Biodegradable Microspheres V: Stimulation of Macrophages with Microparticles Made of Various Polysaccharides

Biodegradable Microspheres V: Stimulation of Macrophages with Microparticles Made of Various Polysaccharides PERARTURSSON'~', ERIKARRO*, PETEREDMANS, JANL. E. ERICSSON*, AND INGVAR

~OHOLM~

Received June 2, 1986, from the 'Department of Pharmaceutical 6iochemistry, University of Uppsala, U psala, Sweden, the *Department of

Pathology, University of Uppsala, Sweden, and the gNationalBoard of Health and Welfare, Division of Bharmacy, Department of Drugs, Uppsala, Accepted for publication December 29, 1986. Sweden.

treated male NMRI-mice (A-lab, Stockholm, Sweden) that were 8-12 Abatmct 0 The interaction between four different microparticulate drug weeks old. The peritoneal lavage generally yielded 1-2 x lo6 carriers and macrophages was investigated in vitro. The microparticles, consistlng of crosslinked starch (1 ,Gu-oglucan with 1,6-~-branches), macrophages from each animal. All cell cultures were maintained in dextran (1,6-cr-o-glucan with 1,%@branches),lichenan (1,J-p-oglu- F-10 medium (Gibco Biocult Ltd, Paisley, Scotland), supplemented can), or mannan (1,6-&-mannan with 1,2-a- and 1,Swbranches), with 20% heat-inactivated, sterile-filtered newborn calf serum,benzyl penicillin (100 U/mL), and streptomycin (10 pg/mL). The medium were investigated for their macrophage stimulatoty properties. Macrowas changed every second day. Elicited (inflammatory) macrophages phage stimulation was assayed by the uptake of ['4C]glucosamineand were harvested from mice that had been treated with an ip injection stimulatoty indices were calculated. Microparticles made of crosslinked of 1.0 mL of thioglycolate broth (Difco, Detroit, MI) 3 d earlier. This lichenan were most stimulatoty, followed by the biologically inert manprocedure yielded -2 x lo' macrophages. Cultured cells were nan and dextran microparticles. Biodegradable starch microparticles continuously studied in a phase-contrast microscope. In order to were less stirnulatoty to the macrophages than the other microparticles. obtain homogeneous monolayers, the macrophages were allowed to Ail micropartides were phagocytosed to the same extent and stimulated adhere to the plastic petri dishes (diameter 5.5 cm; Nunc, Roskilde, the macrophages to release oxygen radicals. Lichenan, mannan, and Denmark) for only 45 min. This waa followed by eight washes with dextran microparticles induced morphological changes in the macrophosphate buffered saline (PBS) to remove the nonadherent cells. phages when given in nontoxic doses. No morphological changes were Twenty-four hours after seeding, the macrophages were characterobserved when the macrophages were exposed to starch microparticles ized morphologically by their ability to phagocytose latex spheres or soluble polysaccharides. (Coulter Electronics Ltd, UK; mean diameter 1.1 pm). With these criteria, 99% of the resident adherent cells were macrophages. The corresponding value for the thioglycolate-elicited cells was 95%. The monolayers were also characterized by spreading,' protein content: When microparticulate drug carriers such as microand T-cell content. The T-cell content w a estimated ~ with anti T-cell antiserum (Cederlane, Ontario, Canada) and a peroxidase staining spheres, nanoparticles, or liposomes are injected intravemethod.g Spleen lymphocytes were used as positive controls. Mononously, they are rapidly opsonized and taken up from the layers of resident and inflammatory macrophages showed a spreadcirculation by the macrophages of the reticuloendothelial ing of 11 7% (n = 6) and 50 6% (n = 61, respectively, &r 1h at system (RES).Although the clearance of particulate materi37 "C, and contained 106 f 18 pg proteidl0' cells (resident cells) als from the circulation is one of the main functions of the and 123 3 pg protein/106 cells (inflammatory cells). The T-cell macrophages, it is now clear that these cells also have content was CO.11 for resident cells and <0.3% for inflammatory several other host defense functions. Upon stimulation (e.g., cells. The number of macrophages waa estimated for each monolayer by phagocytosis of a particulate stimulus), the macrophages by cell counting according to a previously reported method.10 In can release a variety of inflammatory mediators (e.g., oxygen order to facilitate cell counting, the monolayem always had a density of 3.0-5.0 x 10' cells per dish. radicals, proteolytic enzymes, and arachidonic acid metabolites) and growth factors (e.g., interleukin-I and colonyPolysaccharides and Microparticles-The polysaccharides used were: maltodextrin (soluble hydrolysate from potato starch; 1,4-a-m stimulating factors).1.2 As a consequence of the uptake of particles, the macro- glucan with 1,6-a-branches; MW 5000; a gift from Dr. Lars S v e n w n , Stadex AB, Malmb, Sweden); dextran T 70 (1,6-a-ri-glucan with 1,3phages may also differentiate to immunologically more coma-branches; MW 70 000; obtained from AB Pharmacia, Uppsala, petent cells.3 Such stimulated macrophages are often named Sweden); mannan (1,6-a-~-mannanwith 1,2-a- and 1,3-a-branches; inflammatory macrophages due to their presence at sites of prepared from Saccharomyces ceruisiu; obtained from Sigma), and inflammation. Thus, in addition to selective drug delivery, lichenan (1,3- D-glucan; prepared from Cetmria islnndica; obtained the targeting of microparticulate drug carriers to the RES from Signal. &-Labeled microspheres were prepared with acryloymay also result in stimulation of the macrophages and, lated [' Clstarch (MW 7000-8000; specific radioactivity 1.9 mCi/mg; subsequently, enhanced host defense functions of the imobtained from New England Nuclear, Boston, MA) as part of the monomers. ['HIDextran (MW 70 000; specific radioactivity 0.8 mune system. mCi/mg) was a gift from Dr. Anders Uhlin, Pharmacia AB, Uppsala, In this paper, the stirnulatory properties of four different Sweden. Since lichenan is insoluble in water in its native form, this microparticulate drug carriers on macrophages were studied. glucan waa modified according to DiLuzio et al." After acid hydrolyThe microparticles were prepared from structurally related sis, purification, and acryloylation, the high molecular weight fracpolysaccharides. One of these was lichenan (1,3-/3-~-glucan), tion was iaolated using a Sephadex G-25 column, lyophilized, and a polysaccharide known to stimulate macrophages in vitro4 used for microsphere preparation. The yield of the macromolecular ~ . ~other microparticles tested were prepared and in v i v ~ .The product was -18%. Zymosan particles were obtained from Sigma. from biodegradable starch, nondegradable dextran, and manThe acryloyl groups, determined by an NMR method,la did not nan. exceed 5% of the total dry weight (Table I). The polysaccharide microspheres were prepared from acryloylated polysaccharides essentially aa reported earlier.13 The polysaccharides were purified by Experimental Section extraction with toluene and gelfiltration after acryloylation. The lyophilized material was dissolved in 0.2 M phosphate buffer (pH 7.5) CellgResident peritoneal macrophages were obtained from un-

