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Biodegradable Microspheres X: Some Properties of Polyacryl Starch Microparticles Prepared from Acrylic Acid-Esterified Starch
Biodegradable Microspheres X: Some Properties of Polyacryl Starch Microparticies Prepared from Acrylic Acid-Esterified Starch T I M O L A A K S O * * A N D INGVAR S J Ö H O L M * *
Received May 15, 1987, from the 'Division of Pharmacy, National Board of Health and Welfare, Box 607, S-751 25 Uppsala, Sweden, and the ^Department of Pharmaceutical Biochemistry, Biomédical Center, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden. Accepted for publication August 25, 1987. Abstract O Acrylic acid-esterified starch was produced by reacting starch with acrylic acid chloride. This reaction was rapid and easy to control. Introduction of acrylic groups into starch reduced the enzymatic degradability of starch (e.g., with 12 acrylic groups/100 glucose resi dues, -75% of the degradation products eluted before glucose on gel filtration). The degradability could be increased to a large extent by preincubation at pH 5.5 in vitro (e.g., after 16 weeks, the corresponding figure was -15%). The acrylic acid-esterified starch was used to prepare polyacryl starch microparticies. These were rapidly eliminated from the circulation after iv injection in mice, mainly by uptake in the liver. The elimination of the microparticies from the liver, monitored with [uC]starch, displayed a half-life of -3.5-4.5 months. After 5 and 6 months, -30% of the initial radioactivity remained in the liver. This is equivalent to the amount anticipated from the enzymatic degradation of the monomer (acrylic acid-esterified starch) in vitro and the innate nondegradability of the 14C-marker. These results, taken together, indicate that the ester bond between starch and the hydrocarbon chain in polyacryl starch microparticies is hydrolyzed at lysosomal pH.
During the past 20 years considerable interest has been devoted to the development of carriers for the targeting of drugs and enzymes. Liposome-encapsulated drugs have thus been shown to be effective in animal models (e.g., for the treatment of lysosomal parasitic diseases 1 2 and fungal dis eases 3 ). Solid carriers" offer some advantages as far as carrier capacity, stability, or preparation is concerned, but present other problems essentially related to their degradation and metabolism in vivo. Polyisobutyl cyanoacrylate nanoparticles are biodegradable, 6 but formaldehyde is produced during their metabolism. 6 · 7 Polyacrylamide 8 and polyacryldextran 9 microparticies are degraded only slowly in vivo. Polyacryl starch microparticies are currently investigated as carriers for macromolecules 10 and low molecular weight drugs in vivo. 11 They are essentially made up by starch, which is biocompatible and degradable. However, prior to the preparation of the microparticies by radical polymerization in a water-in-oil emulsion, the starch has to be acryloylated. 12 This has previously been carried out by reacting it with acrylic acid glycidylester. We have found that this reaction reduces the enzymatic degradability of the starch, so that the resulting microparticies are not completely eliminated from the liver. 13 The reduced degradability may be due to the ether bond connecting the acrylic derivative to the starch, which probably is cleaved slowly in vivo. In the present investigation, a new method to prepare acryloylated starch is suggested in which the stable ether bond is replaced by an ester bond. The properties of the acryloylated starch and polyacrylstarch microparticies ob tained are studied both in vitro and in vivo.
Experimental Section Materials—Merkotest (for the determination of α-amylase activi 0022-3549/87/1200-0935$01.00/0 © 1987, American Pharmaceutical Association
ty) and acrylic acid chloride (Lot 4212147) were obtained from KABI AB, Stockholm, and Merck, Darmstadt, respectively. Pluronic F 68 was purchased from BASF, Wyandotte, and amyloglucosidase (Lot 31-F0305) from Sigma Chemical Company. Soluble starch (Lot 58583) (maltodextrin, mol wt 5000), obtained as a gift from Dr. Lars Svensson, Stadex AB, Malmö, Sweden, and [14C]starch (Lot 1252132), prepared from Nicotiana tabacum and purchased from New England Nuclear, West Germany, were used without further purifi cation. All other chemicals were of analytical grade. Synthesis of iV-Acryloylalanine—iV-Acryloylalanine was pre pared according to Kaczmar and Traser.14 L-Alanine (6.9 g, 67 mmol) was dissolved in 30 mL of distilled water and the solution was cooled to 0 °C in an ice bath. Acrylic acid chloride (6 g, 67 mmol) was added slowly while maintaining the temperature below 10 °C, and main taining the pH at 8.5-9 by the addition of 10 M NaOH. The reaction mixture was acidified to pH 1-2 with 6 M HC1 and extracted six times with 25 mL of ethylacetate. The product was recrystallized once from ethylacetate and dried, giving a yield of 64%. The structure was confirmed by NMR analysis. The melting point was 161 °C. Preparation of Acryloylated Starch—Acrylic acid chloride was reacted with soluble starch in water at slightly alkaline pH. Freshly distilled acrylic acid chloride was added to the starch solution (0.14 M) in 50-/¿L portions, while the pH was maintained at -8.5-9 by the addition of 0.5 M NaOH. The temperature was kept at 0 "C through out the reaction. When the pH value had been stable for 15 min after the last addition of acrylic acid chloride, the reaction mixture was passed through a Sephadex G25 column using water as the mobile phase. The pooled void volume was freeze-dried and stored at 4 °C. The number of acrylic groups/glucose residues, referred to as the degree of derivatization, was determined by FT-1!! NMR according to the method of Lepistö et al. le Incubation of Acryloylated Starch at pH 5.5—Acryloylated starch was dissolved in 0.2 M phosphate buffer (pH 5.5) to give a final concentration of 0.22 M, and the solution was preserved with gentamicin (50 μβ/mL). After incubation at 37 °C for 8 and 16 weeks, the samples were gel filtrated on a Sephadex G25 column. The void volume was pooled, freeze dried, and subjected to NMR analysis or incubation with amyloglucosidase. Enzymatic Degradation of Starch In Vitro—Starch (30-50 mg) was incubated overnight with -100 U of amyloglucosidase in 400 μί, of 0.1M citrate buffer (pH 4.5) at 37 °C in a water bath with shaking, according to the method of Laakso et al.13 The reaction was stopped by adding 400 fiL of trichloroacetic acid (10% w/v) to the samples. After centrifugaron (3000 x g, 15 min), 400 μΐ- of the supernatant was gel filtrated on a Sephadex G-25 column. Fractions of 1.0 mL were collected and subsequently analyzed for glucose content by the enthrone method. Determination of Glucose Using Anthrone—The method used was a modification of that described by Jermyn.1" The sample (200 ßL) was mixed with 133 ¿iL of concentrated hydrochloric acid, 13 /¿L of formic acid, and 1.05 mL of the freshly prepared anthrone reagent [20 mg of anthrone in 100 mL of 80% (v/v) sulphuric acid]. After mixing gently (to avoid frothing), the samples were boiled for 12 min in a water bath and then cooled in an ice bath. The samples were mixed and sonicated. Each sample (320 /iL) was then transferred to a microtiter plate containing 30 μL of 0.1% Tween 20 in water. The plate was read in a microtiter plate spectrophotometer (Multiscan, Flow Laboratories) at 620 nm against distilled water with 0.1% Journal of Pharmaceutical Sciences / 935 Vol. 76, No. 12, December 1987
