phagocytosis of bioactive microspheres

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1986 1: 32Journal of Bioactive and Compatible PolymersY. Ikada and Y. Tabata

Phagocytosis of Bioactive Microspheres

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Phagocytosis of Bioactive Microspheres

Y. IKADA AND Y. TABATAResearch Center for Medical Polymers and Biomaterials

Kyoto University53 Kawahara-cho, Shogoirc, Sakyo-ku, Kyoto 606, JAPAN

ABSTRACT

The influence of several proteins on the uptake of microspheres was investi-gated using mouse peritoneal macrophages. Thioglycollate-stimulated mac-rophages were cultivated for 3 h with protein-grafted and protein-coated cellu-lose microspheres smaller than 2 μm in the presence and the absence of serum.Bovine serum albumin reduced the phagocytosis of microspheres, while y-globu-lin, human fibronectin, bovine tuftsin, and gelatin enhanced the phagocytosis.This trend was not influenced substantially by the presence of serum and themode of surface binding of the proteins; that is, covalent grafting or physical ad-sorption (coating). However, in the case of gelatin binding, phagocytosis wasgreatly enhanced by the presence of serum as compared with the other proteins.

INTRODUCTION

hen a f oreign material is exposed to a living environment, the liv-W ing system initiates defense reactions against the invading foreignbody. One of the most important cells in the initial non-specific defenseis the phagocytic macrophage. Therefore, it is of prime importance tostudy the behavior of macrophages with foreign materials in order todevelop bioactive and compatible polymers which are truly applicablefor clinical medicine.

Roughly speaking, medical polymers can be divided into two classes.One is the bio-inert polymer which should not be recognized as a foreignbody by the living defense system. The other is the bioactive polymerwhich should positively react with the biological environment. Thisclassification may be more clearly explained by taking a polymeric drugdelivery system as an example. Consider a microsphere-drug composite

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whose size is susceptible to phagocytosis. If the drug becomes effectiveonly after uptake by phagocytic cells, the microsphere composite musthave the surface structure which is readily recognized by the cells as atarget to engulf. However, if the phagocytic digestion of the drug mustbe avoided, then the microsphere should have a surface to which thedefense system is quite indifferent.Until now, most of the work on phagocytosis of foreign microspheres

have been carried out using polystyrene latex beads [1-3]. They are,however, very difficult to modify in order to prepare microspheres hav-ing different surface characteristics starting from the same material. Inthe present work, we have prepared cellulose microspheres with diame-ters smaller than 2 Nm, which is a size very susceptible to macrophagephagocytosis. Cellulose was selected as the starting material becausethis hydrophilic but water-insoluble polymer can be chemically modifiedwith ease. The present report describes the results on phagocytosis bymouse peritoneal macrophages of the cellulose microspheres bound withdifferent proteins.

EXPERIMENTAL

Reagents

Bovine serum albumin (BSA) and bovine IgG (Cohn fraction II) wereobtained from Sigma Chemical Co. (St. Louis, MO). Tuftsin was ob-tained from Cambridge Research Biochemicals, Ltd. (Harston, England).Gelatin was kindly supplied by Nitta Gelatine Co., Ltd., Japan. Humanplasma fibronectin (FN) was isolated in our laboratory from frozenhuman plasma by affinity chromatography with a gelatin-Sepharosecolumn [4]. Cyanogen bromide (CNBr) and n-hexylamine were purchasedfrom Nakarai Chemicals Ltd., Japan. Poly(vinyl alcohol) (PVA) andtriacetyl cellulose were kindly supplied by Unichika Kasei Ltd., andDaicel Chemical Industries, Ltd., Japan, respectively and used withoutfurther purification.

Media

Culture medium (EMEM-FCS) was prepared by supplementing EagleMEM MEDIUM (Nissui Seiyaku Co., Ltd.) with 10% fetal calf serum(FCS, M.A. Bioproducts, Walkersville, MD), 2 mM L-glutamine, and18 mM NaHC03 at pH 7.4. Calcium and magnesium-free Hanks’ bal-anced salt solution (HBSS) and PBS were obtained from Nissui SeiyakuCo., Ltd.



