects. In fact themainproblems associated with systemic drug administration areessentially related to the bio-distribution of pharmaceuticalsthroughout the body. This indiscriminate distribution meansthat, to achieve a required therapeutic concentration the drug hasto be administered in large quantities, the major part of which isjust wasted in normal tissues. Ideally, a “perfect” drug shouldexert its pharmacological activity only at the target site, using thelowest concentration possible and without negative effects onnon-target compartments. The delivery systems currentlyavailable enlist carriers that are either simple, soluble macro-molecules (such as monoclonal antibodies, soluble syntheticpolymers, polysaccharides and biodegradable polymers) ormore complex particulate multicomponent structures (micro-capsules, microparticles, cells, cell ghosts, lipoproteins, lipo-somes, erythrocytes).

Drug delivery either by means of soluble or particulatecomponents can affect drug pharmacokinetic, drug bioavailability,sometime safety and efficacy however only rarely reach the maingoal of having the drug targeted only to the site were its actionshould be exerted. Thus, an additional advantage ofmodern deliverysystem is sometime associatedwith the possibility of having selectedtargeting properties useful to increase drug selectivity and improvedrug efficacy.Unfortunately, sometime the body recognizes the drugtargeting system as non-self and unexpected toxicities could hamperthe use of the same. This is the case of the first generation ofmonoclonal antibodies coupled to cytotoxic drugs or of other solublecarriers experimented at preclinical level.

It has been envisaged that ideal drug delivery systems shouldbe made of self-powered, computer-controlled medical nanor-obot system, named pharmacyte [1], capable of precisetransport, timing, and targeted delivery of pharmaceuticalagents to specific target in the body. This ideal drug deliverysystem is not yet available but significant progress has beenmade in the last years over the traditional drug formulationsand, in our opinion, the cell based delivery systems are theclosest ones to the ideal drug delivery system named above.

Among the cell based delivery systems two categories couldbe identified:

• Transduced cells, capable of expressing pharmaceuticallyrelevant agents

• Cell carriers which could be loaded with drugs ortherapeutics. In this category the carrier cells could releasethe drug content in circulation or at selected sites or couldtarget the drug to other relevant cells in the body.

Transduced cells have been recently reported as capable ofdelivering immunomodulatory molecules at sites of interest (i.e.the skin) as in Ref. [2] or to be able to advantageously targetmelanoma brain metastases upon systemic 5-fluorocytosineadministration as when neural stem/progenitor cells were used[3]. Cell carriers so far investigated include bacteria cells andanimal cells. Bacterial cells have been used as nonliving cell

envelope preparations from gram-negative cells, devoid ofcytoplasmic content, while preserving morphology and surfaceantigenic structures [4]. These ghosts have been successfullyinvestigated mainly as adjuvant particles to improve an immuneresponse against the ghost-derived target antigens. Among theanimal cells of special relevance are macrophages and red bloodcells. Macrophages could be loaded with drugs by way ofingested nanoparticles. This approach require that macrophagesbe adoptively transferred to the recipient after ex vivo loadingwithnanoparticles formulated drugs and/or contrasting agents [5,6].The majority of these macrophages (74–81%) are immediatelysequestered in the liver and in the spleen (13–18%) [6],suggesting that the system is best suited for as a targeting systemthan as a drug delivery system. Erythrocytes (red blood cells,RBCs) constitute potential biocompatible carriers for differentbioactive substances, including protein drugs, as well asconventional therapeutics. They feature unique properties suchas biodegradability, biocompatibility and large carrier volumesand thus are well suited to be used for drug encapsulation [7–9].

2. Transduced cells as a drug delivery system

Different cells have been transduced with selected genes andwith different vectors. Usually gene transfer is performed tointroduce a gene expressing a fluorescent protein to track thebehaviour of the cell in vivo or to correct a genetic defect (i.e. a genemutation or deletion) or to make the target cell susceptible to theaction of a selected drug (i.e. by expressing Tymidine kinase) [10].These transduced cells are not discussed in this paper andconsidered out of the scope of this review. Instead, we haveselected few examples that in our opinion illustrate the possibilitythat cells can be transduced to efficiently release a therapeutic agent.Until now the examples we have found are all related to theproduction of biologics, mainly cytokines, but in some cases also asfor the production and/or release of selected enzymes useful asreplacement therapeutics in patients carrying a specific gene defect(as in the case of arylsulfataseA) or to release protective proteins (asfor superoxide dismutase). A partial list, provided as an example, oftransduced cells that are able to release a biologic agent is shown inTable 1. From this list it is immediately evident that interleukins andinterferons have been widely investigated. The main problemsassociatedwith this approach are related to the duration of the effectof the transduced cells (that also in the best cases does not persist formore that few months), to the safety of the construct (the vectorused for gene transfer is still a matter of concern especially for itsintegration sites in the human genome) and to the distribution in thebody of the transduced cells that is difficult to control. However, thepossibility of engineering stem cells for the in vivo production andrelease of selected therapeutics appear promising and this area willcertainly enjoy new applications hopefully up to the clinic.