* *

OO22-3549/87/02OO-O 127M 1.0010 1987,American Pharmaceutical Association

0

*

Journal of Pharmaceutical Sciences / 127 Vol. 76,No. 2,February 1987

Table CCharacterlstlcs of the Mlcropartlcles D-T-C

Microparticle Starch (1,6-a-branched 1,4-aglucan)' Dextran (1,&a-branched 1.6a-glucan)' Mannan (1.2-a- and 1,3-abranched 1,6-a-mannan)' Lichenan (1 ,%p-glucan)

Degradation, YO

=

Size distribution, Yod

<0.5 f l

0.5-2.2

p-ll

>2.2

p-ll

15-0.8-0

100

*4

0

89

11

15-0.5-0

15

*6

12

88

0

15-0.8-0 15-0.7-0

17+4 13 ? 2

0 0

93

7

91

9

'Taken from refs 4,22, and 33;see Experimental Section. bThe D-T-C values denote the amounts of the components in the monomer solution used for particle formation: D denotes the concentration of derivatized macromolecules ( i a , polysaccharides;g per 100 mL); T denotes the total concentration of acryloyl groups (g per 100 mL); and C denotes the relative amount of the crossllnker, expressed as the percentage (w/w) of the total amount of acrylic monomers. CMicrosphereswere degraded with amyloglucosidase as described previously (ref 13). dThe size of the autoclaved spheres was determined from photographs taken by scanning electron microscopy.

containing 1 x lo-' M EDTA. After addition of ammonium peroxydisulfate (0.44 M; pH 7.4; 200 pL), the oxygen-free mixture (5 mL) was homogenized with 300 mL of ch1oroform:toluene (1:4) containing poloxamer 188 (Pluronic F68) to produce a water-in-oil emulsion. Upon addition of N,N,N',N'-tetramethylethylenediamine, the water phase droplets polymerized to microspheres. The microsphere composition was characterized by the D-T-C nomenclature as sug gested by Hjert6n1' and modified by Edman et al.16 (Table I). Most (88-93%) of the polysaccharide microspheres had a diameter of 0.52.2 pm. The microspheres and native polysaccharides were suspended, alternatively dissolved in physiological saline in appropriate concentrations, autoclaved, and stored in iqjection vials a t 4 "C until further use. Electron Microscopy--Resident macrophages were grown on rectangular glass pieces (12 x 3 mm) and placed in nunclone plastic petri dishes. The cells were exposed to starch, dextran, mannan, or lichenan microspheres (1.0 mg/mL) for 2 h (see below) and kept in culture for 5 d before harvest. Unstimulated resident macrophages were used as controls. Preparation for scanning electron microscopy was carried out as follows. The cells were fixed in 2% purified glutaraldehyde in 0.1 M Na cacody1ate:HCl buffer with 0.1 M sucrose (pH 7.2) for 48 h. Following washing in buffer:sucrose solution, the cells were postfixed in 1%OsO, in 0.15 M cacodylate buffer (pH 7.2) for 90 min a t room temperature. Dehydration in acetone and critical-point drying was carried out as described previously.ls A Polaron E 5100 cool, triode sputter apparatus was used to coat the specimens with 5 nm of go1d:palladium (80:ZO). The cells on the glass were examined and photographed in a Jeol 100-C transmission electron microscope equipped with a side entry goniometer and a scanning attachment. The microscope was operated a t 40 kV with a beam current of 100

4.