Tween 20. Standard curves were obtained from glucose solutions ranging from 10 to 250 /¿g/niL. Underivatized starch gave the same color yield as glucose, while acryloylated starch gave a yield of -80%. Block Polymerization of Acrylic Acid-Esterified Starch—Acryl ic acid-esterified starch was dissolved in 0.5 mL of phosphate buffer (pH 7.5) in test tubes, and 20 μ-L of ammoniumperoxydisulphate was added to give a final concentration of 0.08 M. The samples were rapidly mixed, and JV^^'^'-tetramethylethylenediamine (TEMED) was added to give a final concentration of 0.25 M. After renewed mixing, the samples were allowed to polymerize. The formation ofa mechanically stable gel within 30 s was considered as a successful polymerization. The size of the microparticles was measured from photographs taken by scanning electron microscopy according to Höglund and Morein,19 as described by Sjöholm and Edman.8 Enzymatic Degradation of Microparticles—Microparticles were incubated in pooled mouse serum obtained from heparinized mouse plasma by freezing and thawing. The α-amylase activity of the sera was determined with the Merkotest reagent kit and found to be in the range 5.000-7.000 IU/L. Incubations were carried out at 37 °C in a water bath with reciprocal shaking. 14C-Labeled microparticles (0.5 mg in 100 juL of physiological saline) and mouse serum (100 μ&, preheated to 37 °C, were mixed and incubated for 5, 15, or 60 min. Ice-cold physiological saline (800 /xL) was then added and the particles were centrifugea (3000 x g) for 15 min at +4°C. The supernatant (500 /iL) was carefully withdrawn and the radioactivity was counted in a scintillation counter (TriCarb 2405 Spectrometer, Packard) after addition of 2 mL of a scintillation cocktail (Instagel, Packard). The counting efficiency was calculated with an external standard. 14 C-Labeled microparticles (0.5 mg in 0.2 mL of 0.2 M citrate buffer, pH 4.5) were incubated with either amyloglucosidase (~100 units) or a lysosome-enriched fraction (0.2 mL) at 37 °C with recipro cal shaking. The lysosome-enriched fraction was prepared as de scribed earlier.10 The radioactivity in the supernatant was deter mined after centrifugation at 3000 x g for 15 min. Distribution and Elimination of Poly aery lstarch Microparticles In Vivo—The l4C-labeled microparticles (0.1 or 0.5 mg), in 0.2 mL of physiological saline, were administered iv in male NMRI mice (2025 g). Blood samples (75 μ-h) were taken from the orbital plexus 5 min after administration, whereupon the mice were killed by cervi cal dislocation. The spleen, lungs, one kidney, and 0.15-0.20 g of the liver were removed and prepared for liquid scintillation counting as described earlier.20
mixture was recovered in the acryloylated starch. In a control experiment, acrylic acid was substituted for acrylic acid chloride in the derivatization step. No acrylic groups could be detected in the starch by NMR. Hydrolysis of the Ester Bond between Acrylic Acid and Starch at pH 5.5 In Vitro—The enzymatic degradability of the acrylic acid-esterified starch was studied after incubation in vitro at pH 5.5, a pH close to that prevalent in lysosomes, as an indirect measure of the hydrolysis of the ester bond connecting the acrylic group to the starch (see Scheme I). This procedure is based on the assumption that complete hydrolysis of the ester bond will regenerate underivatized starch and thus restore its complete degradability by en zymes such as amyloglucosidase. The extent of degradation was analyzed by gel filtration of the degradation products on Sephadex G25 and subsequent determination of the glucose content in the fractions. Underivatized starch was complete ly degraded in this experimental system, with all degrada tion products appearing in the low molecular weight frac tions after gel filtration. When acrylic acid-esterified starch containing —12 acylic groups/100 glucose residues (D.D. = 0.12) was digested by amyloglucosidase, ~75% of the degradation products eluted before the low molecular weight fractions (Figure 2).
Results Reaction of Starch with Acrylic Acid Chloride—Soluble starch was reacted with acrylic acid chloride, at 0 °C and pH 8.5-9 in water, to obtain acrylic acid-esterified starch. The proposed chemical reaction is shown in Scheme I. The acrylic group is bound directly to starch through an ester bond and the derivatized starch will be hereafter referred to as acrylic acid-esterified starch. Previously, the starch was derivatized by reacting it with acrylic acid glycidylester, in which case an ether bond is formed adjacent to the starch. (See Scheme II.) The obtained acrylic acid-esterified starch was purified by gel filtration and subsequently freeze dried. The content of acrylic groups in the starch was determined by FT- 1 !! NMR. Figure 1 shows the derivatization degree versus the amount of acrylic acid chloride added during the reaction. Within the range studied, the relationship is almost linear. Thus, the reaction can easily be controlled so that a desired acrylic group content (D.D. = degree of derivatiza tion; acrylic groups/glucose residue) can be obtained. About 20-30% of the acrylic acid chloride added to the reaction
Polysaccharide
PS -
OH + C l
-
0 II C -
Acrylic acid chloride added, μ\Flgure ^Derivatization of starch with acrylic acid chloride. Starch was reacted with acrylic acid chloride at pH 8.5-9, the derivatized starch was purified by gel filtration, and the degree of derivatization (acrylic groups/glucose residue) was determined with FT-1H NMR. The mean ± SD (n = 3) is given.
pH CH = CH_
8.5-9 ►
PS
Scheme I—Reaction of starch with acrylic acid chloride giving acrylic acid-esterified starch. 936 /Journal of Pharmaceutical Sciences Vol. 76, No. 12, December 1987
CH = CH,
After eight weeks of incubation at pH 5.5, the soluble acrylic acid-esterified starch was purified from low molecular weight substances, such as the preservative, by gel filtration. The void fractions were pooled and freeze dried. This materi al was incubated with amyloglucosidase. The amount of degradation products recovered before the low molecular weight fractions in gel filtration was —25% (Figure 2). After a further eight weeks of incubation at pH 5.5, this figure was -15%. In a control experiment, starch was exposed at pH 9 for 1 h to simulate the reaction conditions during acryloylation, and thereafter digested by amyloglucosidase. The size distribu tion of the degradation products was almost identical to that obtained after 16 weeks of incubation of the acrylic acidesterified starch at pH 5.5, as described above, with - 1 5 % of the material eluting before glucose (data not shown). The same results were obtained when acrylic acid was included during the acryloylation reaction. This demonstrates that the enzymatic degradability of starch is reduced by the reaction conditions per se, and not by any side reaction(s) caused by the acrylic acid formed during the esterification. In summary, the enzymatic degradability of the acrylic
