Drug Delivery, 14:295–300, 2007Copyright c© Informa HealthcareISSN: 1071-7544 print / 1521-0464 onlineDOI: 10.1080/10717540701203000

Preparation and Validation of Carrier Human ErythrocytesLoaded by Bovine Serum Albumin as a ModelAntigen/Protein

Mehrdad Hamidi, Najmeh Zarei, Abdolhossein Zarrin,and Soleiman Mohammadi-SamaniDepartment of Pharmaceutics, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

Erythrocytes as the most readily available and abundant cellswithin the body have been studied extensively for their potentialapplication as drug delivery carries. In this study, human erythro-cytes were loaded by bovine serum albumin (BSA) as a model anti-gen/protein using hypotonic preswelling method for targeted deliv-ery of this antigen-to antigen-presenting cells. The average loadedamount, efficiency of entrapment, and cell recovery upon loadingprocedure were 1979.25 ± 9.4 µg, 30.06 ± 0.20%, and 87.53 ±0.66%, respectively. The total BSA recovery upon loading proce-dure was 97.20 ± 4.90%. The apparent mechanism of entrapmentwas simple concentration-based gradient in/out the cells with someminor limiting factors against protein entry into the cells. We haveshown that the intra- and intersubject variations of the methodwere interestingly low (i.e., less than 5% in all cases).

Keywords Bovine Serum Albumin, Carrier Erythrocytes, HypotonicPreswelling, Vaccine Delivery

Cellular carriers, including erythrocytes, leukocytes,platelets, islets, hepatocytes, and fibroblasts, all have beensuggested as potential delivery systems for drugs and otherbioactive agents (Banker and Rhodes 2002, 560; Rossi et al.2005). They can be used to provide slow release of entrappeddrugs in the circulatory system, to deliver drugs to a specificsite within the body, as cellular transplants to provide missingenzymes and hormones (in enzyme or hormone replacementtherapy), or as endogenous bioreactors to synthesize and secretmolecules capable of affecting the metabolism and functionof other cells (Banker and Rhodes 2002, 560; Highley and DeBruijn 1996; Jain, Jain, and Dixit 1997). Erythrocytes, as themost abundant and readily available cells in the body, havegained the highest degree of interest among the aforementioned

Received 16 August 2006; accepted 10 December 2006.Address correspondence to Mehrdad Hamidi, Department of Phar-

maceutics, Faculty of Pharmacy, Shiraz University of Medical Sciences,P.O. Box 71345–1583, Shiraz, Iran. E-mail:hamidim@sums.ac.ir

cells, owing to a series of advantages. These cells are nonim-munogenic, biodegradable, and biocompatible and may havenearly normal life-span in circulation, among other advantages(Gutierrez Millan et al. 2004a, 2004b; Hamidi and Tajerzadeh2003; Magnani et al. 2002). One of the main advantage oferythrocytes is that they act as a true drug targeting systemthat achieve selective drug distribution to different organsand tissues, specially the phagocytic cells of the mononuclearphagocyte system (MPS). The selective accumulation oftherapeutic agents in phagocytic cells, such as macrophages,by the use of carrier erythrocytes has been reported for antibi-otics (Jaitely et al. 1996), antileshmanials (Summers 1983),antimalarials (Talwar and Jain 1992), antineoplasm agents(Al-Achi and Boroujerdi 1990), anti-HIV peptides (Rossi et al.1998), and enzymes such as asparaginase, uricase, urokinase,and arginase (Gutierrez Millan et al. 2004b).

In recent years, a major goal of researchers in vaccine designand formulation has been the development of sustained-releaseor pulsed-release delivery systems capable of eliminating the re-quirement for a multiple dosing schedule inherent to the admin-istration of conventional vaccines (Storni et al. 2005; Coombeset al. 1996). This would result in the decreased number of vac-cination periods and therefore reduced problems with patientnoncompliance and population coverage (Powell 1996; Zhaoand Leong 1996). Aside from this, a new generation of vaccinesare poorly immunogenic and need potent adjuvants to inducean effective protective immunity (Zhou et al. 2003; O’Hagan,Singh, and Gupta 1998). Therefore, development of more effi-cient and safe adjuvant/vaccine delivery systems to obtain highimmune responses is of primary importnace. Most of the vaccinedelivery systems, especially those based on polymeric systems,have high immunogenic potency but are toxic and immunogenicsuch that they cause nonspecific stimulation of host immune sys-tems (Giudice, Podda, and Rappuoli, et.al. 2002; Babiuk et al.2000; Gupta and Siber 1995).