Exposure of Macrophageti to Microparticleti-Twenty-four hours after seeding, the monolayers were exposed to the microparticles (100pL; suspension in PBS) or soluble polysaccharides (100 pL; in PBS) for 2 h a t 37 "C. The final concentrations of the particles were 0.1, 1.0, or 10.0 mg/mL (for details, see individual experiments in the Results section). To remove noningested and unbound microspheres, all monolayers were rinsed eight times with phosphate buffered saline. No or few microspheres were visible on the macrophage surface after the washings. Toxicity of Microparticlea-The toxicity of the microparticles was followed by counting the number of spread adherent cells during a period of 5 d.lo Briefly, the mean number of cells was estimated in 10 different parts of the dishes every second day (before the medium was changed). The cells were counted a t the center of 10 circles drawn in the bottom of each dish, using an inverted phase-contrast microscope and a 32 x objective lens. Phagocytosis of Microparticlea-Microparticles labeled with L"C1starch (specific activity 1.2-2.0 x 10' d p d m g dry weight) were used in the phagocytosis experiments. After incubation and counting, the rinsed monolayers were digested overnight in 1.0 mL of 0.5 M NaOH and aliquots were withdrawn for determination of radioactivity in a scintillation counter.17 Nonspecific binding was found to be 10-15% when the experiments were repeated in Hank's Balanced Salt Solution. ~'4ClClucosamineUptak4"CI-Labeled glucosamine (0.5 pCi; 128 /Journal of Pharmaceutical Sciences Vol. 76,No. 2, February 1987

specific radioactivity 54.2 mCi/mmol; New England Nuclear, Boston, MA) was added to the monolayers 3 d after stimulation with the polysaccherides.l8 At day 5 (i.e., 48 h after [14Clglucosamineexposure), the monolayers were counted, washed eight times with PBS containing 0.05 M unlabeled glucosamine (to remove free ["Clglucosamine), and their radioactivity content was determined. The glucosamine uptake was linear over a wide range of macrophage densities. For a density range of 1.5 x 10' to 1.7 x lo6 cells/well, linear regression analysis showed a correlation coefficient of 0.97 for six experiments performed with mannan-stimulated macrophages over a period of several months. The experiments were usually performed in conventional culture medium containing 0.1-0.5 ng of endotoxin per milliliter. The endotoxin was determined with a chromogenic method.19 The autoclaved microsphere suspensions contained c0.5 ng endotoxinlml. Control experiments were performed in endotoxin-free medium. The culture medium and the microspheres were supplemented with polymyxin B sulfate (10 pg/mL; Sigma). After incubation at 37 "C for 30 min, samples were withdrawn for analysis of endotoxin content. The polymyxin B sulfate-supplemented solutions and particle suspensions contained ~ 0 . ng 1 of endotoxin and were not toxic to the macrophages a s judged by the number of well spread cells. Hydrogen Peroxide Assay-The HzOzrelease from microparticle-stimulated macrophages was estimated according to Ruch et al.', Briefly, resident macrophages were exposed to 1mg/mL of microparticles for 2 h in the presence of homovanillinic acid (100 p M ;Sigma) and horseradish peroxidase (1 U/mL; Sigma). When HzOz was released from the macrophages, homovallinic acid was oxidized to a highly fluorescent dimer (2,2'-dihydroxy-3,3'-dimethoxydiphenyl5,5'diacetic acid) in the presence of peroxidase. The fluorescence intensity (Aex = 312 nm; Aem = 420 nm) was then utilized to assay the amount of H202 released from the macrophages. In another set of experiments, the microparticle-stimulated macrophages were cultured for 24 or 96 h before the release of HzOz was estimated. At these time points, the cells were exposed to zymosan particles (1 mg/mL; 2 h) and the release of hydrogen peroxide was assayed as described above. Statistic-The t test for the comparison of two independent means was used to analyze the results. A difference was considered as significant if p < 0.05. All figures are expressed as mean values f SEM (n = 4-6).

