Biodegradation studies on periodate oxidized cellulose

Biodegradationstudies on periodate oxidizedcellulose Maninder Sigh*, AlokR Ray”?and PadmaVasudevan** Centre for Biomedical (Received

Engineering,

Indian

Institute

of Technology,

New Delhi

110016,

India

10 June 1981)

Sodium periodate oxidized cellulose was found to degrade into small molecules when implanted subcutaneously in rats. Thus the potentiality of using oxidized cellulose as a biodegradable drug carrier is envisaged. Keywords:

cellulose,

biodegradability,

drug diffusion,

oxidation.

It is well known that cellulose does not degrade enzymatitally in the human body and no detailed report on its hydrolytic cleavage exists, although cellulose fibres have been cited to be partially biodegradable’,‘. By itself, the &I,4 linkage between the glucose units in cellulose, which is poly (1.4 fl-D-glucopyranose), is quite stable but could be made susceptible to hydrolysis by introducing chemical modifications. One such product is periodate oxidized cellulose, hereafter referred to as oxidized cellulose or OC for simplicity, which is formed by oxidizing cellulose with sodium metaperiodate leading to the conversion of anhydroD-glucose units to dialdehyde units without the simultaneous occurrence of side reactions to any great extent3. No work on the in viva degradation of oxidized cellulose has been reported in the literature but the hydrolysis of the material in vitro at high pH is known to occur4. Therefore it was thought that it might degrade at the physiological pH which is around 7.4. The present article deals with the degradation of periodate oxidized cellulose in a physiological environment and the term biodegradation has been used in this sense only.

MATERIALS

AND METHODS

*Present Address: Department of Chemistry, Marquette University, Milwaukee, WI 53233, U.S.A. tTo whom all correspondence should be addressed. *‘New Affiliation: Centre for Rural Development and Appropriate Technology, New Delhi 110016, India

16

Butterworth

Biomaterials

& Co. (Publishers)

1982,

Vol3

January

Preparation

Ltd.

0142-9612/82/010016-05

of oxidized cellulose

Cellulose (2 g) was oxidized with sodium metaperiodate solution (0.1-0.4M, 100 ml) at 25°C in the dark for 7-24 h. The product, after the required reaction time, was plunged into an excess of water for 24 h, after which it was washed repeatedly with distilled water until periodate-free washings were obtained. The course of oxidation was followed by estimating the oxygen consumption in terms of the fall in the periodate concentration, found by titration with sodium arsenite3. Products with different degrees of oxidation were prepared by manipulating the concentration of periodate and the reaction time. The oxidized cellulose was finally dried in air. The oxygen consumption, determined as above, was compared with the carbonyl content of the material obtained by its oximation reaction with hydroxylamine5.

Degradation

Cellulose powder was purchased from E. Merck (Germany) and sodium metaperiodate was obtained from B.D.H. (England). “C-Cellulose was supplied by Bhabha Atomic Research Centre, Bombay (India). 2,5-Diphenyloxazole (PPO) and 1,4-bis-(4-methyl, 5-phenyl-2oxazolyl) benzene (dimethyl POPOP) for the preparation of a scintillation cocktail for /I-counting were obtained from SISCO (India). All other chemicals used were of analytical grade. White albino rats of Wistar strain, used for the animal experiments, were housed in the animal house of All India Institute of Medical Sciences, New Delhi. A Unicam SP500 series 2 spectrophotometer was used for U.V. spectroscopic measurements while 14C-activity

0 1982

was counted on a Packard Tricarb liquid scintillation spectrometer. The infrared spectra of the KBr pelleted samples were taken on an Unicam SP 1200 infrared spectrometer.

of oxidized cellulose in vitro

About 200 mg of OC was kept in equilibrium in 5 ml of Ringer phosphate buffer (pH 7.4). The supernatant fluid after centrifugation at 30009 was removed at fixed intervals of time and the rate of OC solubilization was followed by measuring the absorbance due to the carbonyl groups at 240 nm. A fresh 5 ml of the buffer was re-equilibrated with the residue and the process repeated. Similar experiments were performed on an OC-50 sample after reducing 100 mg of it with 100 mg sodium borohydride in tris buffer (pH 8.0). (a) By Spectrophotometry:

lb) By 14C Counting: About 1 mg of “C-labelled cellulose was weighed out accurately and was diluted 1000 fold with unlabelled cellulose with thorough mixing. This was oxidized with 0.4 M metaperiodate solution as earlier. Unlabelled OC was also prepared simultaneously for control measurements. The 14C labelled OC thus prepared was kept in con$03.00

Biodegradation

of cellulose:

M. Singh et al.

tact with 5 ml of Ringer solution (pH 7.4). The supernatant liquid, after centrifugation at 3OOOg, was separated periodically and a fresh 5 ml of the eluting solution was added to the residue. 0.5 ml of the above supernatant was mixed with 15 ml of the scintillation cocktail and the solution was counted in the liquid scintillation counter. The total p-count of the oxidized sample was determined by fully digesting a small weighed portion of it with 25 ml of 0.15 N NaOH solution. A control was simultaneously run in all the above experiments, unlabelled OC being used for this purpose.