Erythrocytes, as the autologous cells of the bost body, canbe regarded as a safe adjuvant/vaccine delivery system. Besidethe general advantages of erythrocytes as an ideal drug delivery

295

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.

296 M. HAMIDI ET AL.

system, they have two remarkable characteristics for use as avaccine delivery system: They can be used for controlled releaseof vaccines with an aim to reduce the number of doses needed forprimary immunization or to develop single dose vaccines; theymay serve as a vehicle to target antigens to antigen-presentingcells (APC); for instance macrophages and dendritic cells, forimproved and potentiated immunity.

Bovine serum albumin (BSA) is one of the most widely usedmodel antigens during vaccine delivery studies (Hoshi et al.1998; Sprott, Tolson, and Patel 1997; Ramaldes et al. 1996)owing to its ease of assay, well-known immunogenicity pattern,well-known physicochemical properties, low cost, and generalavailability (Hoshi et al. 1998). In our study was encapsulatedin human erythrocytes using the hypotonic preswelling method.The encapsulation method was validated and characterized interms of the antigen-loading parameters of the prepared vehicles.

MATERIALS AND METHODSBSA (Sigma, St. Louis, MO, USA; Art. No. A2153) was

purchased locally. Heparin sodium was purchased from IranianPharmaceutical Development and Investment Company (Rasht,Iran). Other chemicals and solvents were from chemical lab orHPLC purity grades, as needed, and were purchased locally.

Preparation of Human ErythrocytesBlood samples were withdrawn by venipuncture from healthy

volunteers aged 25 to 30 years using 19-gauqe hypodermic nee-dles connected to disposable polypropylene syringes. After cen-trifuging at 600 g for 10 min, the plasma and buffy coat wereseparated by aspiration, and the remaining packed erythrocyteswere washed three times with phosphate-buffered saline (PBS;150 mM NaCl and 5 mM K2HPO4; pH = 7.4).

Encapsulation of BSAA hypotonic preswelling method described by Tajerzadeh and

Hamidi (2000) was used for loading the human erythrocytes byBSA. For this purpose, 1 ml of washed packed erythrocytes wastransferred gently to a polypropylene test tube, 4 ml of a hypo-tonic PBS with osmolarity of 0.67 that of the eutonic solutionwas added, and the resulting cell suspension was mixed gen-tly by 10-times inversion. The swollen cells were separated bycentrifugation at 600 g for 10 min and the supernatant was dis-carded. A 200-µl aliquot of a hemolysate, prepared by dilutinganother portion of erythrocytes with distilled water (1:1), wasthen added gently to the remaining swollen cells. We assumedthat this hemolysate layer plays an important role as an osmoticshock barrier and also as a reservoir of cell constituents for un-derlying cells and thus prevents them from substantial loss ofcellular components near the lysis point. Then, 250 µl of anaqueous solution of BSA (8 mg/ml) was gently added to thecell suspension, and the resulting mixture was inverted gentlyseveral times and centrifuged at 600 g for 5 min. Addition of

protein solution, mixing, and centrifuging were repeated threemore times to achieve the lysis point of the cells.

This point was detectable by a sudden increase in trans-parency of the cell suspension and the disappearance of a distinctboundary between cells and supernatant on centrifuging. Then,the erythrocytes were resealed by the rapid addition of 100 µlof hypertonic PBS with an osmolarity of ten times the eutonicsolution, followed by gentle mixing of the suspension by severalinversions. Finally, the resulting mixture was incubated at 37◦Cfor 30 min to reanneal the resealed cells. The carrier erythro-cytes obtained by this manner were washed three times using10 ml aliquots of PBS to wash out the unentrapped BSA andthe released hemoglobin and other cell constituents during theloading process.