ResuI ts Toxicity of Polysaccharides-The macrophage toxicity o f the microparticles was investigated by exposing the macrophages to increasing doses of the microparticles. The number o f adherent cells was estimated immediately after, and 1,3, and 5 d following exposure to 0.1 and 1.0 mg/mL of microspheres. When a microsphere dose o f 10.0 m g / m L was used, the adherence was estimated immediately after and 5 d after microsphere exposure. The viability of the macrophages was not affected when the cells were exposed to 0.1 or 1.0 m g / m L of microspheres (Figure 1). A l l microspheres except those made from biodegradable starch were toxic at the highest

80

-3 60 In-

d

W

0m' 40

5

20

0

0.1

10.0

1.0 dose, mg

flgum 1- Viability of macrophages 5 d after stimulation with different doses of microparticles. The viability was expressed as the percentage of remaining adherent cells after 5 d of cultivation (n = 4; mean values 2 SEM). Key: lichenan (0);mannan (m); dextran (0);starch (0).

dose level (10 mg/mL). When the corresponding soluble polysaccharides were studied in the same way as the microspheres, no effects on the monolayers were observed even at the highest concentration, indicating that these polysaccharides were nontoxic (data not shown). Uptake of Microspheres and Soluble PolysaccharidesThe uptake of different microparticles (1.0 mg/mL) was studied with ['4C]-labeled microspheres (see Table 11).Resident macrophages showed a lower uptake of the microspheres (2.5-4.4 %/lo6 cells) than the inflammatory macrophages (8.3-15.6 pg/106 cells). Thus, the microparticle uptake by inflammatory macrophages was 1.9-4.8 times more efficient than that by resident macrophages. In control experiments, the microspheres were incubated with cell-free medium at 37 "C for 2 h. The biodegradable starch spheres were most affected, and 40 5 3% of the radioactivity was released into the serum-containing medium. The other spheres released -10% of the marker. Since the starch microspheres were degraded in the medium as well as in the macrophages, it may be conceivable that the measured uptake does not reflect the real uptake. However, the microparticles were added to the macrophages in a large excess and, therefore, the degradation by a-amylase in the cell culture medium would not affect the number of microparticles available for each macrophage. Further, most Table blacrophage Uptake of Mlcropartlclecr' Uptake, @/lo6 cellsb Resident Thioglycolate Microparticle 3.0 2 0.2 Starch Dextran 3.2 2 0.4 Mannan 4.4 t 1.1 Lichenan 2.5 2 0.3 Soluble Polysaccharide Starch 0.15 2 0.04' 0.11 ? 0.02 Dextran

14.1 2 15.6 2 8.3 2 11.2?

1.9 1.9 0.9 0.8

Thiogiyco1ate:Resident Ratio

4.7 4.8 1.9 4.5

n.d.d n.d.

'Radiolabeled microparticles were prepared by copolymerization with acryloylated [14C]starch. Uptake was determined as macrophageassociated radioactivityas described in the Expefimental Section. 'The soluble polysaccharides were incubated with the macrophages for 4 h. Not determined.

of the radioactivity remained associated with the macrophages during the experiment (for >2 h), indicating that the cellular metabolism did not affect the results (unpublished results). Thus, the measured uptake of the starch spheres should well reflect the true uptake. The uptake of soluble polysaccharides (1.0 mg/mL) was much lower than that of the particles (see Table 11). If the uptake of the soluble [3H]dextran is expressed in terms of fluid-phase pinocytosis (i.e., 88 the endocytotic index according to Duncan et a1.21),the rate of uptake by the macrophages was 0.03 pL/106 cells per hour. This value corresponds well with the endocytotic indices for poly(vinylpyrro1idine) which were between 0.05 and 0.13 & / l o 6 rat peritoneal macrophages per hour, depending on the molecular weight of the macromolecules.21 The corresponding value for soluble ['4Clstarch was 0.04 & / l o 6 cells per hour. Morphology of Macrophages-The ultrastructural appearance of unstimulated resident macrophages is shown in Figure 2. Following exposures to lichenan, mannan, and to some extent dextran microspheres, a considerable enlargement of the cells was observed (Figure 3). By light microscopy, the cells also showed the presence of numerous vacuoles in their cytoplasm. Sometimes the cells tended to round up (Figure 3). Cells exposed to biodegradable starch microspheres did not show these changes and resembled control cells. Following stimulation with native lichenan particles, the cells became enlarged and vacuolated. Exposure to the water-soluble polysaccharides did not cause any appreciable change in the appearance of the cells (data not shown). Stimulation of the M a c r o p h a g e d i n c e some microparticle-stimulated macrophages showed morphological changes as compared with the unstimulated ones, possible alterations of the metabolism of resident and inflammatory macrophages was investigated by means of ['4Clglucosamine incorporation (Figures 4 and 5). Unstimulated, resident macrophages (given 100 pL of PBS for 2 h) showed a low uptake of [14C]glucosamine,ranging from 138 5 20 to 195 k 20 dpm per lo6 cells. When the macrophages were stimulated with native insoluble lichenan, a n 8-fold increase in glucosamine uptake was observed (Figure 4). ARer acid hydrolysis of the native lichenan, the sterile, filtered, soluble polysaccharide still stimulated the macrophages (p < 0.01). When the soluble lichenan was derivatized and polymerized to microparticles, the macrophage-stimulating capacity increased 2fold (p < 0.01) compared with that of soluble lichenan. Thus, the stimulatory structure in lichenan (i.e., the 1,3-P-~-glucan structure) was presented more effectively to the macrophages in particulate than in soluble form. The macrophage stirnulatory properties of the other polysaccharides in soluble and particulate form were also investigated (Figure 5). The particles were administered at a concentration of 1.0 mg/mL, while the covesponding soluble polysaccharides were given in concentrations of 1.0 and 10 mg/mL. All particulate polysaccharides significantly stimulated the macrophages to increased ['4Clglucosamineuptake. The soluble polysaccharides (1.O mg/mL) did not stimulate the macrophages. However, when given in a 10-fold higher concentration, the soluble, high molecular weight fraction of lichenan stimulated the macrophages (p < 0.01). Since both phagocytosis and glucosamine uptake were quantified, a stimulatory index could be calculated from the expression