3. Cell carriers

3.1. Macrophages

Macrophages are differentiated cells of the immune system thatare able to phagocytise microorganisms as well as nanoparticulate

Table 2Some pathogens with a selective macrophage tropism

Pathogen

Listeria monocytogenes BacteriaBrucella spp. "Salmonella spp. "Mycobacteria spp. "Leishmainia "Herpes simplex VirusVaricella zoster "Cytomegalovirus "HIV-1 "Candida spp. FungiToxoplasma gondi Protozoa

Table 1Transduced cells for the delivery of biologics

Cell type Transduced gene Reference

Myeloid precursors TREM 2 [11]Fibroblasts Brain derived neurotrophic

factor (BDNF)[12]

Mesenchymal stem cells IL-10 [13]Neural progenitor cells Interferon-beta [14]Epidermal stem cells Laminin 5 [15]Hematopoietic stem cells Arylsulfatase A [16]Mesenchymal stem cells IL-12 [17]Fibroblasts Superoxide dismutase [18]Hematopoietic cells TRAIL [19]Muscle progenitor cells Factor IX [20]Neural progenitor cells Arylsulfatase A [21]Platelets Factor VIII [22]Neural stem cells Neurotrophin-3 [23]Fibroblasts Interferon alpha [24]Muscle stem cells Bone morphogenetic protein 4 [25]

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materials and soluble compounds. Because of these reasons,macrophages have been considered as cell targets for the selectivedelivery of drugs using nanoparticles [26–36] as well as cellcarriers for the delivery of therapeutic agents [4–6]. In both casesthe basic mechanisms for the recognition and internalization ofextracellular materials are similar and have been extensivelyexplored.Macrophages byway of selected receptors can recognizepolysaccharides [37], complement [38], endotoxins (i.e. LPS)[39], immunoglobulins (by the Fc receptors) [40], LDLs (byscavenger receptors) [41], etc. Based on the knowledge we havegained on the biology of macrophages and on the expression of awide range of receptors, it is now possible to design drugformulations and/or particulate materials that are recognized andinternalized by these receptors. It is essential to note that severalother cells in the body express one or more of the receptors presentonmacrophages but usually their ability to internalize a recognizedagent is strongly reduced over the phagocyte ability of macro-phages. Thus, because of this reason, nanoparticle drugs thatshould be in vivo delivered by way of macrophages couldunspecifically also distribute into unwanted cells or body districts.As a consequence of the engagement of different receptors theendocytic pathway could localize the particulate material taken upby macrophages into different intracellular compartment withrelevant consequences in terms of drug efficacy and distribution. Infact, engagement of different receptors involves differences in thecytoskeletal elements that mediate ingestion, differences invacuole maturation, and differences in inflammatory responses[42]. Nanoparticulate systems are particularly useful for thedelivery of therapeutic agents to macrophages [43–45]. Howeverthe binding ability of nanoparticles tomacrophages depends on thephysicochemical properties of the particles, in particular surfacecharge, hydrophobicity and hydrophilicity, morphology, andchemical structure. In same cases nanoparticles have shownsome cytotoxicity due to secretion of inflammatory cytokines andmetalloproteinases and activation of respiratory burst [46–49]. Thedelivery of nanoparticles loaded with drugs is of great potentialinterest for the treatment of those parasites, bacteria and virusesthat have a special macrophage tropism (Table 2). Whenmacrophages are used as drug delivery systems, they should be

first loaded with the nanoparticulate drug ex vivo than re-infusedinto the host where their content is distributed to tissues that favorhoming of macrophages. This approach permits to establish thera-peutic drug concentrations in tissue and sera increasing drugbioavailability and the drug therapeutic index [5,6].