acid-esterified starch was largely restored during incubation at pH 5.5. This indicates that the ester bond between acrylic acid and starch is hydrolyzed at this pH. Preparation and Properties of Polyacryl Starch Microparticles In Vitro—In order to obtain a mechanically stable gel, a certain concentration of acrylic groups is necessary. By block-polymerization of acrylic acid-esterified starch, it was found that a T value of 0.9 was needed (see explanation of the D-T-C nomenclature in Experimental Section). This T value is about two times higher than that needed to polymerize the previously studied acrylic acid glycidylester-derivatized starch (Scheme II). The most likely explanation is a lower reactivity of acrylic acid-esterified starch, maybe due to sterical reasons. Microparticles with D-T-C values of 20-1-0 could be pre pared from acrylic acid-esterified starch. However, these microparticles easily agglomerated. This was also the case when microparticles were prepared as co-polymers with acrylamide or bisacrylamide. On the contrary, when acryloylalanine (with T = 0.5) was co-polymerized, the resulting polyacrylstarch microparticles did not show any tendency to aggregate. Probably the presence of negative charges in the microparticles at physiological pH leads to repulsive forces between the microparticles. Consequently, all the micropar ticles used in the subsequent work were co-polymers of 1500 acryloylated starch and acryloylalanine. Enzymatic Degradation of Different I4C-Labeled Poly acryl Starch Microparticles—The efiFect of the microparticle composition and the amount of TEMED, added during the microparticle preparation, on the serum stability was stud ied. The total concentration of acrylic groups (the T value) was varied only with the content of acrylic groups in the E 1000starch, since both the starch and the acryloylalanine concen trations were kept constant. Microparticles prepared from starch having a D.D. of 0.16 or lower were completely dissolved after 5 min of incubation ou in mouse serum, irrespective of whether 0.1 or 0.5 mL of 3 TEMED was used in the microparticle preparation (Figure ¡3 3). With a D.D. of 0.18 and addition of 0.1 mL of TEMED, the resulting microparticles were also degraded to a large extent. More stable microparticles could be obtained by increasing the D.D. to 0.22. The stability was still influenced by TEMED, with 5 or 15% of the radioactivity in soluble form after 5 min when 0.1 and 0.5 mL of TEMED, respectively, was added. The microparticles used in the in vivo studies were also incubated with amyloglucosidase and a lysosome-enriched 60 80 kO fraction in vitro (Table I). Microparticle preparation 1 was degraded to a somewhat lesser extent than preparation 2. Elution volume, ml The lysosome-enriched fraction was not as efficient as amy Figure 2—Gel filtration of the digest obtained after amyloglucosidaseloglucosidase in degrading the microparticles. No synergistic degradation of acrylic acid-esterified starch before and after in vitro efiFect was found between amyloglucosidase and the lyso hydrolysis at pH 5.5. Starch containing 12 acrylic groups/100 glucose some-enriched fraction (data not shown). residues was incubated with amyloglucosidase ( · ) . The degradation Distribution of Different Polyacrylstarch Microparti with amyloglucosidase was repeated after incubation at 37°C and pH cles after Intravenous Injection—The characteristics of the 5.5 for 8 (□) and 16 (■) weeks. The size distribution of the degradation microparticles used in the in vivo studies are summarized in products was determined by gel filtration on a Sephadex G25 column, Table I and their serum stability is given in Figure 3. The Dand the glucose content in each fraction was analyzed with enthrone. The values V0 and V, are the elution volume of Blue dextran and T-C value was 10-1.4-0, and the only difference between the preparations was that either 0.1 or 0.5 mL of TEMED was glucose, respectively.
Polysaccharide PS - OH + CH
PS
CH 2
ΛΗ
OH - CH
CH,
CH 2 - 0 - C
0 II
C - CH = CH,
pH 8.5
CH = CH,
Scheme II—Reaction of starch with acrylic acid glycidylester (previously used method). Journal of Pharmaceutical Sciences / 937 Vol. 76, No. 12, December 1987
added during the microparticle preparation. After iv injec tion in mice, both microparticle preparations were rapidly cleared from the circulation and <10% of the dose was recovered per mL of blood 5 min post injection (Table II). At that time, -75-85% of the dose was found in the liver and
100
i
c n +-
2
*j Q. 3
0.5-1.5% in the spleen. The organ distribution is essentially in accordance with the distribution of polyacrylstarch microparticles prepared from starch derivatized with acrylic acid glycidylester.14 However, slightly more of the dose (-75-85%) is deposited in the liver. This may be due to the presence of negative charges on the particles21 or higher stability to enzymatic degrada tion. The lower kidney values obtained in this study, <1% of the dose compared with 1.7-4.4% in the previous study, also indicate that less of these microparticles were degraded during the distribution. Elimination of Different Polyacrylstarch Microparticles from the Liver—The elimination of microparticles 1 and 2 (see Table II) was followed for six months after iv injection of 0.5 mg of 14C-labeled microparticles in mice. The results are summarized in Figure 4. Both microparticle preparations had a similar elimination profile. After a rapid elimination during the first week, a slower elimination phase, with a half-life of -3.5-4.5 months, was seen up tofivemonths post injection. On average, —20% of the dose was retained in the liver after both five and six months.
50 >
Discussion
Considering the lysosomotropic properties of any solid drug carrier system, not only must the carrier be degraded in n cc vivo, but the degradation products should also be able to diffuse out from the lysosomes. Hydrophilic substances, for example sugars, cannot pass the lysosomal membrane if their molecular weight is above ~200.M We have found that polyacrylstarch microparticles, prepared from starch deriva tized with acrylic acid glycidylester, are not completely eliminated from the liver lysosomes within 16 weeks.14 A major drawback of using acrylic acid glycidylester is the formation of an ether bond adjacent to the glucose molecule (see Scheme II). Even if the ester bond joining the hydrocar 30 60 bon chain in the microparticles (obtained after polymeriza Time, min tion of the acrylic groups) and the starch is hydrolyzed in vivo, dihydroxypropoxy residues will remain bound to the Figure 3— The stability of different 1*C-labelled polyacryl starch micro- starch. This may impair the metabolism of the starch, as particles In mouse serum. The amount of TEMED added during the observed in the case of hydroxyethylstarch.23·24 Thus, it microparticle preparation was 0.1 mL (empty marks) or 0.5 mL (filled should be possible to obtain a completely degradable acryloymarks). The D-T-C of the microparticles and the degrees of derivatizalated starch by derivatizing it in a reversible way, utilizing a tionwere: 10-1.1-0, 0.13(9); 10-1.1-0,0.16(V); 10-1.2-0,0.18(A);and 10-1.4-0, 0.22 ( ■ , D). Acryloylalanine, corresponding toT= 0.5, was covalent bond which is unstable in the lysosomes. The present work has shown that it is possible to connect Included in all the microparticle preparations. Each value is the mean ± SD of three determinations. acrylic groups to the starch via an ester bond by reacting it o
Table II—Organ Distribution of Radioactivity after Intravenous Injection of "4C-Labeled Polyacrylstarch Microparticles In Mice"
Table I—Characteristics of the Microparticles Used In In Vivo Studies"
Microparticle
Characteristic
TEMED, mL" Diameter, μτη, % c so.5 0.6-1.0 1.1-1.5 1.6-2.0 2.1-2.5 >2.5 Degraded by amyloglucosidase, *}>o" Degraded by lysosomal enzymes, %"
Dose, mg
Preparation
Microparticle 1
1
2
0.1
0.5
31 31 18 13 4 2 59 ± 2 20±2
64 13 8 11 2 1 64 ± 2 39 ± 5
• The D-T-C value was 10-1.4-0, of which T = 0.5 was attributed by Nacryloyialanine. "The amount of TEMED added during the microparticle preparation. c The size distribution was determined by scanning electron microscopy. " Incubated overnight with 100 U of amyloglucosidase. * Incubated overnight with a lysosome-enriched fraction. 938 / Journal of Pharmaceutical Sciences Vol. 76, No. 12, December 1987
Percent of Dose
Organ
0.1
0.5
Liver Spleen Lungs Kidneys Blood 1
86.9 0.6 2.2 0.2 3.2
± ± + ± ±
4.8 0.1 0.3 0.1 0.6
Liver Spleen
78.5 1.2 6.1 0.8 6.9
± + ± ± ±
4.0 0.1 0.6 0.2 1.0
Lungs
Kidneys Blood*
Microparticle 2
— — — — 73.4 1.3 3.3 0.9 5.8
± + + ± +
2.2 0.1 0.2 0.1 0.5
"The organ distribution was determined 5 min after iv injection of microparticles 1 and 2; for microparticle characteristics see Table I; each value is the mean ± SE of 5-6 animals; no compensation was made for the radioactivity from the blood content of the organs. " Radioactivity in 1 mL of blood.