Stimulatory Index = dDm (slucosamine)/106cells pg phagocytozed microparticles/106 cells

If the mean values from the experiments depicted in Table I1 Journal of Pharmaceutical Sciences / 129 Vol. 76, No. 2, February 1987

Figure 2-Scanning electron micrograph of resident (unstimulated) macrophages after 5 d in culture. Note that the cells are small and flattened (x 1680).

In

Flgure 3-Scanning electron micrograph of a cell stimulated with mannan microspheresafter 5 d i n culture. The cell is considerably larger than unstimulated cells (compare with Figure 2) and tends to partially attain an elongated, cylindrical shape (x 1400).

1.8

d

I

= vl

1.6

9

3

9

:m

W

1.4

9 ;T

1.2

n E

c

E

0, aJ

3

= I

aJ .C

5$

1.4

-

1.2

-

1.0

Q

1.0

Y ID

z

1.6 7.8

g

0.8

.f

0.6

g

0.4

gu

0.2

1

0.8

0.6

T

F

0.4

d

0

*" 0.2

PBS

r

PBS

N

S

P

Figure 4-Stimulation of resident macrophages with a native ( N ) , soluble (S), and particulate ( P ) 7,3-p-D-glucan (lichenan). Control macrophages were given physiological saline (PBS; n = 4; mean values SEM).

*

and Figure 5 are used, the following stimulatory indices are obtained: 108 dpmlpg for starch spheres; 140 dpmlpg for dextran spheres; 182 dpmlgg for mannan spheres; and 376 dpmlpg for lichenan spheres. From these results it can be concluded that the lichenan microparticles containing 1,s-p glucosidic bonds are more stimulatory than the other spheres. The amount of radioactivity taken up by inflammatory macrophages ranged from 920 f 136 to 1088 2 142 d p d l 0 ' cells for control cells (see Figure 6). No significant increase in ['4C]glucosamine uptake could be detected when the inflammatory cells were stimulated with the microparticles. Thus, these cells were obviously prestimulated and could not be 130 / Journal of Pharmaceutical Sciences Vol. 76, No. 2, February 1987

MD

D

M

L

TG

Figure 5-Stimulation of resident macrophages with starch (MD), dextran (D), mannan (M), and lichenan (L) in soluble (0)and microparticulate (0)form. Control macrophages were given phosphate buffered saline (PBS). Prestimulated inflammatory macrophages (TG) were included as a positive control (n = 4; mean values SEM).

*

further stimulated by the microparticles as detected with the [14C]glucosamineassay. It has been shown that some macrophage-activating agents require endotoxin in order to become stimulatory (e.g., ?interferon), while others can activate macrophages independently of endotoxin (e.g., a-and pinterferon).MSince the endotoxin requirement has not been evaluated for glucans, some experiments were performed under endotoxinfree conditions (see Table 111). No significant differences between endotoxin-positive and endotoxin-negative samples were observed. Thus, the presence of endotoxins can not account for the increased ['4Clglucosamine uptake in the stimulated macrophages. Effect of Soluble Polysaccharides on Uptake and Stimulation of Particulate Polysaccharides-The soluble polysac-

Tabk Ill-kcrophage Stlmulatlon In the Presence and Ab8ence of Endotoxln

Microparticle

-

Mannan Mannan Lichenan Uchenan

dpmll0'

Endotoxin

Stimulus

H202Release, nmoi/lO' Cells

141 2 5 192k 28 86of 166 977 k 80 866 2 115 882? 62

+ + +

Starch Dextran Mannan Lichenan Zymosan

-

0.0 f 0.0 5.3 k 0.6 7.1 f 0.2 8.7 2 0.1 7.2 f 0.4

Polymyxin

d m L

C811S'

10.0

-

10.0

-

10.0

Table V 4 0 Releare by Macrophager During Phagocytorlr of Mlcropartlc?ee?