BSA AssayA reversed-phase HPLC method was developed and used

throughout this study for BSA assay. The method consisted ofa gradient system of 0.1% trifluoroacetic acid (TFA) in water(A) and 0.08% TFA in acetonitrial (B) with initial A/B ratio of70/30 changed linearly to the final ratio of 35/65 (A/B) within 20min. The reversal to the initial condition then occurred within2 min, and finally the system was re-equilibrated over 8 min(total run time of 30 min). The flow rate was 1 ml/min all overthe gradient steps. The analyte separation was carried out usinga wide-pore Symmetry 300

©R C4 protein analysis column (50× 4.6 mm; particle size 5 µm; pore size 300 A; Waters, MA,USA) operated at 40◦C and equipped by the corresponding guardcolumn (Waters).

The solvent delivery system used was a double-reciprocatingpump (mode 600, Waters, MA, USA) and a UV-detector (model746, Waters, MA, USA), with a wavelength of 280 nm was usedfor analyte detection with the outputs processed and record by acompatible integrator (model 486, Waters, MA, USA). Sampleinjection was made by a loop injector (Rheodyne

©R , Cotati, CA,USA) equipped with a 50 µl loop.

To determine the amount of loaded BSA, 0.1 ml of finalwashed erythrocytes was diluted with 0.1 ml of distilled water tocompletely lyse the cells. Then, the suspension was centrifugedat 10000 g for 20 min and the supernatant was filtered througha 0.45 µm syringe filter (Teknokroma, Spain). Finally, 50 µl ofthe filtrate was injected to the chromatograph.

Loading ParametersTo evaluate the effect of any changes in encapsulation method

variables on the loading efficiency, three indices were definedas loading parameters:

1. Loaded amount, the amount of BSA encapsulated in 0.1 mlof the final packed erythrocytes.

2. Efficiency of entrapment, the percentage ratio of the loadedamount of BSA to the amount added during the entire loadingprocess.

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.

PREPARATION AND VALIDATION OF CARRIER HUMAN ERYTHROCYTES 297

3. Cell recovery, the percentage ratio of the hematocrit valueof the final loaded cells to that of the initial packed cells,measured on equal volumes of two suspensions.

Methodological TestsIncubation of Intact Erythrocytes with Isotonic BSA Solution

To investigate the possible uptake and/or degradation of BSAby intact human erythrocytes, 0.5 ml of washed packed erythro-cytes was incubated at 37◦C with 0.5 ml of BSA solutions inPBS with a concentration of 8 mg/ml (the same as used in load-ing procedure). At 15, 30, and 60 min, equal volumes of thecell suspension were harvested and centrifuged at 1000 g for10 min, and the concentrations of remaining BSA in the super-natant were determined, as described, by direct injection to thechromatograph. In addition, the BSA concentration in the cel-lular fraction also was determined at each time point after lysis,as described above.

Concentration and Volume of Added BSA SolutionThe encapsulation procedure was performed using aqueous

solutions of BSA with concentrations of 1, 2, 4, 6, 8, 10, and 15mg/ml. The loading parameters were determined in each case.

To verify the observed point of lysis and also to optimize thevolume of the protein solution used during the loading proce-dure, the process was performed on four separate erythrocytesamples such that to each of the cell suspensions, 2, 3, 4, and5 successive 250 µl aliquots of BSA solution were added. Af-ter completion of the procedure, the loading parameters weredetermined in each case.

Mechanism of EntrapmentTo investigate the possible mechanism of BSA entrapment by

erythrocytes, an encapsulation procedure was performed and theconcentrations of protein in each of three final washing solutionsas well as in final packed cells were determined by HPLC. Then,the total amount of washed out (unentrapped) protein was cal-culated by considering the total volume of discarded solutions.Also, the total amount of entrapped protein was determined us-ing the loaded amount multiplied by the cell recovery of the

TABLE 1Concentration of BSA determined in supernatant and cell lysate upon incubation of intact erythrocytes with isotonic

BSA solutiona(n = 3)

Measured BSA concentration (mg/ml)

15 min 30 min 60 min

Initial added concentration Lysate Supernatant Lysate Supernatant Lysate Supernatant

8 mg/ml NDb 7.78 (0.20)c ND 7.69 (0.13) ND 7.55 (0.18)

aIncubation temperature: 25◦C.bNot detectable.cMean (SD).