with acrylic acid chloride at alkaline pH. The degree of derivatization of the starch could be controlled by varying the amount of acrylic acid chloride added during the reaction. An ester bond can be hydrolyzed by two different mecha nisms in the lysosomes: by enzymatic activity or by the low pH. However, an enriched lysosomal fraction, shown to contain considerable esterase activity toward p-nitrophenylacetate, failed to degrade acrylic acid-esterified starch to a greater extent than amyloglucosidase. The same was also true for microparticles prepared from this starch. This sug gests that the acryloyl ester bond in the soluble starch and the ester bond between the hydrocarbon chains and the starch in the particles are not susceptible to hydrolysis by the lysosome preparation used in vitro. However, it cannot be excluded that a low enzymatic activity may have contributed to the degradation of the microparticles in vivo during the time period studied (six months). At any rate, we have unequivocally shown that the ester bond in the soluble acryloylated starch prepared with acrylic acid chloride is hydrolyzed at pH 5.5, which is close to the lysosomal pH.28·26 The in vivo elimination rate of microparticles prepared from acrylic acid-esterified starch was similar to that of the most degradable microparticles in our previous study,14 with a half-life of -3.5-4.5 months. However, the starch used cannot be expected to be completely eliminated from the lysosomes for two reasons: the innate nondegradability of the [Ï4C]starch used as a tracer,14 and the nonspecific reduction in degradability obtained during the acryloylation of the starch. Considering these two factors, -30% of the radio activity can be anticipated to remain in the lysosomes for long periods of time. On the average, —30% of the initial radioactivity was also found in the liver six months after microparticle administration. This strongly indicates that the ester bond is cleaved in vivo, since considerably more radioactivity would have been retained in the liver if the 14C-
80 -
60 ai
ΙΛ O
Ό
U0 -
20
-//8 16 Time,weeks
24
label had not been hydrolyzed from the hydrocarbon chain (formed by the polymerization of the acrylic groups of the modified starch). The microparticles prepared in the present study are not likely to become widely used in vivo, mainly because it is necessary to co-polymerize acryloylalanine in order to pre vent microparticle agglomeration. Clearly, the use of higher amounts of acrylic groups in the microparticle preparation will decrease the rate of microparticle degradation in vivo and will also result in a higher proportion of synthetic hydrocarbon chains in the microparticles. However, it can be concluded that the ester bond formed after the derivatization of starch with acrylic acid chloride is hydrolyzed in vitro at lysosomal conditions, and that microparticles prepared thereof are metabolized in vivo to the expected extent. Thus, the results can form a basis for a rational design of biode gradable starch microparticles with crosslinks containing ester bonds.
References and Notes 1. Alving, C. R.; Steck, E. A.; Hanson, P. S.; Loizeaux, W. L.; Chap man, W. C; Waits, V. B. Life Sei. 1978, 22, 1021-1026. 2. New, R. R.; Chance, M.L·.;Thomas, S. C; Peters, W. Nature 1978,272, 55-56. 3. Lopez-Berestein, G.; Fainsiein, V.; Hopfer, R.; Menta, K.; Sulli van, M. P.; Keatin, M.; Rosenblum, M. G.; Mehta, R.; Luna, M.; Hersh, E. M.; Veuben, J.; Juliano, R. L.; Bodey, G. P. J. Infect. Dis. 1985,151, 704-710. 4. Microspheres and Drug Therapy. Pharmaceutical, Immunological and Medical Aspects; Davis, S. S.; Ilium, L.; McVie, J. G.; Tomlinson, E.; Eds.; Elsevier Science: Amsterdam, 1984. 5. Grislain, L.; Couvreur, O.; Lenaerts, V.; Roland, M.; DeprezDecampeneere, D.; Speiser, P. Int. J. Pharm. 1983, 25, 335-345. 6. Leonard, F.; Kulkarni, R. K.; Brandes, G.; Nelson, J.; Cameron, J. J. J. Appl. Polym. Sei. 1966,10, 259-272. 7. Leanerts, V.; Couvreur, P.; Christiaens-Leyh, D.; Joins, E.; Roland, M.; Rollman, B.; Speiser, P. Biomaterials 1984,5,65-68. 8. Sjöholm, I.; Edman, P. J. Pharmacol. Exp. Ther. 1979,211, 656662. 9. Edman, P.; Sjöholm, I. J. Pharm. Sei. 1983, 72, 796-799. 10. Artursson, P.; Edman, P.; Sjöholm, I. J. Pharmacol. Exp. Ther. 1984,232,705-712. 11. Laakso, T.; Stjärnkvist, P.; Sjöholm, I. J. Pharm. Sei. 1987, 76, 134-140. 12. Artursson, P.; Edman, P.; Laakso, T.; Sjöholm, J. J. Pharm. Sei. 1984, 73, 1507-1513. 13. Laakso, T.; Artursson, P.; Sjöholm, J. J. Pharm. Sei. 1986, 75, 962-967. 14. Kaczmar, B. U.; Traser, S. Makromol. Chem. 1976,177(7), 19811989. 15. Lepistö, M.; Artursson, P.; Edman, P.; Laakso, T.; Sjöholm, I. Anal. Biochem. 1983, 133, 132-135. 16. Jermyn, M. A. Anal. Biochem. 1975, 68, 332-335. 17. Hjertén, S. Arch. Biochem. Biophys. Suppl. 1962,1, 147-151. 18. Edman, P.; Ekman, B.; Sjöholm, I. J. Pharm. Sei. 1980, 69, 838842. 19. Höglund, S.; Morein, B. J. Gen. Virol. 1973, 21, 359-369. 20. Artursson, P.; Laakso, T.; Edman, P. J. Pharm. Sei. 1981, 72, 1415-1420. 21. Wilkins, D. J.; Myers, P. A. Brit. J. Exp. Pathol. 1966, 47, 568576. 22. Reijngoud, D. J. K.; Tager, J. M. Biochim. Biophys. Acta 1977, 472, 419-424. 23. Thompson, W. L.; Fukushima, T.; Rutherford, R. B.; Walton, R. P. Sure. Gynecol. Obstet. 1970, 131, 965-972. 24. Mishler, I. V.; John, M. Pharmacology of Hydroxyethylstarch; Oxford University: Oxford, 1982; p 45. 25. Ohkuma, S.; Poole, B. Proc. Nati. Acad. Sei. USA 1978, 75, 3327-3331. 26. Reijngoud, D-J.; Oud, P. S.; Tager, J. M. Biochim. Biophys. Acta 1976, 448, 303-313.