-

'p > 0.05 for all pairs. bAn endotoxin content of < 0.1 ng/mL was

9.3 f 0.6

'One milligram of microparticles or zymosan per mL were incubated = 4).

with resident macrophages for 2 h (n

considered as negative (ref. 19).

charides were added at a concentration of 1-10 mg/mL 15 min before addition of the microspheres. A significant inhibition on the uptake of mannan microspheres was obtained with soluble mannan (1.0 mg/mL), indicating that these spheres were partly phagocytosed through the mannose/fucose receptor (Table IV, experiment no. llg4 No inhibition of the uptake of dextran and lichenan microspheres was observed with soluble mannan (data not shown). Soluble dextran (10 mg/mL) or lichenan (10 mg/mL) did not inhibit the uptake of the microspheres (data not shown). Thus, specific receptors for dextran and lichenan do not contribute significantly to the uptake of dextran and lichenan particles, respectively. This does not mean that such receptors are absent from the macrophage surface. For instance, a receptor for 1,3-/3-~glucanhas recently been identified on human monocytes.2" Other receptors (e.g., receptors for various serum opsonins) may be quantitatively more important for the uptake of the polysaccharide microparticles.26 The same inhibition experiments were performed with unlabeled microspheres and the uptake of ["C]glucosamine was measured. It was shown that inhibition with soluble mannan of the mannose/fucose receptor was followed by a decrease in ['4C]glucosamine uptake (see Table IV, experiment no. 2). Release of H302from the M a c r o p h a g e d i n c e the release of inflammatory mediators may be a potential side effect of the targeting of microparticulate drug camera to macrophages, the release of one such mediator, HzOz, from microparticle-stimulated macrophages was investigated (Table V).All microparticles, independent of structure, induced a Significant release of HzOz from the macrophages ranging from 5.3 2 0.6 nmoYl0' cells for starch microparticlea to 8.7 2 0.1 nmoY106 cells for mannan microparticles. Zymosm particles, previously shown to stimulate macrophages,20 were used as positive controls. In separate experiments, macrophages stimulated by different microparticles were maintained in culture for 24 or 96 h. A t these time points, the cells were restimulated with release was estimated (Table zymosan particles and the Hz02 VI). Twenty-four hours after microparticle stimulation, the control macrophages responded to the zymosan particles with

Table Vl-Zymoran-induced Stimulated Macrophager'

Release of H201 by MlcroparticleH202Release, nmol/lOecellsb

Stimulus

Starch Dextran Mannan Lichenan Zymosan

24 h

96 h

12.1 f 1.8 9.1 f 1.3 11.O f 0.5 1.7 k 0.4 4.9 f 1.3 0.5 f 0.8

5.7 f 0.9 5.9

f 2.2

6.6 t 0.7 0.0

f 0.0

0.0

f

0.9 f 0.6 0.0

'Macrophages were stimulated as in Table V; 24 or 96 h after the stimulation, 1 .O mg/mL of zymosan particles was added to the macrophages for 2 h and the H202 release was measured (n = 4). bThe spontaneous reiease of H202 from nonstimulated and microparticlestimulated macrophages varied between 0.0 2 0.0 and 0.3 f 0.6 nmolll oBcells. a release of 12.1 2 1.8 nmol of H202/106cells. Macrophages previously stimulated with starch or dextran microparticles released similar quantities of HzO2, while the mannan-, lichenan- and zymosan-stimulated macrophages showed a decreased capacity to release H202. Ninety-six hours after microparticle stimulation, all macrophages showed a decreased response to the zymosan particles. Again, the macrophages stimulated with starch and dextran microparticles released approximately the same amounts of H202 as the control macrophages, while the mannan-, lichenan-, and zymosan-stimulated macrophages had lost their capacity to release detectable amounts of H202. Similar results have been obtained by Murray, who studied the effects of phorbolmyristate-acetate and zymosan on the release of oxygen radicals from macrophages.27

Discussion In this paper, the macrophage stirnulatory properties of a microparticulate drug carrier system based on starch was studied. Microparticles were also prepared from three other polysaccharides in order to study some structural require-

Table IV-Effect of Soluble Polyraccherlder on Phagocytorlr o?Mannan Mlcrorphems and Uptake of ['4C]Glucoeamlne

Experiment 1

2

Soluble Polysaccharide

Dextran Mannan Lichenan

-

Dextran Mannan Lichenan

Uptake of Microspheres, w/1Os cells 3.7 f 0.3 3.6 ? 0.4 2.3 f 0.2 3.6 k 0.4

Uptake of ['4C]glucosamine, dpm/lO' cells

663 k 61 635 f 77 421 f 46

Yo of Total

Uptake

P

100 97 61 96 100 96

<0.05

64

ns.'

n.s: ns.'

<0.01

n.d.