FIG. 1. Effect of concentration of BSA solution on loaded amount and effi-ciency of entrapment (n = 3).

method. Finally, taking the volume fraction of cells in wholesuspension at the point of lysis, the mechanistic behavior oferythrocytes against the protein molecules was exploited.

Process Validation TestsTo validate the encapsulation process, the following three

tests were carried out:

1. Intrasubject variations. Three blood samples were collectedfrom a healthy volunteer and the loading procedure was car-ried out on each sample separately. The loading parametersfor each sample as well as the corresponding coefficients ofvariations (CV %) were determined.

2. Intersubject variations. Blood samples were collected from6 healthy volunteers (3 male and 3 female subjects), and theloading procedure was carried out in each case. The loadingparameters for each of the subjects as well as the correspond-ing coefficients of variations were determined.

3. Recovery. The measured entrapped, unentrapped, and to-tal amount of BSA recovered after completion of the

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.

298 M. HAMIDI ET AL.

FIG. 2. Effect of volume of added BSA solution (8 mg/ml) on loaded amountand efficiency of entrapment (n = 3).

encapsulation procedure were compared with those calcu-lated by considering the total added amount and the volumefraction of cells and supernatant at the point of lysis.

Statistical AnalysisThe experiments were carried out in triplicate (n = 3) and

the differences between the results were judged using T-test orANOVA parametric tests at a significance level of 0.05.

RESULTS AND DISCUSSION

BSA Uptake by ErythrocytesThe results of incubation of intact human erythrocytes with

BSA isotonic solutions are shown in Table 1. As can be seen,no detectable amount of protein was taken up by intact erythro-cytes. Therefore, we considered that the erythrocyte membranehad no significant active role in the encapsulation of BSA inhuman erythrocytes. On the other hand, a small amount of BSAwas degraded by intact erythrocytes. Therefore, caution shouldbe taken when calculating the protein loading or interpreting the

TABLE 2Entrapped and unentrapped amounts of BSA at the end of the encapsulation process (n = 3)

BSA concentration BSA total amountFraction Volume (ml) (µg/ml) (µg)

Total washing solution 30 193.36 (13.47)a 5903.83 (401.46)Final packed cells 0.8227 (0.0142) 2405.8 (11.53) 1979.25 (9.45)

Total amount recovered 7776.58 (392.71)

a Mean (SD).

results of loading experiments with respect to this slight degra-dation, whenever applicable.

Concentration of BSA SolutionThe effect of BSA concentration on the loaded amount and

efficiency of entrapment is shown in Figure 1. According to thesedata, it is clear that the loaded amount of protein is related di-rectly to the concentration of BSA solution used, throughout theconcentration range of 1–8 mg/ml and approaches a plateau be-yond this range. The efficiency of entrapment increases slightlyup to 4 mg/ml, beyond which a declining trend is evident. Whilethe use of concentrations higher than 4 mg/ml resulted in somelower efficiency of entrapment, the concentration of 8 mg/mlwas selected to be used during the process. We chose 8 mg/mlmainly because of the suitable (not optimal) entrapment effi-ciency with optimal absolute amount of loaded protein in unitvolume of packed carrier cells, a parameter that is critical fordose adjustment during in vivo studies on this delivery system.

Volume of Added BSA SolutionThe effect of the volume of protein solution added during

the encapsulation process on the loading parameters is shown inFigure 2. An interesting consistency was found between thesefindings and the macroscopically detected point of lysis, at whicha sudden increase in loaded amount and efficiency of entrapmentoccurs exactly when the transparency of erythrocyte suspensionincreases remarkably. Thus, the macroscopic evidence for thepoint of lysis can be used reliably for the detection of the lysispoint. Furthermore, since both the loaded amount and the effi-ciency of entrapment were shown to be optimum with 1000 µlof protein solution, which is high enough to ensure the achieve-ment of the lysis point, this volume was selected as the optimumvolume to be added during the process.