3.2. Red blood cells

Human RBCs are the most common cells of blood, areresponsible for oxygen transport and have a typical biconcaveshape. Normal human RBCs have a diameter of 7–8 μm and anaverage volume of 90 fl (Fig. 1). In mammals, RBCs areanucleated and lose their organelles during maturation. Oxygentransport is guaranteed by about 270 million haemoglobinmolecules per cell each of which with four heme groups. Ahuman body is commonly endowed with 2–3 1013 RBCscontinuously produced at a rate of 2 million per second. In fact,RBCs spent its 100–120 day life-span travelling the circulatorysystem before being selectively removed by macrophages in thereticuloendothelial system (RES) [50].

The surface area of mature, biconcave RBCs is about136 μm2 but can swell to a sphere of approx 150 fl. It isnoteworthy that RBCs can also cross undamaged capillaries of2–3 μm in diameter. The RBC membrane is strictly connectedwith the membrane skeletal proteins which are organized in auniform shell. The RBC shape can undergo a number ofreversible transformations. An important determinant of RBCsurvival is its deformability. Key factors affecting deformabilityare internal viscosity (mainly contributed by RBC haemoglo-bin), the surface/volume of the cell and the intrinsic deform-ability of the membrane. The RBCs have other very interestingproperties namely they behave as an osmometer since theyshrink when placed into a hypertonic solution or swell whenplaced into a hypotonic solution. The RBCs can reach a criticalhaemolytic volume giving rise to holes on the membraneranging from 10 nm up to 500 nm. These processes are usuallyreversible and following haemolysis the holes close and the cellresumes its biconcave shape.

Red blood cells constitute potential biocompatible carriersfor different bioactive substances, including protein drugs, sincethey feature some unique advantages [7–9], for examples, theyare completely biodegradable without generation of toxic

Fig. 1. Scanning electron microscopy of human red blood cells. Bar: thedimension is 10 μm.

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products and show high biocompatibility especially whenautologous erythrocytes are employed; they can be easilyhandled ex vivo by means of several techniques for theencapsulation of different molecules, after which one canobtain loaded erythrocytes with morphological, immunologicaland biochemical properties similar to those of native cells;lacking a nucleus and other organelles, most of their volume isavailable for the encapsulation of drugs; they protect theencapsulated substance from premature inactivation anddegradation by endogenous factors and, at the same time, theorganism against the toxic effects of the drugs thus avoidingimmunological reactions. Potentially a wide variety of chemi-cals can be entrapped, even peptides of high molecular weight,presenting significant biotechnological applications. RBCshave a longer life-span in circulation as compared to othersynthetic carriers and could act as bioreactors due to thepresence of several enzymatic activities that can directly affectthe loaded molecules and, in the case of loaded prodrugs, giverise to the active drug itself.

Furthermore, exploiting another main feature of the RBCs, aselective targeting of drugs directly to macrophages withoutaffecting the non-targeted compartments, could be achieved.After their natural life-span (approx 120 days) in systemiccirculation, the senescent RBCs are recognized by the cells ofthe phagocytic system (RES, otherwise known as themonocyte–macrophage system) and removed from circulationto be destroyed. It is possible to artificially induce thesesenescent signals on the RBCmembrane, in order to specifically

target the drug-containing erythrocytes to the phagocytic cells,in particular to the monocyte-derived macrophages [8].