Acknowledgments
This work was financially supported by the Swedish Medical Figure 4—Elimination of radioactivity from the liver after iv injection of Research Council (Project 03X-07138), the Foundation "Bengt Lundqvists Minne", and the I. F. Foundation for Pharmaceutical u C-labeled polyacryl starch microparticles. The radioactivity content of Research. the liver after injection of 0.5 mg of microparticle preparations 1 (O) andThe authors wish to thank Dr. Lennart Meurling and Dr. Bo 2 ( · ) in mice. Microparticle characteristics are given in Table I. Each Jonsson for fruitful discussions, and Mrs. Elisabeth Tidare and Mrs. value is the mean ± SE of 4-6 animals. Ingela Stadenberg for technical assistance. Journal of Pharmaceutical Sciences /939 Vol. 76, No. 12, December 1987
Received May 15, 1987, from the 'Division of Pharmacy, National Board of Health and Welfare, Box 607, S-751 25 Uppsala, Sweden, and the ^Department of Pharmaceutical Biochemistry, Biomédical Center, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden. Accepted for publication August 25, 1987. Abstract O Acrylic acid-esterified starch was produced by reacting starch with acrylic acid chloride. This reaction was rapid and easy to control. Introduction of acrylic groups into starch reduced the enzymatic degradability of starch (e.g., with 12 acrylic groups/100 glucose resi dues, -75% of the degradation products eluted before glucose on gel filtration). The degradability could be increased to a large extent by preincubation at pH 5.5 in vitro (e.g., after 16 weeks, the corresponding figure was -15%). The acrylic acid-esterified starch was used to prepare polyacryl starch microparticies. These were rapidly eliminated from the circulation after iv injection in mice, mainly by uptake in the liver. The elimination of the microparticies from the liver, monitored with [uC]starch, displayed a half-life of -3.5-4.5 months. After 5 and 6 months, -30% of the initial radioactivity remained in the liver. This is equivalent to the amount anticipated from the enzymatic degradation of the monomer (acrylic acid-esterified starch) in vitro and the innate nondegradability of the 14C-marker. These results, taken together, indicate that the ester bond between starch and the hydrocarbon chain in polyacryl starch microparticies is hydrolyzed at lysosomal pH.
During the past 20 years considerable interest has been devoted to the development of carriers for the targeting of drugs and enzymes. Liposome-encapsulated drugs have thus been shown to be effective in animal models (e.g., for the treatment of lysosomal parasitic diseases 1 2 and fungal dis eases 3 ). Solid carriers" offer some advantages as far as carrier capacity, stability, or preparation is concerned, but present other problems essentially related to their degradation and metabolism in vivo. Polyisobutyl cyanoacrylate nanoparticles are biodegradable, 6 but formaldehyde is produced during their metabolism. 6 · 7 Polyacrylamide 8 and polyacryldextran 9 microparticies are degraded only slowly in vivo. Polyacryl starch microparticies are currently investigated as carriers for macromolecules 10 and low molecular weight drugs in vivo. 11 They are essentially made up by starch, which is biocompatible and degradable. However, prior to the preparation of the microparticies by radical polymerization in a water-in-oil emulsion, the starch has to be acryloylated. 12 This has previously been carried out by reacting it with acrylic acid glycidylester. We have found that this reaction reduces the enzymatic degradability of the starch, so that the resulting microparticies are not completely eliminated from the liver. 13 The reduced degradability may be due to the ether bond connecting the acrylic derivative to the starch, which probably is cleaved slowly in vivo. In the present investigation, a new method to prepare acryloylated starch is suggested in which the stable ether bond is replaced by an ester bond. The properties of the acryloylated starch and polyacrylstarch microparticies ob tained are studied both in vitro and in vivo.
Experimental Section Materials—Merkotest (for the determination of α-amylase activi 0022-3549/87/1200-0935$01.00/0 © 1987, American Pharmaceutical Association
ty) and acrylic acid chloride (Lot 4212147) were obtained from KABI AB, Stockholm, and Merck, Darmstadt, respectively. Pluronic F 68 was purchased from BASF, Wyandotte, and amyloglucosidase (Lot 31-F0305) from Sigma Chemical Company. Soluble starch (Lot 58583) (maltodextrin, mol wt 5000), obtained as a gift from Dr. Lars Svensson, Stadex AB, Malmö, Sweden, and [14C]starch (Lot 1252132), prepared from Nicotiana tabacum and purchased from New England Nuclear, West Germany, were used without further purifi cation. All other chemicals were of analytical grade. Synthesis of iV-Acryloylalanine—iV-Acryloylalanine was pre pared according to Kaczmar and Traser.14 L-Alanine (6.9 g, 67 mmol) was dissolved in 30 mL of distilled water and the solution was cooled to 0 °C in an ice bath. Acrylic acid chloride (6 g, 67 mmol) was added slowly while maintaining the temperature below 10 °C, and main taining the pH at 8.5-9 by the addition of 10 M NaOH. The reaction mixture was acidified to pH 1-2 with 6 M HC1 and extracted six times with 25 mL of ethylacetate. The product was recrystallized once from ethylacetate and dried, giving a yield of 64%. The structure was confirmed by NMR analysis. The melting point was 161 °C. Preparation of Acryloylated Starch—Acrylic acid chloride was reacted with soluble starch in water at slightly alkaline pH. Freshly distilled acrylic acid chloride was added to the starch solution (0.14 M) in 50-/¿L portions, while the pH was maintained at -8.5-9 by the addition of 0.5 M NaOH. The temperature was kept at 0 "C through out the reaction. When the pH value had been stable for 15 min after the last addition of acrylic acid chloride, the reaction mixture was passed through a Sephadex G25 column using water as the mobile phase. The pooled void volume was freeze-dried and stored at 4 °C. The number of acrylic groups/glucose residues, referred to as the degree of derivatization, was determined by FT-1!! NMR according to the method of Lepistö et al. le Incubation of Acryloylated Starch at pH 5.5—Acryloylated starch was dissolved in 0.2 M phosphate buffer (pH 5.5) to give a final concentration of 0.22 M, and the solution was preserved with gentamicin (50 μβ/mL). After incubation at 37 °C for 8 and 16 weeks, the samples were gel filtrated on a Sephadex G25 column. The void volume was pooled, freeze dried, and subjected to NMR analysis or incubation with amyloglucosidase. Enzymatic Degradation of Starch In Vitro—Starch (30-50 mg) was incubated overnight with -100 U of amyloglucosidase in 400 μί, of 0.1M citrate buffer (pH 4.5) at 37 °C in a water bath with shaking, according to the method of Laakso et al.13 The reaction was stopped by adding 400 fiL of trichloroacetic acid (10% w/v) to the samples. After centrifugaron (3000 x g, 15 min), 400 μΐ- of the supernatant was gel filtrated on a Sephadex G-25 column. Fractions of 1.0 mL were collected and subsequently analyzed for glucose content by the enthrone method. Determination of Glucose Using Anthrone—The method used was a modification of that described by Jermyn.1" The sample (200 ßL) was mixed with 133 ¿iL of concentrated hydrochloric acid, 13 /¿L of formic acid, and 1.05 mL of the freshly prepared anthrone reagent [20 mg of anthrone in 100 mL of 80% (v/v) sulphuric acid]. After mixing gently (to avoid frothing), the samples were boiled for 12 min in a water bath and then cooled in an ice bath. The samples were mixed and sonicated. Each sample (320 /iL) was then transferred to a microtiter plate containing 30 μL of 0.1% Tween 20 in water. The plate was read in a microtiter plate spectrophotometer (Multiscan, Flow Laboratories) at 620 nm against distilled water with 0.1% Journal of Pharmaceutical Sciences / 935 Vol. 76, No. 12, December 1987