Mechanism of EntrapmentAs shown in Table 2, for an experimental run, ∼5.8 mg of the

total 8 mg BSA added during the encapsulation procedure wasdiscarded as three washing solutions. At the same time, the totalamount of BSA remaining in the erythrocytes was ∼1.9 mg pertotal packed cells recovered after the loading process. The totalvolume of the reaction mixture was 2.5 ml at the point of reseal-ing. This volume consist of 1.2 ml for swollen cells, 0.2 ml for

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.

PREPARATION AND VALIDATION OF CARRIER HUMAN ERYTHROCYTES 299

TABLE 3Recovery of BSA as entrapped, unentrapped, and total protein after the encapsulation process in human erythrocytes (n = 3)

Fraction Expected amount (µg) Measured amount (µg) Recoverya

Entrapped fraction 2624.00 1979.25 (9.45)b 75.18% (0.36)Unentrapped fraction 5376.00 5803.83 (401.46) 107.95% (7.46)

Total 8000 7776.58 (392.71) 97.20% (4.90)

aRatio of measured amount to expected amount of BSA in each fraction at erythrocyte lysis point (see text for details).b Mean (SD).

TABLE 4Loading parameters of encapsulation method of BSA in human

intact erythrocytes (n = 3)

Parameter Mean SD

Loaded amount (µg) 1979.25 9.45Entrapment efficiency (%) 30.06 0.20Cell recovery (%) 87.53 0.66

TABLE 5Intrasubject variations of loading parameters of BSA in human

intact erythrocytes (n = 3)

Loaded amount Efficiency of entrapmentSample No. (µg) (%)

1 237.58 29.692 229.23 28.653 229.11 28.63Mean 231.97 28.99SD 4.85 0.61CV% 2.09 2.10

TABLE 6Intersubject variations of loading parameters of BSA in intact human erythrocytes of three male and female volunteers

Male Female

Sample No. Loaded amount (µg) Efficiency of entrapment (%) Loaded Amount (µg) Efficiency of entrapment (%)

1 231.97 28.99 210.97 26.372 245.42 30.67 222.48 27.813 240.15 30.01 229.23 28.65Mean 239.18 29.89 220.90 27.61SD 6.78 0.85 9.23 1.15CV% 2.83 2.84 4.17 4.16

hemolysate, 1 ml for total protein solution added in four steps,and 0.1 ml for hypertonic resealing solution. From this volume,0.82 ml belongs to the carrier cells. Accordingly, we expectedthat if the distribution of protein between the intracellular andextracellular fractions would be governed only by a simple con-centration gradient-based diffusions, from the total amount of 8mg of added protein during the process, about 2.62 mg would beentrapped in the erythrocytes, and the rest (i.e., 5.38 mg) wouldbe discarded as unentrapped protein.

In fact, as shown in Table 3, we can say that the partitioning ofprotein at the lysis point, mainly (∼75%) depends on the volumefraction of cells in the suspension, considering the fact that themain part (i.e., more than 65%) of the 25% difference betweenthe expected and measured protein mass inside erythrocytes,has been found as extra protein mass in washing solution. Weconcluded that there has been a limiting factor for completeprotein entry to erythrocyte in samples at lysis point (e.g., largesize, high polarity, limited diffusion time, and adsorption of BSAto erythrocyte surface). However, the results of the incubationtest of intact erythrocytes with BSA showed that the erythrocytemembrane had no significant active role in the uptake of BSA,the protein only passed via the pores made in the membrane,upon hemolysis, inward and outward the erythrocyte.

Loading ParametersThe average loading parameters of BSA in human erythro-

cytes are shown in Table 4. The loaded amount of BSA is

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.