3.2.1. Preparation of carrier RBCsDifferent methods have been proposed to encapsulate

therapeutic agents into RBCs in order to obtain an appropriatedelivery of drugs, enzymes or peptides [51–54]. These methodsare performed to reach a dual objective, that is to seek anenhanced performance of the encapsulated substance whileensuring that the RBCs undergo the fewest possible alterations.It is important to obtain erythrocytes as similar as possible tonormal ones in order to ensure their proper survival andcirculation in functional terms. The methods most widely usedare commonly based on the remarkable property of the RBCs toincrease in volume when placed under condition of reducedosmotic pressure, such as in the presence of a hypotonicsolution. Three variations of the hypotonic haemolysisprocedures are available: the dilutional, preswell dilutionaland dialysis methods. The hypotonic dialysis method (Fig. 2) isthe best of these because it preserves the biochemical andphysiological characteristics of the erythrocytes resulting fromthe process and furthermore, it results in the highest percentageof encapsulation. More specifically, erythrocytes at a hematocritof 70% are placed in a dialysis tube and then immersed in ahypotonic solution. During the incubation, the membrane poresappear and open and, at this point, a drug added externally canbe incorporated inside the erythrocyte by a passive mechanism.A drug can be added to the erythrocytes before dialysis only ifthe molecular weight of the substance to be encapsulated isgreater than the cut-off of the dialysis tube. On the contrary, ifthe substance is rapidly dialyzable, it should be added to theexternal dialysing buffer if it is available in large amounts or,after the dialysis step, incubating the dialysed RBCs with thesubstance directly, when it is available in limited quantities. Inthe latter situation, the maximum concentration to be loadedmay be limited by the need to avoid high osmolalities whichinterfere with the lysis procedure. Finally, by raising the saltconcentration to its original level, the pores close, the RBCsreassume their normal biconcave shape and the substanceremains encapsulated inside the cells at a suitable concentration.Successively, the non-entrapped substance is washed out andthe loaded RBCs are ready to be used as carriers for the deliveryof the encapsulated drugs. Furthermore, by inducing theformation of an antigenic site on the RBC membrane, it ispossible to specifically target the drug-containing erythrocytesto macrophages. In particular, when ZnCl2 is externally addedto loaded RBCs, it is able to induce the reversible clusterizationof the band 3 protein (an anion transporter on the RBC surface)that, once stabilized by addition of the cross-linker agent bis(sulfosuccinimidyl)suberate (BS3), becomes an antigenic sitereadily recognized by autologous IgG and complement. Thismimics the physiological senescent process and promotesphagocytosis of the red blood cell by macrophages [55].Phagocytised RBCs enter the endocytic pathway that leads atlast to the degradation of the carriers in the lysosomalcompartment and the release of their content within themacrophages [56]. Moreover, it is possible to modulate the in

Table 3Active substances encapsulated into red blood cells

Category ofdrug

Aim Potential application References

Nucleoside/nucleotideanalogues

Drug release incirculation

Antivirals; antimicrobials [58–61]

Nucleoside/nucleotideanalogues

Drug targeting tomacrophages

Antimicrobials [62–66]

Glucocorticoidanalogues

Drug release incirculation

Inflammatory diseases [67–70]Cistic Fibrosis; ChronDisease; Ulcerative Colitis

Enzymes Degradation of toxicmetabolites; enzymereplacement therapy

Leukaemia; detoxificationof xenobiotics; ADAdeficiency; Gaucherdisease

[71–74]

Toxins Kill/treatintracellularpathogens

M. avium infections [75,76]

Peptides Affect proteinturnover

Inflammation [77–80]

Glutathione Immunomodulation;antivirals

Optimize immuneresponse; antiviral agent incombination therapy

[81–88]

AntisensePNA

Inhibit transcription Immunomodulatory;antiviral

[89–92]

Fig. 2. Pharmacokinetic of dexamethasone in plasma. A representative patient receiving 10 (♦) or 15 (▪) mg of Dex-21-P encapsulated into autologous RBCs wasinvestigated. Semi-logarithmic plot of plasma concentration of dexamethasone in patient at times 0 (immediately post-infusions), 1, 9, 16 and 28 days post-twosuccessive infusions of erythrocytes loaded with 10.76 and 15.47 mg of Dex-21-P, respectively. The glucocorticoid receptor percentage of saturation at steady state iscalculated by Eq. θ=[L]⁎100/(kd+[L]), where Kd is the dissociation constant of GR (glucocorticoid receptor) that for dexamethasone on lymphocytes is assumed5.5 nM and [L] is the dexamethasome plasma concentration at steady state, (modified from Ref. [70]).

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vivo survival of the treated cells by controlling the extension ofband 3 clustering by varying the amount of Zn2+ used [57]. Thismethod allows an estimation of the amount of drug to bedelivered to phagocytic cells, controlling the rate of red bloodcell removal from circulation.