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Preparation of Cellulose Microspheres

Microspheres were prepared by the solvent evaporation process at37 °C [5]. The standard procedure used was as follows; 200 ml of 2 wt %aqueous solution of PVA was prepared in a 300 ml glass beaker, a solu-tion, consisting of 20 ml methylene chloride and 400 mg triacetyl cellu-lose, was poured rapidly into the PVA solution and the mixture wasemulsified by sonication at 64 W for 2 min. The resulting emulsion wasagitated continuously at 37 °C for a predetermined period of time toevaporate methylene chloride. When the methylene chloride evapora-tion seemed completed, the emulsion was centrifuged at 8,000 rpm for20 min and the supernatant discarded. The triacetyl cellulose micro-spheres were washed four times with distilled water. Between washingthe microspheres were centrifuged at 8,000 rpm for 20 min, the superna-tant discarded, and the pellet resuspended. Pure cellulose microsphereswere regenerated by the following alkaline saponification of the triacetylcellulose microspheres prepared as described above. The suspension of1.4 g of triacetyl cellulose microspheres in 100 ml methanol was addedto a 200 ml flat-bottomed flask with a stopper. The suspension wasstirred at 40 °C for 30 min. Then, 4 ml of 5 N NaOH aqueous solutionwas added and the suspension was stirred for 5 h. The regenerated cellu-lose microspheres were centrifuged at 8,000 rpm for 20 min and thesupernatant discarded. Microspheres were then washed four times withdistilled water by centrifugation. The washed microspheres were dis-persed in distilled water and stored at 4 °C until use. The hydrolysis oftriacetyl cellulose to cellulose was confirmed by the swelling behavior ofmicrospheres with an alkaline aqueous solution.The microspheres are spherical with an average diameter less than 2

~m, when observed by scanning electron microscopy. The scanning elec-tron microscope used was a Hitachi Model S-450.

Grafting of Proteins onto Cellulose Microspheres

The preparation scheme of protein-grafted cellulose microspheres isshown in Figure 1. The proteins were coupled to cellulose microsphereswith the CNBr activation method by Cuatrecasas, et al. [6]. 20 mg ofcellulose microspheres was placed in a glass tube containing 16 ml of0.2 N sodium carbonate, cooled to 5 °C, and then 0.4 g of CNBr in 0.2 mlof acetonitrile was added all at once under gentle stirring. The reactionwas allowed to proceed for 12 min. After that, the microspheres werecentrifuged at 8,000 rpm for 5 min and the supernatant discarded.Microspheres were then washed three times with 0.1 N sodium bicar-



35

1111 L. r-u~fJllen:~

Figure 1. Preparation scheme for the protein-grafted cellulose microspheres.

bonate of pH 9.5. Between washing, microspheres were centrifuged at8,000 rpm for 5 min, the supernatant discarded. 20 mg of the cellulosemicrospheres activated by CNBr was suspended in 10 ml of 0.1 Nsodium bicarbonate aqueous solution (pH 9.5) containing 10 mg of a pro-tein. The coupling reaction was carried out at room temperature for 5 h.Then, the protein-grafted cellulose microspheres were centrifuged at8,000 rpm for 5 min. The microspheres were washed successively with0.1 N sodium acetate and distilled water by centrifugation. The SEMphotographs of the resulting microspheres are shown in Figure 2. As isseen, the morphology of microspheres is not changed during the cou-pling reaction.

Cellulose Microspheres Coated with Adsorbed Proteins

To enhance protein coating through adsorption, the hydrophilic cellu-lose surface was converted to a hydrophobic one by allowing n-hexylamineto link to the cellulose microspheres using the CNBr activation method.The procedure for activation by CNBr is the same as described above.The microspheres activated with CNBr were resuspended in 20 ml of25% N,N-dimethylformamide/0.1 N sodium bicarbonate (pH 9.5) con-taining 76.4 mg of n-hexylamine. The coupling reaction was carried outat room temperature for 10 h and then the microspheres were washedsuccessively with 0.1 N sodium acetate and distilled water by centrifu-gation. The resulting modified cellulose microspheres (Cell-C6) were dis-persed in distilled water and stored at 4 °C until use. No morphologicalchange of the microspheres by the reaction was observed with SEM.The Cell-C6 microspheres were added to 1 ml of PBS (pH 7.4) containinga protein at 37 °C. The proteins used were BSA, IgG, tuftsin, FN, andgelatin and their solution concentrations were in the range of about 1.5 x10-9 to 1.5 mug - mlm. The number of microspheres added was 1 x 108 in a



36

well. After adsorption for 1 h, 100 N1 of the suspension was added to 9000 of the culture media.