300 M. HAMIDI ET AL.

comparable to those values reported in the literature for a vari-ety of proteins (Gutierrez Millan et al. 2004). This can ensuresufficient entry of BSA into the body on reinjection of fairlylow volumes of the packed cells. A cell recovery of ∼87.53%,being practically appreciable, is comparable to recovery resultsof other drugs and proteins reported by others (Magnani et al.2002; Rossi et al. 2005).

Process Validation TestsTable 5 shows that the intrasubject variations of the loading

procedure are fairly low. Also, as shown in Table 6, the intersub-ject variations of the loading procedure are low too, even whensamples taken from subjects of different sexes were included inthe study. Considering the biological nature of the carriers usedin this study, these markedly low variations of loading param-eters are highly interesting and promising with respect to thepopular use of this carrier system.

The recovery of the encapsulation process is shown in Ta-ble 3 with respect to entrapped, unentrapped, and total protein.These data may provide a reasonable basis for investigation ofthe mechanism of entrapment, as discussed earlier, and indi-cate a remarkable degree of accuracy and validity for the wholeprocess, particularly considering the high total protein recovery(i.e., 97.2%).

CONCLUSIONThe hypotonic preswelling method for encapsulation of

drugs/proteins in human erythrocytes was evaluated using BSAas a model antigen. The effect of different variables such asvolume, concentration, and method of addition of protein solu-tion were studied. A knowledge of the optimal conditions of theloading process to yield the best results allows the design of asuitable encapsulation procedure for a particular antigen and/orprotein with some minor modification, if necessary, and thisstudy can serve as a model for such evaluations. In addition, themechanism of entrpment of BSA in intact human erythrocyteswas exploited and we showed that the probable mechanism isthe entry of protein into the cells via the pores made on the cellmembrane at the point of hemolysis. Then, on closure of poresby hypertonic resealing, the protein is trapped in the cells.

Development of this delivery system for BSA may not onlybe of remarkable importance in vaccine delivery, but also pro-vide some valuable information on the possibility of controlledrelease of proteins using this delivery system. Finally, validationtests on the loading method indicated a remarkable degree of ac-curacy, precision, and reproducibility for the loading method inpreparation of carrier erythrocytes for antigen delivery studiesusing BSA as a model.

REFERENCESAl-Achi, A., and Boroujerdi, M. 1990. Pharmacokinetics and tissue uptake of

doxorubicin associated with erythrocyte-membrane: Erythrocytes-ghosts vs.erythrocytes-vesicles. Drug Devel. Ind. Pharm. 16:2199–2219.

Babiuk, S., Baca-Estrada, M., Babuik, L. A., Ewen, C., and Foldvari, M. 2000.Cutaneous vaccination: the skin as an immunologically active tissue and thechallenge of antigen delivery. J. Control. Rel. 66:199–214.

Banker, G. S., and Rhodes, C. T. 2002. Modern Pharmaceutics. New York:Marcel Dekker Inc.

Coombes, A. G. A., Lavelle, E. C., Jenkins, P. G., and Davis, S. S. 1996. Singledose, polymeric, microparticle-based vaccines: the influence of formulationconditions on the magnitude and duration of the immune response to a proteinantigen. Vaccine 14:1429–1438.

Giudice, G. D., Podda, A., and Rappuoli, R. 2002. What are the limits of adju-vanticity? Vaccine 20:S38–S41.

Gupta, R. K., and Siber, G. R. 1995. Adjuvants for human vaccines-currentstatus, problems and future prospects. Vaccine 13:1263–1276.

Gutierrez Millan, C., Zarzuelo Castaneda, A., Sayalero Marinero, M. L., andLanao, J. M. 2004a. Factors associated with the performance of carrier ery-throcytes obtained by hypotonic dialysis. Blood Cells Mol. Dis. 33:132–140.

Gutierrez Millan, C., Sayalero Marinero, M. L., Zarzuelo Castaneda, A., andLanao, J. M. 2004b. Drug, enzyme and peptide delivery using erythrocytes ascarriers. J. Control. Rel. 95:27–49.