Identification

of the in vitro products

The products of degradation were expected to be glycolic and 2,4_dihydroxybutyric acids and were identified by a paper chromatographic procedure described in detail in Reference 4. The starting solution for the paper chromatography was the supernatant from a sample of OC equilibrated with 5 ml of Ringer phosphate buffer (pH 7.4) for one month. Glycolic acid was separately identified in a similar supernatant by a colour reaction with 2,7dihydroxynaphthalene. The method followed was taken from References 6, 7.

In vivo degradation

by 14C counting

“C-labelled OC samples prepared as for the in vitro experiments were implanted subcutaneously on both sides of white albino rats. Two such rats were housed in one metabolic cage; control animals with unlabelled OC and with no implant were kept similarly. Urine of all these animals was collected periodically, every day for the first IOdays and at 2-3 days’ intervals subsequently and were tested for “C-activity. Samples for 14C counting were prepared by taking 0.1-0.3 ml of the filtered urine (collected) the total volume of which was also noted for calculations, and dissolving it in 15 ml of the scintillation cocktail. The total P-activity of the samples were determined by taking a known weight of the “C-labelled OC sample, digesting it completely with 25 ml of 0.15 N NaOH solution, and counting the resulting solution in the fl-counter.

Table 1 No.

1 2 3 4 5 6 7

Infrared

spectra of cellulose and oxidized

Sample

Cellulose oc-20 oc-37 oc-50 oc-75 Cellulose reduced by NaBH4 OC-50 reduced by NaBH4

Assignment

of the bands

OC - 20 = 10% oxidized cellulose and similarly for OC-37,OC-50 and OC-75 also.

CH,OH

ti Figure

CH,OH

dH

1

Oxidation

RESULTS Preparation

of cellulose

to periodate

II 0 oxidized

i cellulose

AND DISCUSSION and characterization

of oxidized

cellulose

The oxidation of cellulose by periodate is shown in Figure 1. According to this reaction, the oxygen consumption of OC is equal to the percentage of the chain units that have been oxidized. Fibrous material with any oxygen consumption between 0 and 100 can be prepared by treating cellulose with unbuffered sodium metaperiodate solution at 25°C in the dark (to prevent any side reaction). The choice of conditions for the preparation depends upon the desired degree of oxidation. This is controlled by choosing a suitable concentration of periodate, the ratio of cellulose to solution, and the reaction time. Extensive data for obtaining OC of various degrees of oxidation are available in literature3 and were used in the present experiments. The possible structure of oxidized cellulose is shown in Figure 1. The oxidation by periodate occurs specifically at positions 2 and 3 to form the dialdehyde. The infrared spectral peaks of various cellulose and OC samples are listed in Tab/e 7. In the table, and in the discussion subsequently, OC-20, OC-37, OC-50, OC-75, etc. represent cellulose oxidized to 20, 37,50 and 75 percent respectively, i.e., 20, 37,50 and 75 glucose units oxidized respectively out of 100 glucose units in the samples. The unoxidized samples as well as oxidized samples after reduction differ slightly in their infrared absorptions. The band at 1760 cm-’ is due to the -C = 0 stretching of the aldehyde groups. This is absent from cellulose as well as from reducing OC indicating the absence of a free -CHO group. One may expect this to be strong in OC. This is not so because of partial hemiacetal formation between the -CHpOH and -CHO groups. In fact, other authors have reported’ that particularly in the presence of moisture, practically all the aldehyde groups exist in the hemiacetal form, so much so that no free -CHO group is detected.

cellulose samples

Band frequency

cm-t.

Appearance

and intensity

of the band

3340

2890

1760

1640

1170

1060

894

W W W VW VW W W

S S S M W S S

-

s S S S S s S

M

W W W W -

VW VW VW VW M M

W VW W W W W W

W W W W W W W

OH stretching

CH stretching

c=o bending (adsorbed water)

H-O-H bending (adsorbed water)

Ring frequency

(1065) O-H bending

Pglycosidic linkages

S M W VW

= = = =

Strong Medium Weak Very weak.