Collection of Macrophages and Phagocytosis Assay

Four days prior to harvest, 5-7 week-old, male BALB/c mice were in-jected aseptically with 2 ml thioglycollate broth (Brewer’s Medium,Difco Laboratories, Detroit, MI) intraperitoneally. On the day of har-vest, mice were exsanguinated by decapitation. Sacrificed mice wereinjected i.p. with cold HBSS and the peritoneal cells were collected bysyringe aspiration. After centrifugation (1,000 rpm, 5 min, 0 °C), pelletedperitoneal cells were resuspended in EMEM-FCS, counted on a hemo-cytometer, and brought to a cell density of 1 x 105 macrophages/mlEMEM-FCS. 1 ml of cell suspension was seeded into 16 mm wells, witha round glass cover slip on each bottom, on tissue culture plates (A/S.

Figure 2. SEM photographs of protein-grafted microspheres: (1) Cellulose, (2) BSA-g-cellulose, (3) IgG-g-cellulose, (4) FN-g-cellulose; (5) Tuftsin-g-cellulose, and (6) Gelatin-g-cellulose.



37

Nunc, Kamstrup, Roskilde). After incubating at 37 °C in a 5% CO~, 95%air atmosphere for 2 h, the cover slips were washed thoroughly withEMEM (EMEM-FCS without FCS) to remove non-adhering cells andFCS. After that, 1 ml of EMEM or EMEM-FCS containing 1 x 107 ofmicrospheres was added into each well, and incubated for 3 h. After in-cubation, the cover glass slips were washed several times with shakingand fixed with 2.5% glutaraldehyde in EMEM. After washing in dis-tilled water, the cover glass slips were reversed upside down and em-bedded on slide glasses with glycerin jelly. Macrophages were observedby phase-contrast microscopy. In each test at least 400 macrophageswere examined and the average number of the microspheres phago-cytized by a macrophage was counted. Experiments were repeated atleast three times for each type of microsphere.

RESULTS AND DISCUSSION

Phagocytosis at 4 °C

One of the problems in the studies of phagocytosis is how unambigu-ously one can count the number of microspheres internalized by a mac-rophage. Sometimes, phase-contrast microscopy fails to distinguishclearly the microspheres merely attached to the macrophage surfacefrom those engulfed into the macrophage interior. Because the biologicalability of macrophages to engulf microspheres are greatly suppressed atlow temperatures [7], and the cell is only able to attach to the micro-sphere surface through physicochemical forces, we studied the phago-cytosis on microspheres at 4 °C.Regardless of the nature of the microsphere surface or the presence or

absence of FCS, the number of microspheres associated with eachmacrophage was below 0.3 (Table 1). This is quite small in comparisonwith that observed by phagocytosis at 37 °C (Table 2). This finding sug-gests that the microspheres counted by the phase-contrast microscopeafter washing the cover glass on which macrophages had been placedand incubated with microspheres, are those which have not merely at-tached to the macrophage, but have been actually taken up into the in-terior of the cell.

Phagocytosis at 37 °C of Protein Grafted Microspheres

The cellulose microspheres grafted with BSA showed the least phago-cytosis, irrespective of the presence of FCS (Table 2). The next to leastphagocytosis was observed for the virgin microspheres of cellulose. The



38

Table 1a. Phagocytosis at 4°C of the protein-grafted microspheres by macrophages.

aln the absence of FCS.bln the presence of FCS.cdiameter is 1.73 pm.dCellulose-NH( CH2)sCHJ.

Table lb. Phagocytosis at 4°C of the protein-coated Cell-C6microspheres by macrophages.

aThe values denote the number of microspheres phagocytized by one macrophage in the presence of FCS,while those in parentheses denote the number of microspheres phagocytized in the absence of FCS.



39

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surface modification with C6 chains greatly enhanced phagocytosis ofthe cellulose microspheres by macrophages, almost to a level similar tothat of polystyrene microspheres. Macrophage phagocytosis was alsomarkedly accelerated by surface grafting of the cellulose microsphereswith a variety of proteins except for BSA. Addition of FCS to theculture medium increased the uptake of gelatin-grafted cellulose micro-spheres to a remarkable extent. Added FCS had no effect on the phago-cytosis of the other protein-grafted microspheres and actually decreasedphagocytosis for microspheres of polystyrene and C6-coupled cellulose.The effect of FCS addition can be seen distinctly in Table 2, where the