Not significant. Journal of Pharmaceutical Sciences / 131 Vol. 76, No. 2,February 7987

ments for macrophage stimulation. Lichenan, a 1,3-pglucan, was used since polysaccharides with this structure are known to stimulate macrophages.28Mannan, a 1,6-a-mannan, was used since macrophages express a specific receptor for the monomer of this polysaccharide (i.e., mannosez4). Dextran (1,Ba-glucan) was used since it is biologically inert, biocompatible, and has a structure similar to that of starch. All the polysaccharide microspheres used were stimulatory to macrophages as judged by morphology, macrophage adherence to the monolayers, HzOzrelease, and [14Clglucosamine uptake. However, although the uptake of particles was about the same, their effects on the cells were quantitatively different. The following conclusions may be drawn. First, the spheres containing a-glucosidic bonds were less stimulatory than those with /3-glucosidicbonds. Thus, lichenan-stimulated macrophages enhanced the uptake of [14Clglucosamineup to 4-fold compared with the other spheres. Second, the biodegradable starch microspheres caused no significant morphological alterations and stimulated the macrophages to a lesser extent than the inert spheres. The low stimulatory effect of the starch spheres may be attributed to their degradation in the vacuolar apparatus. Indeed, it has been shown that these starch spheres are rapidly digested by a purified lysosomal fraction.20 Third, the higher stimulatory effect obtained with the mannan microspheres, as compared with the starch and dextran spheres, was related to an increased uptake of the former through the mannosetfucose receptor. Fourth, all soluble polysaccharides became stimulating when presented to the macrophages as microparticles, whereas the only soluble polysaccharide that stimulated the macrophages was the 1,3-pglucan (lichenan). The inability of the polysaccharides in solution (with the exception of lichenan) to stimulate the macrophages may be due to the low intracellular concentration of these s~bstances.~O Alternatively, the high degree of rigidity of the particulate polysaccharides may be critical for the stimulatory effect on macrophages in vitro.31 The stimulatory effect of lichenan was observed even at the low concentrations obtained with the soluble compound. This was unexpected since soluble glucans are not assumed to have a fixed, but rather a random, coil conformation.22 It is notable that all the microparticles, independent of structure, stimulated the macrophages. This stimulation may be a consequence of the internalization process, which evokes a membrane flow associated with the filling of the vacuolar apparatus of the cell. If this holds true, all types of microparticulate carriers may atimulate the macrophages to some extent. This is supported by the results obtained with the hydrogen peroxide assay. Whether this stimulation will result in a subsequent inflammation may be a question of the properties of the particle surface as well as of the degree of stimulation (i.e., the number of particles presented to the macrophages). However, it should be noted that Seljelid et al.,4 in studies of the effect on macrophages of 42 different glucans in vitro, demonstrated that not all insoluble compounds (e.g., chitin and cellulose) were stimulatory. The morphologic changes in the cells exposed to the inert microparticles were quite striking. Vacuolization, as seen by light microscopy, evidently reflects the filling and expansion of the vacuolar apparatus with microspheres. However, the mechanisms underlying the changes in size and shape are uncertain. It is unlikely that the simple filling of the vacuolar apparatus would cause these changes. The vacuolization can be observed immediately after phagocytosis, while the increase in size is maximal after 5 d in culture. It remains to be shown whether or not other macrophage-stimulating compounds, which are not taken up in bulk or do not expand the vacuolar apparatus, also induce morphologic changes. The polyacryl starch microspheres were originally devel132 / Journal of Pharmaceutical Sciences Vol. 76, No. 2, February 1987

oped as a targeting device for delivery of macromolecular drugs to the RES.23 More recently this system has been modified to be suitable for the delivery of low molecular weight drugs as well.32In this paper we show that microparticles can be tailored from any soluble polysaccharide. This increases the number of potential applications of this carrier system. By incorporating polysaccharides with specificity for unique macrophage receptors (e.g., the mannose/fucose receptor), it may be possible to further increase the distribution of these particles to the macrophages. By inclusion of macrophage-stimulating polysaccharides, such as lichenan, in the particle matrix, it may be possible to activate the macrophages to express increased host defense functions. In this case, the carrier may be used alone as an immunostimulating agent or as a n adjuvant in combination with the appropriate entrapped drug. On the other hand (as illustrated by the release of reactive oxygen metabolites), the use of microparticulate carriers for the above mentioned purposes is associated with certain risks for an “overreaction” of the target cells (the macrophages), with side effects, such as inflammation, as a potential result. Whether the in vitro results obtained here can be used to predict the in vivo properties of a certain microparticulate carrier remains to be seen.