Tween 20. Standard curves were obtained from glucose solutions ranging from 10 to 250 /¿g/niL. Underivatized starch gave the same color yield as glucose, while acryloylated starch gave a yield of -80%. Block Polymerization of Acrylic Acid-Esterified Starch—Acryl ic acid-esterified starch was dissolved in 0.5 mL of phosphate buffer (pH 7.5) in test tubes, and 20 μ-L of ammoniumperoxydisulphate was added to give a final concentration of 0.08 M. The samples were rapidly mixed, and JV^^'^'-tetramethylethylenediamine (TEMED) was added to give a final concentration of 0.25 M. After renewed mixing, the samples were allowed to polymerize. The formation ofa mechanically stable gel within 30 s was considered as a successful polymerization. The size of the microparticles was measured from photographs taken by scanning electron microscopy according to Höglund and Morein,19 as described by Sjöholm and Edman.8 Enzymatic Degradation of Microparticles—Microparticles were incubated in pooled mouse serum obtained from heparinized mouse plasma by freezing and thawing. The α-amylase activity of the sera was determined with the Merkotest reagent kit and found to be in the range 5.000-7.000 IU/L. Incubations were carried out at 37 °C in a water bath with reciprocal shaking. 14C-Labeled microparticles (0.5 mg in 100 juL of physiological saline) and mouse serum (100 μ&, preheated to 37 °C, were mixed and incubated for 5, 15, or 60 min. Ice-cold physiological saline (800 /xL) was then added and the particles were centrifugea (3000 x g) for 15 min at +4°C. The supernatant (500 /iL) was carefully withdrawn and the radioactivity was counted in a scintillation counter (TriCarb 2405 Spectrometer, Packard) after addition of 2 mL of a scintillation cocktail (Instagel, Packard). The counting efficiency was calculated with an external standard. 14 C-Labeled microparticles (0.5 mg in 0.2 mL of 0.2 M citrate buffer, pH 4.5) were incubated with either amyloglucosidase (~100 units) or a lysosome-enriched fraction (0.2 mL) at 37 °C with recipro cal shaking. The lysosome-enriched fraction was prepared as de scribed earlier.10 The radioactivity in the supernatant was deter mined after centrifugation at 3000 x g for 15 min. Distribution and Elimination of Poly aery lstarch Microparticles In Vivo—The l4C-labeled microparticles (0.1 or 0.5 mg), in 0.2 mL of physiological saline, were administered iv in male NMRI mice (2025 g). Blood samples (75 μ-h) were taken from the orbital plexus 5 min after administration, whereupon the mice were killed by cervi cal dislocation. The spleen, lungs, one kidney, and 0.15-0.20 g of the liver were removed and prepared for liquid scintillation counting as described earlier.20
mixture was recovered in the acryloylated starch. In a control experiment, acrylic acid was substituted for acrylic acid chloride in the derivatization step. No acrylic groups could be detected in the starch by NMR. Hydrolysis of the Ester Bond between Acrylic Acid and Starch at pH 5.5 In Vitro—The enzymatic degradability of the acrylic acid-esterified starch was studied after incubation in vitro at pH 5.5, a pH close to that prevalent in lysosomes, as an indirect measure of the hydrolysis of the ester bond connecting the acrylic group to the starch (see Scheme I). This procedure is based on the assumption that complete hydrolysis of the ester bond will regenerate underivatized starch and thus restore its complete degradability by en zymes such as amyloglucosidase. The extent of degradation was analyzed by gel filtration of the degradation products on Sephadex G25 and subsequent determination of the glucose content in the fractions. Underivatized starch was complete ly degraded in this experimental system, with all degrada tion products appearing in the low molecular weight frac tions after gel filtration. When acrylic acid-esterified starch containing —12 acylic groups/100 glucose residues (D.D. = 0.12) was digested by amyloglucosidase, ~75% of the degradation products eluted before the low molecular weight fractions (Figure 2).
Results Reaction of Starch with Acrylic Acid Chloride—Soluble starch was reacted with acrylic acid chloride, at 0 °C and pH 8.5-9 in water, to obtain acrylic acid-esterified starch. The proposed chemical reaction is shown in Scheme I. The acrylic group is bound directly to starch through an ester bond and the derivatized starch will be hereafter referred to as acrylic acid-esterified starch. Previously, the starch was derivatized by reacting it with acrylic acid glycidylester, in which case an ether bond is formed adjacent to the starch. (See Scheme II.) The obtained acrylic acid-esterified starch was purified by gel filtration and subsequently freeze dried. The content of acrylic groups in the starch was determined by FT- 1 !! NMR. Figure 1 shows the derivatization degree versus the amount of acrylic acid chloride added during the reaction. Within the range studied, the relationship is almost linear. Thus, the reaction can easily be controlled so that a desired acrylic group content (D.D. = degree of derivatiza tion; acrylic groups/glucose residue) can be obtained. About 20-30% of the acrylic acid chloride added to the reaction
Polysaccharide
PS -
OH + C l
-
0 II C -
Acrylic acid chloride added, μ\Flgure ^Derivatization of starch with acrylic acid chloride. Starch was reacted with acrylic acid chloride at pH 8.5-9, the derivatized starch was purified by gel filtration, and the degree of derivatization (acrylic groups/glucose residue) was determined with FT-1H NMR. The mean ± SD (n = 3) is given.
pH CH = CH_
8.5-9 ►
PS
Scheme I—Reaction of starch with acrylic acid chloride giving acrylic acid-esterified starch. 936 /Journal of Pharmaceutical Sciences Vol. 76, No. 12, December 1987
CH = CH,
After eight weeks of incubation at pH 5.5, the soluble acrylic acid-esterified starch was purified from low molecular weight substances, such as the preservative, by gel filtration. The void fractions were pooled and freeze dried. This materi al was incubated with amyloglucosidase. The amount of degradation products recovered before the low molecular weight fractions in gel filtration was —25% (Figure 2). After a further eight weeks of incubation at pH 5.5, this figure was -15%. In a control experiment, starch was exposed at pH 9 for 1 h to simulate the reaction conditions during acryloylation, and thereafter digested by amyloglucosidase. The size distribu tion of the degradation products was almost identical to that obtained after 16 weeks of incubation of the acrylic acidesterified starch at pH 5.5, as described above, with - 1 5 % of the material eluting before glucose (data not shown). The same results were obtained when acrylic acid was included during the acryloylation reaction. This demonstrates that the enzymatic degradability of starch is reduced by the reaction conditions per se, and not by any side reaction(s) caused by the acrylic acid formed during the esterification. In summary, the enzymatic degradability of the acrylic