number of microspheres phagocytized and the FCS concentration of themedium for some typical microspheres are compared. Phagocytosis forthe virgin and BSA-grafted cellulose was insignificant and hardly influ-enced by the presence of FCS, while the addition of FCS apparently in-creased the phagocytosis for the gelatin-grafted cellulose, in markedcontrast with that for the C6-coupled microspheres.Some specific proteins remarkably enhance the phagocytic rate by a

phenomenon called opsonization [8,9]. Among those known to be respon-sible for opsonization are immune proteins like IgG and complementcomponents [10,11]. These opsonins may be present on the particle sur-face, strongly bound or weakly adsorbed, and act as a ligand for thereceptor which exists on the phagocytic cell surface. This specific inter-action between the ligand immune protein distinguishing foreign an-tigens and the macrophage receptor results in the promotion of phago-cytosis. One opsonin of them, which is identical to cold-insoluble

glycoprotein or fibronectin, has been found to function as an adhesive tocells and is distributed in plasma in a free state or bound to cell surfaces[14,15]. Fibronectin is known to have binding sites specific for gelatin,collagen, fibrinogen, fibrin, and glycosaminoglycans that are similar toheparin, in addition to cells [16,17].Many workers have revealed that the particle uptake by phagocytic

cells is also affected by the physicochemical properties of the particlesurface, in particular, its hydrophobicity [18-21]. In general, an increasein hydrophobicity of a particle surface leads to enhanced uptake, unlessthe surface is too hydrophobic such as fluorinated polymers. For in-stance, Van Oss and Gillman [18] have shown that the bacteria, whosesurface is more hydrophobic than that of neutrophils, can be readilyphagocytized by the phagocytic neutrophils. Other microspheres arealso known to undergo phagocytosis even though soluble or surface-bound opsonizing immune proteins are not involved.The results given in Table 2 may be interpreted in terms of opsoniza-

tion and hydrophobicity of the microsphere surface. There is no reason



41

to suspect that the resistance of the virgin cellulose microsphere againstphagocytosis is due to its high hydrophilicity (or low hydrophobicity).BSA has neither the opsonizing ability nor the property to alter thehydrophilicity of the cellulose and consequently does not increasephagocytosis when it is grafted to the cellulose surface. BSA is the pro-tein in the highest concentration among the serum proteins in FCS.Therefore, the microspheres, when placed in the medium containingFCS, will be preferably coated by the most abundant protein. It followsthat the C6 coupled cellulose with a very hydrophobic surface is easilyadsorbed by BSA [22] when mixed with FCS and hence shows the sup-pressed phagocytosis [23]. The exception is the gelatin-grafted micro-sphere. In this case, fibronectin or other cell-adhesive proteins containedin FCS may be predominently bound to the microsphere surface, sincesuch proteins have a high affinity to gelatin. As a result, the gelatin-grafted cellulose becomes opsonized in the presence of FCS [24,25]. Itcan be seen from Table 2 that the FCS addition has no marked effect onthe microspheres grafted with the native proteins. This indicates thatthese surfaces are neither coated nor bound by any of the serum proteinsin FCS which is in contrast to that observed for microspheres that haveno native proteins on the surface.

Phagocytosis at 37 °C of Microspheres Coupled with C6-Chains

Protein adsorption generally takes place on foreign surfaces, especial-ly if they are moderately hydrophobic. Protein desorption also dependson the hydrophobicity of the surface to which the protein has been ad-sorbed. From the results in Figure 3 it is suggested that the C6-coupledcellulose is probably hydrophobic enough to be strongly adsorbed byproteins and to be highly resistant against protein desorption. Thus, wehave attempted to study the effect of pre-coated with different proteinson the phagocytosis of C6-coupled microsphere in the absence andpresence of FCS. The proteins were previously adsorbed on the micro-sphere surfaces in PBS without serum at 37 °C for 1 h at different pro-tein concentrations.The number of the pre-coated microspheres phagocytized at 37 °C in

the absence of FCS is illustrated in Figure 4. No appreciable effect wasobserved when the protein coating concentration was less than 10-1mg - ml-’; however, a significant influence appeared as the coating con-centration became greater than 10-8 mg-ml-1, except for tuftsin. Themost prominent change was observed for IgG and BSA; the former in-creased phagocytosis, while the latter decreased phagocytosis with in-creasing coating concentration. The results were similar to that found



42

Figure 3. SEM photographs of TGC-stimulated mouse peritoneal macrophages afterphagocytosis of gelatin-grafted microspheres: (1) control macrophages and (2) macro-phages after 3 h incubation with gelatin-grafted microspheres in Eagle MEM containing10% FCS.