Hamidi, M., and Tajerzadeh, H. 2003. Carrier erythrocytes: an overview. DrugDel. 10:9–20.

Highley, M. S., and De Bruijn, E. A. 1996. Erythrocytes and the transport ofdrugs and endogenous compounds. Pharm. Res. 13:186–195.

Hoshi, S., Saito, N., Kusanagi, K., Ihara, T., and Ueda, S. 1998. Adjuvant effectsof fluoride on oral immunization of chickens. Vet. Immunol. Immunopathol.63:253–263.

Jain, S., Jain, S. K., and Dixit, V. K. 1997. Magnetically guided rat erythrocytesbearing isoniazid: preparation, characterization, and evaluation. Drug Devel.Ind. Pharm. 23:999–1006.

Jaitely, V., Kanaujia, P., Venkatesan, N., Jain, S., and Vyas, S. P. 1996. Resealederythrocytes: drug carrier potentials and biomedical applications. Ind. Drugs.33:589–549.

Magnani, M., Rossi, L., Fraternale, A., Bianchi, M., Antonelli, A., Crinelli, R.,and Chiarantini, L. 2002. Erythrocyte-mediated delivery of drugs, peptidesand modified oligonucleotides. Gene Ther. 9:749–751.

O’Hagan, D. T., Singh, M., and Gupta, R. K. 1998. Poly(lactide-co-glycolide)microparticles for the development of single-dose controlled-release vaccines.Adv. Drug Deliv. Rev. 32:225–246.

Powell, M. F. 1996. Drug delivery issues in vaccine development. Pharm. Res.13:1777–1785.

Ramaldes, G. A., Deverre, J.-R., Grognet, J.-M., Puisieux, F., and Fattal, E. 1996.Use of an enzyme immunoassay for the evaluation of entrapment efficiencyand in vitro stability in intestinal fluids of liposomal bovine serum albumin.Int. J. Pharm. 143:1–11.

Rossi, L., Brandi, G., Schiavano, G. F., Balestra, E., Millo, E., Scarfi, S., Da-monte, G., Gasparini, A., Magnani, M., Perno, C. F., Benatti, V., and De Flora,A. 1998. Macrophage protection against human immunodeficiency virus orherpes simplex virus by red blood cell-mediated delivery of a heterodin-ucleotide of azidothymidine and acyclovir. AIDS Res. Hum. Retroviruses.14:435–444.

Rossi, L., Serafini, S., Pierige, F., Antonelli, A., Cerasi, A., Fraternale, A.,Chiarantini, L., and Magnani, M. 2005. Erythrocyte-based drug delivery. Ex-pert Opin. Drug Del. 2:311–322.

Sprott, G. D., Tolson, D. L., and Patel, G. B. 1997. Archaeosomes as novelantigen delivery systems. FEMS Microbiol. Lett. 154:17–22.

Storni, T., Kundig, T. M., Senti, G., and Johansen, P. 2005. Immunity in re-sponse to particulate antigen-delivery systems. Adv. Drug Del. Rev. 57:333–355.

Summers, M. P. 1983. Recent advances in drug delivery. Pharm. J. 230:643–645.Tajerzadeh, H., and Hamidi, M. 2000. Evaluation of hypotonic preswelling

method for encapsulation of enalaprilat in intact human erythrocytes. DrugDev. Ind. Pharm. 26:1247–1257.

Talwar, N., and Jain, N. K. 1992. Erythrocytes as carrier of primaquin prepara-tion: characterization and evaluation. J. Control. Rel. 20:133–142.

Zhao, Z., and Leong, K. W. 1996. Controlled delivery of antigens and adjuvantsin vaccine development. J. Pharm. Sci. 85:1261–1270.

Zhou, S., Liao, X., Li, X., Deng, X., and Li, H. 2003. Poly-D,L-lactide-co-poly(ethylene glycol) microspheres as potential vaccine delivery systems.J. Control. Rel. 86:195–205.

Dru

g D

eliv

ery

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

CD

L-U

C S

anta

Cru

z on

10/

26/1

4Fo

r pe

rson

al u

se o

nly.