acid-esterified starch was largely restored during incubation at pH 5.5. This indicates that the ester bond between acrylic acid and starch is hydrolyzed at this pH. Preparation and Properties of Polyacryl Starch Microparticles In Vitro—In order to obtain a mechanically stable gel, a certain concentration of acrylic groups is necessary. By block-polymerization of acrylic acid-esterified starch, it was found that a T value of 0.9 was needed (see explanation of the D-T-C nomenclature in Experimental Section). This T value is about two times higher than that needed to polymerize the previously studied acrylic acid glycidylester-derivatized starch (Scheme II). The most likely explanation is a lower reactivity of acrylic acid-esterified starch, maybe due to sterical reasons. Microparticles with D-T-C values of 20-1-0 could be pre pared from acrylic acid-esterified starch. However, these microparticles easily agglomerated. This was also the case when microparticles were prepared as co-polymers with acrylamide or bisacrylamide. On the contrary, when acryloylalanine (with T = 0.5) was co-polymerized, the resulting polyacrylstarch microparticles did not show any tendency to aggregate. Probably the presence of negative charges in the microparticles at physiological pH leads to repulsive forces between the microparticles. Consequently, all the micropar ticles used in the subsequent work were co-polymers of 1500 acryloylated starch and acryloylalanine. Enzymatic Degradation of Different I4C-Labeled Poly acryl Starch Microparticles—The efiFect of the microparticle composition and the amount of TEMED, added during the microparticle preparation, on the serum stability was stud ied. The total concentration of acrylic groups (the T value) was varied only with the content of acrylic groups in the E 1000starch, since both the starch and the acryloylalanine concen trations were kept constant. Microparticles prepared from starch having a D.D. of 0.16 or lower were completely dissolved after 5 min of incubation ou in mouse serum, irrespective of whether 0.1 or 0.5 mL of 3 TEMED was used in the microparticle preparation (Figure ¡3 3). With a D.D. of 0.18 and addition of 0.1 mL of TEMED, the resulting microparticles were also degraded to a large extent. More stable microparticles could be obtained by increasing the D.D. to 0.22. The stability was still influenced by TEMED, with 5 or 15% of the radioactivity in soluble form after 5 min when 0.1 and 0.5 mL of TEMED, respectively, was added. The microparticles used in the in vivo studies were also incubated with amyloglucosidase and a lysosome-enriched 60 80 kO fraction in vitro (Table I). Microparticle preparation 1 was degraded to a somewhat lesser extent than preparation 2. Elution volume, ml The lysosome-enriched fraction was not as efficient as amy Figure 2—Gel filtration of the digest obtained after amyloglucosidaseloglucosidase in degrading the microparticles. No synergistic degradation of acrylic acid-esterified starch before and after in vitro efiFect was found between amyloglucosidase and the lyso hydrolysis at pH 5.5. Starch containing 12 acrylic groups/100 glucose some-enriched fraction (data not shown). residues was incubated with amyloglucosidase ( · ) . The degradation Distribution of Different Polyacrylstarch Microparti with amyloglucosidase was repeated after incubation at 37°C and pH cles after Intravenous Injection—The characteristics of the 5.5 for 8 (□) and 16 (■) weeks. The size distribution of the degradation microparticles used in the in vivo studies are summarized in products was determined by gel filtration on a Sephadex G25 column, Table I and their serum stability is given in Figure 3. The Dand the glucose content in each fraction was analyzed with enthrone. The values V0 and V, are the elution volume of Blue dextran and T-C value was 10-1.4-0, and the only difference between the preparations was that either 0.1 or 0.5 mL of TEMED was glucose, respectively.
Polysaccharide PS - OH + CH
PS
CH 2
ΛΗ
OH - CH
CH,
CH 2 - 0 - C
0 II
C - CH = CH,
pH 8.5
CH = CH,
Scheme II—Reaction of starch with acrylic acid glycidylester (previously used method). Journal of Pharmaceutical Sciences / 937 Vol. 76, No. 12, December 1987
added during the microparticle preparation. After iv injec tion in mice, both microparticle preparations were rapidly cleared from the circulation and <10% of the dose was recovered per mL of blood 5 min post injection (Table II). At that time, -75-85% of the dose was found in the liver and
100
i
c n +-
2
*j Q. 3
0.5-1.5% in the spleen. The organ distribution is essentially in accordance with the distribution of polyacrylstarch microparticles prepared from starch derivatized with acrylic acid glycidylester.14 However, slightly more of the dose (-75-85%) is deposited in the liver. This may be due to the presence of negative charges on the particles21 or higher stability to enzymatic degrada tion. The lower kidney values obtained in this study, <1% of the dose compared with 1.7-4.4% in the previous study, also indicate that less of these microparticles were degraded during the distribution. Elimination of Different Polyacrylstarch Microparticles from the Liver—The elimination of microparticles 1 and 2 (see Table II) was followed for six months after iv injection of 0.5 mg of 14C-labeled microparticles in mice. The results are summarized in Figure 4. Both microparticle preparations had a similar elimination profile. After a rapid elimination during the first week, a slower elimination phase, with a half-life of -3.5-4.5 months, was seen up tofivemonths post injection. On average, —20% of the dose was retained in the liver after both five and six months.
50 >
Discussion
Considering the lysosomotropic properties of any solid drug carrier system, not only must the carrier be degraded in n cc vivo, but the degradation products should also be able to diffuse out from the lysosomes. Hydrophilic substances, for example sugars, cannot pass the lysosomal membrane if their molecular weight is above ~200.M We have found that polyacrylstarch microparticles, prepared from starch deriva tized with acrylic acid glycidylester, are not completely eliminated from the liver lysosomes within 16 weeks.14 A major drawback of using acrylic acid glycidylester is the formation of an ether bond adjacent to the glucose molecule (see Scheme II). Even if the ester bond joining the hydrocar 30 60 bon chain in the microparticles (obtained after polymeriza Time, min tion of the acrylic groups) and the starch is hydrolyzed in vivo, dihydroxypropoxy residues will remain bound to the Figure 3— The stability of different 1*C-labelled polyacryl starch micro- starch. This may impair the metabolism of the starch, as particles In mouse serum. The amount of TEMED added during the observed in the case of hydroxyethylstarch.23·24 Thus, it microparticle preparation was 0.1 mL (empty marks) or 0.5 mL (filled should be possible to obtain a completely degradable acryloymarks). The D-T-C of the microparticles and the degrees of derivatizalated starch by derivatizing it in a reversible way, utilizing a tionwere: 10-1.1-0, 0.13(9); 10-1.1-0,0.16(V); 10-1.2-0,0.18(A);and 10-1.4-0, 0.22 ( ■ , D). Acryloylalanine, corresponding toT= 0.5, was covalent bond which is unstable in the lysosomes. The present work has shown that it is possible to connect Included in all the microparticle preparations. Each value is the mean ± SD of three determinations. acrylic groups to the starch via an ester bond by reacting it o
Table II—Organ Distribution of Radioactivity after Intravenous Injection of "4C-Labeled Polyacrylstarch Microparticles In Mice"
Table I—Characteristics of the Microparticles Used In In Vivo Studies"
Microparticle
Characteristic
TEMED, mL" Diameter, μτη, % c so.5 0.6-1.0 1.1-1.5 1.6-2.0 2.1-2.5 >2.5 Degraded by amyloglucosidase, *}>o" Degraded by lysosomal enzymes, %"
Dose, mg
Preparation
Microparticle 1
1
2
0.1
0.5
31 31 18 13 4 2 59 ± 2 20±2
64 13 8 11 2 1 64 ± 2 39 ± 5
• The D-T-C value was 10-1.4-0, of which T = 0.5 was attributed by Nacryloyialanine. "The amount of TEMED added during the microparticle preparation. c The size distribution was determined by scanning electron microscopy. " Incubated overnight with 100 U of amyloglucosidase. * Incubated overnight with a lysosome-enriched fraction. 938 / Journal of Pharmaceutical Sciences Vol. 76, No. 12, December 1987
Percent of Dose
Organ
0.1
0.5
Liver Spleen Lungs Kidneys Blood 1
86.9 0.6 2.2 0.2 3.2
± ± + ± ±
4.8 0.1 0.3 0.1 0.6
Liver Spleen
78.5 1.2 6.1 0.8 6.9
± + ± ± ±
4.0 0.1 0.6 0.2 1.0
Lungs
Kidneys Blood*
Microparticle 2
— — — — 73.4 1.3 3.3 0.9 5.8
± + + ± +
2.2 0.1 0.2 0.1 0.5
"The organ distribution was determined 5 min after iv injection of microparticles 1 and 2; for microparticle characteristics see Table I; each value is the mean ± SE of 5-6 animals; no compensation was made for the radioactivity from the blood content of the organs. " Radioactivity in 1 mL of blood.