43

Figure 4. Influence of protein pre-coating on phagocytosis of Cell-C6 microspheres bymacrophages in the absence of FCS. (0) IgG, ((I) FN, (0) Gelatin, (O) Tuftsin, (8) BSA,and (A) None.

for the protein-grafted microspheres. This fact strongly indicates thatthe proteins are adsorbed on the surface of C6-coupled microspheres andthat the adsorption appears to be as stable as covalently grafted pro-tein. On the other hand, no enhancement of the limited degree phago-cytosis (Table 2) was observed for the protein coated virgin cellulosemicrospheres. It is apparent that the hydrophilic nature of cellulose re-jected protein adsorption.The remarkable promotion of phagocytosis by IgG coating seen in

Figure 4 was ascribed to the opsonization effect. Gelatin [26] andfibronectin [27] may also be capable of opsonization, but do not seem aseffective as IgG. BSA molecules adsorbed on the microspheres alsomake the surface hydrophilic which leads to a reduction in phagocytosis[23].When FCS was added to the incubation medium, the protein pre-

coating affected phagocytosis differently from that observed in theabsence of FCS. In Figure 5 is shown the influence of protein pre-coatingon phagocytosis in the presence of 10% FCS. If the surface is not pre-coated, the number of microspheres phagocytized is suppressed; this ef-fect is also shown in Figure 3. However, coating with IgG or fibronectin



44

Figure 5. Influence of protein pre-coating on phagocytosis of Cell-C6 microspheres bymacrophages in the presence of 10% FCS. (<D) IgG, (0) FN, (0) Gelatin, (*) BSA, and (A)None.

increased the phagocytosis almost to a level similar to that observedwithout FCS. This indicates that the addition of 10% FCS does not giverise to any significant desorption of coated proteins nor to any proteinsubstitution during incubation for 3 h.The most striking finding is that the gelatin pre-coating markedly

promoted microsphere phagocytosis (Figure 5). This may be due to thebinding of cell-adhesive proteins present in FCS by the pre-coatedgelatin [24,25].As is shown in Figures 4 and 5, the effect of protein concentration at

pre-coating becomes more prominent with the increasing concentrationuntil it reaches about 10-5 mg’ ml-1. This may be due to enhanced proteincoating with concentration and an experiment is currently being carriedout to confirm our assumption.

CONCLUSIONS

Phagocytosis of cellulosic microspheres by mouse peritoneal macro-phages has been found to be greatly affected, both positively and



45

negatively, by the modification of the microsphere surface and by thepresence of FCS.The preferred hydrophilic surface that is resistant to uptake by

phagocytic cells is non-ionic in nature, because a strong interactiontakes place between the cell surface and the foreign materials possess-ing cationic or anionic groups [28]. Therefore, to protect a microspherefrom being attacked by macrophages, one can use a microsphere with ahydrophilic surface or modify a hydrophobic microsphere surface to ahydrophilic one.Phagocytic attack may also be retarded by utilizing serum albumin

which exerts no specific interaction with any cell but exists in plasma inhigh concentration. A stable serum albumin coating can be non-covalently bound to the microsphere surface by physical adsorption.Alternatively, serum albumin can be bound to the microsphere surfacethrough covalent grafting if functional groups for grafting are availableon the microsphere surface. It is not presently clear whether a reductionin phagocytosis observed by immobilizing the serum albumin on themicrosphere surface is due to the hydrophilic nature of the protein or tothe covering the surface with native biomaterial that masks antigensrecognition sites.

If however, the aim is to promote phagocytosis of microspheres, thenthe surface should be covered by specific proteins that act as opsoninsor that can selectively bind some opsonizing biomolecules from the liv-ing fluids. The opsonic protein most readily available for this purpose isIgG, which can be used either by coating a hydrophobic microsphere orby covalent grafting to the macrosphere surface.Gelatin is also very effective in enhancing the phagocytosis. This

denatured protein when bound on the microsphere surface seems toselect cell-adhesive proteins like fibronectin from the surroundingmedium and to bind them through bioaffinity interaction. Gelatin itselfalso has an opsonizing effect, since the microspheres grafted withgelatin are relatively readily phagocytized even in the absence of FCS.There are other methods for surface modification that accelerate

microsphere uptake [29-31]. For example, Sunamoto and his coworkers[31] have demonstrated that surface binding of specific carbohydrateson liposomes enables these microspheres to be increasingly and selec-tively taken up by mouse alveolar macrophages.

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