References and Notes 1. Cohn, 2. A. J.Immunol. 1978,121,813. 2. Baggiolini, M. In Handbook of Inflammation, vol 5;The Phurmacolo y of Inflammation; Bonta, I. L., Bray, M. A., Parnham, M. J., lfd8.; Elsevier: Amsterdam, 1985;p 123. 3. Keller, R. L mphokines 1981,3,283. 4. Sebelid, R.; h g w a l d , J.; Lundwall, A. Exp. Cell. Res. 1981,131, 121. 5. DiLuzio, N. R. TIPS 1983,4,344. 6. Chikara, G.EOS-Riv. Immunol. Immunofarm. 1984,4,85. 7. Rabinovitch. M.: De Stefano. J. EXD.Cell. Res. 1973. 77.323. 8. Lowry, 0.H.; Rosebrough, N. I.; Parr, A. L.; Randall, R. J. J . Biol. Chem. 1951,193,265. 9. van Nooreden, S.;Polak, J. M. In Immunohistochemisty; Polak, J. M., van Noorden, S., Eds.; John Wright and Sons: Bristol, 1983;p 11. 10. Edman, P.; Sjoholm, I.; Brunk, U. J . Pharm. Sci. 1984,73, 153. 11. DiLuzio, N. R.; Williams, D. L.; McNamee, R. B.;Edwards, B. F.; Kitahama, A. Int. J . Cancer 1979,24,773. 12. Lepisto, M.; Artursson, P.; Edman, P.; Laakso, T.; Sjoholm, I. Anal. Biochem. 1983,133,132. 13. Artursson, P.; Edman, P.; Laakso, T.; Sj(lholm,I. J . Phurm. Sci. 1984,73,1507. 14. Hjertbn, S.Arch. Biochem. Biophys. Suppl. 1 1962,197. 15. Edman, P.; Ekman, B.; S oholm, I. J . P h r r n . Sci. 1980,69,838. 16. Collins, V.P.; Arro, E.; hlomquist, E.; Brunk, U.; Fredriksson, B.-A.;Westermark, B. Scanning Electron Microsc.1979,3,411. 17. Artursson, P.; Laakso, T.; Edman, P. J . Pharm. Sci. 1983,72, 1415. 18. Hammond, M. E.; Dvorak, H. F.J . Exp. Med. 1972,136,1618. 19. Friberger, P.; KnBs, M.; Mellstam, L. In Endotoxins and Their Detection with the Limulus Amebocyte Lysate Test; Watson, S . W., Levin, J., Novitaky, T. J., Eds.; Alan R. Liss: New York, 1982;p 195. 20. Ruch, W.; Cooper, P. H.; Baggiolini, M. J. Immun. Meth. 1983, 63,347. 21. Duncan, R.; Pratten, M. K.; Cable, H. C.; Ringsdorf, H.; Lloyd, J. B. Biochem. J . 1981,196,49. 22. Rees, D.A. Polysaccharide Shupes; John Wiley and Sons: New York, 1977. 23. Artursson,P.; Edman, P.; Sjoholm, I. J . Phurmncol. Exp. Ther. 1984,231,705. 24. Sung, Sun-Sang, J.; Nelson, R. S.; Silverstein, S . C. J . Cell Bwl. 1983,96,160. 25. Czop, J. K.;Auaten, K. F. J.Immunol. 1985,135,3388. 26. Artursson, P.; S’Bholm,I. Int. J . Phurm. 1986,32,165. 27. Murray, H.W. Reticuloendothel. Soc. 1982,31,479. 28. Whistler, R. L.; Bushway, A. A.; Singh, P. P.; Nakahara, V.; Tokuzen, R. Adv. Carbohydr. Chem. 1976,32,235.

3.

29. La&, T.; Arturseon, P.; Sjbholm, I., J. Pharm. Sci. 1986, 75, 962. 30. Johnson, E.; BSgwald, J.; Seljelid, R. Scand. J.Immunol. 1982,

16, 525. 31. Bd ald, J: Gouda, I.; Hoffman, J.; Larm,0.; Larseon, R.; S e g i d , R. &and. J. Immunol. 1984,20,355. 32. Leakso, T.;S 'timkvist, P ; Sjbholm, I. J.Phurm. Sci. 1987, 76. 33. Lee,Y.C.; Ballon, C. E. B'iochemistry, 1965,4, 257. 34. Borachi, D.; Censini, 9.; Tagliabue, A. Eur. J. Zmmunol. 1984, 14, 1061.

dote Added in Proof The effects of the various polysaccharide microspheres on macrophage mediated immune functiom, such as tumor cell cytotoxicity,

release of interleukin-1 and prostaglandin E2 is presented elsewhere: Artursson, P.; Edman, P.;Ericsson, J. L. E. Scand. J.Immunol., in press.

Acknowledgments This work was sup rted by grants from the Swedish Medical Research Council, the YF.Foundation for Pharmaceutical Research, and the Swedish Cancer Society. The authors thank Mrs. Elisabeth dare and Miss Marie Malm en for technical assistance, and Dr. & i a n Eriksson for help with endotoxin analyses.

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Journal of Pharmaceutical Sciences / 133 Vol. 76, No. 2, Februaty 1987