Biomaterials

1982,

Vol3

January

17

Biodegradation

of cellulose:

M. Singh et al.

The number of aldehyde groups were, however, estimated from the oxygen consumption as measured by the remaining periodate concentration in the reaction mixture3. Also, the carbonyl groups were estimated by oxime formation with hydroxylamine and the results by the two methods for the same sample are shown in Tab/e 2. It is seen that the estimates from these methods are in good agreement (? 5%).

Studies on degradation

Table 2

I II III IV

18

Percentof glucose units oxidized per 100 glucose by arsenite

Units by hydroxylamine

Designation of the sample

20.3 38.2 52.0 78.2

23 .OO 36.6 50.3 74.3

oc.-20 oc-37 oc-50 oc-75

Biomaterials

1982,

Vol3

January

HC

-“voG’

(a) Degradation by spectrophotometric method: The degradation of OC in Ringer phosphate buffer (pH 7.4) was followed by spectrophotometric absorption at 240 nm which is probably due to the n-n* transition of the carbonyl groups. Since the exact composition of the mixture after degradation is not known, the optical density of the supernatant was taken to be an estimate of the extent of degradation. Figure 3 shows the absorbance vs. time plots. This figure does not include the initial spikes which generally occur in the first few days, probably due to the material which had degraded during preparation and washing of the samples. Also, the degradable groups at the surface are initially more easily available. After this initial spurt, the degradation rate falls partly because the accessibility of the sites is limited by diffusion of eluent into the matrix and there is a decrease in the concentration of -CHO groups. The role played by -CHO groups in inducing degradation was established by measuring the absorbance at 240 nm of supernatant from unoxidized cellulose as well as OC reduced with sodium borohydride. In cellulose, there are

no.

G1OF-oho,G, CH

in vitro

As noted earlier, it is known that oxidized cellulose is hydrolysed in alkaline media at high pH4. Figure 2 shows the reaction according to which the scission occurs, in part, by elimination of alkoxyl-type grouping in the position with respect to a carbonyl group, as shown. The intermediates may then undergo rearrangement to yield glycolic and 2,4_dihydroxybutyric acids. It was expected that OC would degrade to give the same products at pH 7.4 also, possibly at a slower rate. An OC-50 sample was equilibrated with Ringer phosphate buffer (pH 7.4) for a month and the supernatant was used for the identification of products by paper chromatography. Two reference determinations were carried out simultaneously. In one of them, glycolic acid was used as a standard. The second reference was obtained by digesting a small amount of OC-50 completely with 0.15 N NaOH solution. Paper chromatography of the test samples as well as the second reference showed two matching spots. One of the spots in each case corresponded to the standard glycolic acid spot from the first reference. Thus it was concluded that the second spot is probably due to 2,4dihydroxybutyric acid. The presence of glycolic acid was also confirmed by a colour reaction with 2,7_dihydroxynaphthalene. The course of degradation was followed by two methods: spectrophotometric and “C-labelling. The results are reported as follows:

Sample

CH,OH

HC’ ‘H

COOH

COOH

I

I

CH,OH

C(H)(OH)

I CHZ I

6~~0~ Figure 2 Degradation of perioda te oxidized 2,4-Dihydroxybutyric acid and glycolic acid

0

cellulose in to

20

IO

30

Time (days) Figure 3 Degradation of oxidized cellulose atpH 7.4 by spectrophotometry (oJ OC-75, (xl OC-50 and I*J OC-20 1OOmg samples

no -CHO groups while the NaBH4 treatment reconverts the -CHO groups to alcoholic groups. In neither case did the supernatant show any absorption at 240 nm. Figure 3 also compared the degradation rates of three OC samples with varying degrees of oxidation. It is evident that the degradation is faster with higher degrees of oxidation. Also, the initial high degradation attains a steady rate after a longer time in the case of higher degrees of oxidation. In all cases, some material is left even after a month but the U.V. absorption of the supernatant becomes too low to be measured accurately. (6) Degradation by “C-labelling: As seen above, the spectrophotometric method is not suitable for the quantitative determination of kinetic parameters of the degradation. Hence, studies on l4 C-labelled OC were undertaken. The results of these investigations are presented in Figure 4. Tab/e 3 shows supplementary data for the figure. Plot A in the figure represents an OC-90 sample from which it is seen that the sample dissolves completely in about 13 days. Plot B and plot C represent the degradation patterns of OC-50 and OC-20 respectively. In both these cases, the degradation is prolonged to about 40 days. Beyond this, some of the material still remains indicating that complete dissolution has not occurred, but the p-count was too

Biodegradation

of cellulose:

M. Singh et al.

5c

P 3 D : _I

4c

-

3c

,L

4!