3.2.2. Active substances encapsulated into red blood cellsA number of active substances have been encapsulated into

RBCs with the specific aim of using carrier RBCs as a slowdelivery system. In this case the drug is encapsulated as a non-diffusible pro-drug that is converted into a diffusible drug byRBC resident enzymes and released in circulation. Alternativelythe RBCs are used as a drug carrier system. In this case the drugis maintained into the RBCs until these are targeted to andphagocytised by macrophages where their content is released.RBCs have been also used as circulating bioreactors for thedegradation of metabolites or xenobiotics. In this case anenzyme is encapsulated into RBCs where it remains catalyti-cally active as long as the cell circulates. These modified RBCsare able to perform as circulating bioreactors when a metabolite,and/or a xenobiotic able to cross the RBC membrane reach theenzyme within the cell. A list of representative constructs isshown in Table 3.

3.2.3. Coupled molecules to RBC membranesFor several years RBCs have been used as diagnostic tools

for in vitro agglutination studies taking advantage from thepresence of external bound molecules. More recently severalmethods have been developed to couple molecules of interest toRBCs for their in vivo use. Selected examples include thebiotinylation of RBCs: this was useful for the evaluation ofRBC survival or blood volume determination [93–95] but hasbeen also extensively used for the coupling of other biotinylatedmolecules by way of an avidin bridge.

The coupling of fibrinolytic agents, such as clinicallyapplicable tissue-type plasminogen activator (tPA) and uroki-nase (uPA), could maintaining high fibrinolytic activity withoutcompromising the biocompatibility of the carrier. These were

tested in vivo in an animal model in which it was demonstratedthat blood level and tissue distribution of RBCs carrying tPA orurokinase were similar to those of control RBCs [96].Furthermore, when compared to plasminogen activators,RBC-coupled enzymes showed a dramatically prolonged life-time and a higher bioavailability in the blood stream of intactanimals [96,97]. As further examples of plasminogen activatorscoupled to RBCs, streptokinase retains its ability to convert itssubstrate, plasminogen, into plasmin in vitro, while RBCscarrying both collagen antibody and streptokinase were evenable to bind immobilized collagen and degrade fibrin clotsformed over the collagen target [98].

Table 4Results of intra-erythrocytic Dex-21-P therapy on FEV1 in the CF pilot study(raw values)

Intra-erythrocytic dexamethasomeplus conventional therapy

Conventional therapy alone(control)

Patients(No.)

FEV1 (%) ΔFEV1

(%)Patients(No.)

FEV1 (%) ΔFEV1

(%)T=0 T=15 T=0 T=15

1 81 96 +15 1 77 65 −122 60 69 +9 2 73 75 +23 56 62 +6 3 73 68 −54 55 70 +15 4 68 72 +45 51 50 −1 5 59 55 −46 45 55 +10 6 53 44 −97 43 53 +10 7 48 45 −38 41 47 +6 8 44 40 −49 39 43 +15 9 43 36 −7Mean +8.22 Mean −4.22Range +15, −1 Range +4, −12

(modified from Ref. [70]).FEV1 is the forced expiratory volume in 1 s. T=0 is the FEV1 value inpercentage at the beginning of treatment; T=15 is the FEV1 value in percentageat month 15 from the beginning of treatment. All treated patients have received asingle autologous RBC administration every month with an average amount of8.9±3.8 mg of Dex-21-P encapsulated.

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3.2.4. Encapsulation of drugs and contrasting agentsThe methods and procedures described above could in some

cases be adapted to co-encapsulate within RBCs differententities comprising drugs and contrasting agents. Althoughthese developments are limited, it is envisaged that they willincrease in the near future due to the possibility of visualizingthe in vivo behaviour of said erythrocytes and at the same time,reached the goal of delivering proper active substances [99].

The drugs to be encapsulated could be the same discussedabove but could also be new chemical entities. Envisaged drugsthat could benefit from the procedure of co-encapsulation arethose that could interact with the contrasting agent when saidcompound is for example a nanoparticle with a mesoporoussurface that facilitates the encapsulation of hydrophobic drugsor poorly soluble drugs. The contrasting agents that could beencapsulated include fluorescent agents with excitation andemission spectra in the infra red region of the spectra and/orsuperparamagnetic nanoparticles. Said nanoparticles could alsobe surface optimized to interact preferentially with the drug ofinterest [100].