with acrylic acid chloride at alkaline pH. The degree of derivatization of the starch could be controlled by varying the amount of acrylic acid chloride added during the reaction. An ester bond can be hydrolyzed by two different mecha nisms in the lysosomes: by enzymatic activity or by the low pH. However, an enriched lysosomal fraction, shown to contain considerable esterase activity toward p-nitrophenylacetate, failed to degrade acrylic acid-esterified starch to a greater extent than amyloglucosidase. The same was also true for microparticles prepared from this starch. This sug gests that the acryloyl ester bond in the soluble starch and the ester bond between the hydrocarbon chains and the starch in the particles are not susceptible to hydrolysis by the lysosome preparation used in vitro. However, it cannot be excluded that a low enzymatic activity may have contributed to the degradation of the microparticles in vivo during the time period studied (six months). At any rate, we have unequivocally shown that the ester bond in the soluble acryloylated starch prepared with acrylic acid chloride is hydrolyzed at pH 5.5, which is close to the lysosomal pH.28·26 The in vivo elimination rate of microparticles prepared from acrylic acid-esterified starch was similar to that of the most degradable microparticles in our previous study,14 with a half-life of -3.5-4.5 months. However, the starch used cannot be expected to be completely eliminated from the lysosomes for two reasons: the innate nondegradability of the [Ï4C]starch used as a tracer,14 and the nonspecific reduction in degradability obtained during the acryloylation of the starch. Considering these two factors, -30% of the radio activity can be anticipated to remain in the lysosomes for long periods of time. On the average, —30% of the initial radioactivity was also found in the liver six months after microparticle administration. This strongly indicates that the ester bond is cleaved in vivo, since considerably more radioactivity would have been retained in the liver if the 14C-
80 -
60 ai
ΙΛ O
Ό
U0 -
20
-//8 16 Time,weeks
24
label had not been hydrolyzed from the hydrocarbon chain (formed by the polymerization of the acrylic groups of the modified starch). The microparticles prepared in the present study are not likely to become widely used in vivo, mainly because it is necessary to co-polymerize acryloylalanine in order to pre vent microparticle agglomeration. Clearly, the use of higher amounts of acrylic groups in the microparticle preparation will decrease the rate of microparticle degradation in vivo and will also result in a higher proportion of synthetic hydrocarbon chains in the microparticles. However, it can be concluded that the ester bond formed after the derivatization of starch with acrylic acid chloride is hydrolyzed in vitro at lysosomal conditions, and that microparticles prepared thereof are metabolized in vivo to the expected extent. Thus, the results can form a basis for a rational design of biode gradable starch microparticles with crosslinks containing ester bonds.
References and Notes 1. Alving, C. R.; Steck, E. A.; Hanson, P. S.; Loizeaux, W. L.; Chap man, W. C; Waits, V. B. Life Sei. 1978, 22, 1021-1026. 2. New, R. R.; Chance, M.L·.;Thomas, S. C; Peters, W. Nature 1978,272, 55-56. 3. Lopez-Berestein, G.; Fainsiein, V.; Hopfer, R.; Menta, K.; Sulli van, M. P.; Keatin, M.; Rosenblum, M. G.; Mehta, R.; Luna, M.; Hersh, E. M.; Veuben, J.; Juliano, R. L.; Bodey, G. P. J. Infect. Dis. 1985,151, 704-710. 4. Microspheres and Drug Therapy. Pharmaceutical, Immunological and Medical Aspects; Davis, S. S.; Ilium, L.; McVie, J. G.; Tomlinson, E.; Eds.; Elsevier Science: Amsterdam, 1984. 5. Grislain, L.; Couvreur, O.; Lenaerts, V.; Roland, M.; DeprezDecampeneere, D.; Speiser, P. Int. J. Pharm. 1983, 25, 335-345. 6. Leonard, F.; Kulkarni, R. K.; Brandes, G.; Nelson, J.; Cameron, J. J. J. Appl. Polym. Sei. 1966,10, 259-272. 7. Leanerts, V.; Couvreur, P.; Christiaens-Leyh, D.; Joins, E.; Roland, M.; Rollman, B.; Speiser, P. Biomaterials 1984,5,65-68. 8. Sjöholm, I.; Edman, P. J. Pharmacol. Exp. Ther. 1979,211, 656662. 9. Edman, P.; Sjöholm, I. J. Pharm. Sei. 1983, 72, 796-799. 10. Artursson, P.; Edman, P.; Sjöholm, I. J. Pharmacol. Exp. Ther. 1984,232,705-712. 11. Laakso, T.; Stjärnkvist, P.; Sjöholm, I. J. Pharm. Sei. 1987, 76, 134-140. 12. Artursson, P.; Edman, P.; Laakso, T.; Sjöholm, J. J. Pharm. Sei. 1984, 73, 1507-1513. 13. Laakso, T.; Artursson, P.; Sjöholm, J. J. Pharm. Sei. 1986, 75, 962-967. 14. Kaczmar, B. U.; Traser, S. Makromol. Chem. 1976,177(7), 19811989. 15. Lepistö, M.; Artursson, P.; Edman, P.; Laakso, T.; Sjöholm, I. Anal. Biochem. 1983, 133, 132-135. 16. Jermyn, M. A. Anal. Biochem. 1975, 68, 332-335. 17. Hjertén, S. Arch. Biochem. Biophys. Suppl. 1962,1, 147-151. 18. Edman, P.; Ekman, B.; Sjöholm, I. J. Pharm. Sei. 1980, 69, 838842. 19. Höglund, S.; Morein, B. J. Gen. Virol. 1973, 21, 359-369. 20. Artursson, P.; Laakso, T.; Edman, P. J. Pharm. Sei. 1981, 72, 1415-1420. 21. Wilkins, D. J.; Myers, P. A. Brit. J. Exp. Pathol. 1966, 47, 568576. 22. Reijngoud, D. J. K.; Tager, J. M. Biochim. Biophys. Acta 1977, 472, 419-424. 23. Thompson, W. L.; Fukushima, T.; Rutherford, R. B.; Walton, R. P. Sure. Gynecol. Obstet. 1970, 131, 965-972. 24. Mishler, I. V.; John, M. Pharmacology of Hydroxyethylstarch; Oxford University: Oxford, 1982; p 45. 25. Ohkuma, S.; Poole, B. Proc. Nati. Acad. Sei. USA 1978, 75, 3327-3331. 26. Reijngoud, D-J.; Oud, P. S.; Tager, J. M. Biochim. Biophys. Acta 1976, 448, 303-313.
Acknowledgments
This work was financially supported by the Swedish Medical Figure 4—Elimination of radioactivity from the liver after iv injection of Research Council (Project 03X-07138), the Foundation "Bengt Lundqvists Minne", and the I. F. Foundation for Pharmaceutical u C-labeled polyacryl starch microparticles. The radioactivity content of Research. the liver after injection of 0.5 mg of microparticle preparations 1 (O) andThe authors wish to thank Dr. Lennart Meurling and Dr. Bo 2 ( · ) in mice. Microparticle characteristics are given in Table I. Each Jonsson for fruitful discussions, and Mrs. Elisabeth Tidare and Mrs. value is the mean ± SE of 4-6 animals. Ingela Stadenberg for technical assistance. Journal of Pharmaceutical Sciences /939 Vol. 76, No. 12, December 1987