4. Clinical experiences with cell based drug deliverysystems

To the best of our knowledge the cell based drug deliverysystems that have experienced most of the clinical applicationsare those based on the use of RBCs. The first clinicalapplications of RBCs as carriers were in enzyme replacementtherapy. Beutler and coworkers were the first to use placentalglucocerebrosidase entrapped into RBCs for a selective deliveryto macrophages [101]. The treatment was successful but theapproach was later substituted by a recombinant enzymemodified to be recognized by the mannose receptor ofmacrophages and RBCs no longer used [102]. In contrast,entrapped adenosine deaminase introduced by Bax [103] is stillin use and part of a validated clinical protocol. Another enzymewas successfully entrapped into RBCs and evaluated in pilotclinical trials namely asparaginase. This enzyme removes thenon-essential aminoacid asparagines from blood that is essentialfor lymphoblastic proliferation and thus is useful in thetreatment of lymphoblastic leukaemia [104]. Our laboratorywas the first to introduce in the clinic the use of RBCs for theslow delivery of dexamethasone. Dexamethasone is a gluco-corticoid analogue. Corticosteroids (glucocorticoids) are pow-erful, though non-specific, anti-inflammatory agents whichhave been widely used in a variety of inflammatory disorders.The clinical use of corticosteroids has been limited by a numberof important side effects including: development of a “moonface”, re-distribution of fat, muscle wasting, acne, bruising,thinning of the skin, osteoporosis, can exacerbate diabetesmellitus, suppression of growth in children, cataracts.

Systemic adverse effects are related to both dose andduration of treatment. Doses lower than 0.3 mg/day cause nosuppression of plasma cortisol. Low doses of corticosteroidsconstantly delivered may be beneficial and without side effects.Based on these data we have encapsulated into autologousRBCs dexamethasone-21-phosphate (Dex-21-P) as a non-

diffusible pro-drug. Dex-21-P into RBCs is slowly de-phosphorylated by RBC resident enzymes and slowly releasedin circulation as dexamethasone. Pharmacokinetic investiga-tions in different groups of patients have shown that a singleadministration of autologous RBCs loaded by 8–15 mg of Dex-21-P can release dexamethasone for at least 1 month maintain-ing therapeutically relevant concentrations of drug in plasmasufficient to saturate the glucocorticoid receptors by 80–85%(Fig. 2).

These concentrations do not affect cortisolemia and arebeneficial to the patients. During the years we have investigatedthe efficacy and safety of the treatment in chronic obstructivepulmonary disease (COPD) patients [69], in cystic fibrosispatients [70] (Table 4), and in the bowel inflammatory diseases(IBD) [105–108]. In all cases the patients have experienced thebenefit of the treatments without adverse effects of thecorticosteroids analogues. Furthermore, in steroid-dependentIBD patients, it was also possible to free the same from thesystem assumption of oral steroids with disappearance of alltypical side effects of the same.

Until now almost 2000 treatments have been performed indifferent clinical centres without relevant side effects, excep-tional tolerability and acceptability of the treatments by thepatients, and documented efficacy [69,70,105–108]. TheEuropean Medicinal Agency (EMEA) has also granted thestate of orphan drug designation to this treatment for cysticfibrosis patients (Orphan Drug Designation EMEA/OD/039/04-EU/3/04/230).

5. Conclusions

The results summarized in this review show some of thenumerous potential biomedical applications of cell based drug

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delivery systems opening new perspectives to the possibility ofusing our cells for therapeutic purposes. Among these RBCsfeature some unique advantages compared to other deliverysystems making them not only natural, safe and abundantcarriers but, being endowed with enzymes involved inbioconversion reactions, also active bioreactors. Recently, therole of erythrocytes as drug carriers have been documented inseveral reviews [9,109–113]. Herein we report, initially, someinformation regarding the preparation of drug-loaded RBCs andsuccessively, numerous examples of RBCs as drug carriers,prevalently obtained through experiences in our laboratory. Inparticular, RBCs as carriers of nucleotide analogues, proteins,glucocorticoid analogues and antisense peptide nucleic acids arereviewed. Altogether, from the analysis of the data, thefollowing conclusions can be drawn: RBCs are safe carriersthat persist in circulation for months (unless specificallymodified) and could release in circulation active pharmacolog-ical agents for an equivalent period. This delivery systempermits to have in the blood a low and constant drugconcentration that once specifically selected could be therapeu-tically useful without side effects.

Starting from the results obtained from our and other labo-ratories [114,115], we are confident that erythrocytes have greatpotentialities in the field of drug delivery, since the key to successof many therapeutics greatly depends on the development ofnovel technologies to improve and control the delivery of drugs.

Acknowledgment

This work was partially supported by EU NACBO Project500804-2 (2004).

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