50

55 Log

-!

I

L_______L

IO

20

40

Degradation of t?jabelled oxidized cellulose; (*I 90% oxidized cellulose (0.33249, total activity - 226315cpml sample dissolves in 13 days; (al 50% oxidized cellulose 10.26209, total activity - 366300cpml sample visually remains even after 38 days; (0) 20% oxidized cellulose 10.29129, total activity 198264 cpml sample still remains even after 40 days; 4: counts tend to zero at this point

feeble to be measured. Possibly, the unoxidized cellulose in the sample remains intact even after 40 days while practically all the oxidized part has degraded. An attempt was made to fit this data into a rate equation by assuming the per day degradation is dx/dt where x is the amount of OC degraded. Thus the number of counts in the eluent per day is a measure of dxldt. The total counts that could be obtained in an equal weight of the sample completely digested by 0.15 N NaOH, as described earlier, was taken as a measure of the initial concentration ‘a’ of the OC sample. The number of counts remaining in the sample at any given time was determined by subtracting the amount of total counts eluted up to that time from the total initial activity ‘a’. Log dx/dt was then plotted against log (a-x) as shown in Figure 5. The slope of the curve, omitting the initial spurt, is approximately 2.0. However, it is evident from the curve that the reaction is not simple enough to be classified as second or third order. A clear cut interpretation of the kinetics of the process can not be drawn because the matrix is heterogeneous with amorphous and crystalline regions and nondistribution

In vivo degradation

of the oxidized

sites.

of oxidized cellulose

From the preceeding discussion it is concluded that oxidized cellulose degrades at the physiological pH (i.e. 7.4) in vitro Table 3 No.

Sample

1.

OC-90

2. 3.

OC-50 oc-20

Sample size (g)

0.3324 0.2624 0.2912

Total expected counts

226315 366300 198264

Total activity released in Days

counts

13 38 40

158120 185830 115704

Kinetics

of degradation

of OC-50

by C14iabelling

J

30

Tome (days)

uniform

Figure 5

60

(0-x)

Percent recovery

JO

51 58

and the products include small molecules like glycolic and 2,4-dihydroxybutyric acids. In our earlier reports on biocompatibility and biodegradation of oxidized celluloseg*‘O, we had reported that oxidized cellulose degrades slowly in rats and almost disappeared from the implantation sites in a few months as seen by morphological and histopathological methods. However, these studies did not completely rule out the possibility of a simple disintegration of the material as opposed to degradation. In order to see whether it actually breaks up into small, absorbable molecules, we investigated the degradation of 14C labelled OC by implanting it in rats and followed it by measuring the 14C activity in urine samples collected periodically. The results are shown in Figure 6. It is seen that the P-activity of the urine was high in the first few days after which the 14C counts decreased indicating that the degradation is faster to begin with and becomes slower after the initial spurt. As discussed earlier, this may be because initially the degradation sites on the surface are easily accessible to the body fluids. Also, some material may already be present in partially degraded form as a result of repeated washings and other treatments prior to implantation. This pattern matches well with the in vitro and in vivo results. However, a quantitative comparison between in vitro and in vivo results from the 14C-labelled OC experiments is not possible because the disintegrated material (which results after the initial swelling by the bodyfluids) and its degraded products are distributed in the body and are eliminated via urine and other routes. Nevertheless, these experiments clearly show that oxidized cellulose degrades into products which are metabolized and are excreted.

CONCLUSION Periodate oxidized cellulose, which is known to degrade at high pH, is found to degrade into small molecules like glycolic and 2,4-dihyroxybutyric acids at physiological pH. Results of both the in vitro and in viva (in rats) studies indicate that the degradation, which is faster in the first few days, attains a steady value and is sustained for about a month. The suitability of the material for immobilization and release of enzymes and proteins had already been

Biomaterials

1982,

Vol3

January

19

Biodegradation

of cellulose:

M. Singh et al.

showng-“. Thus oxidized cellulose could be used as a carrier for the sustained delivery of enzymes and other drugs in the body.

ACKNOWLEDGEMENT The financial support by Bhabha Atomic Research Centre, India, is gratefully acknowledged. One of the authors (MS) would like to thank Ms. Jasbir Hora for her help in the preparation of the manuscript.

REFERENCES 1 2

3

4 5 6

7

6

9

I

I

I

IO

20

30

Time

(days)

Figure 6 C%ctivity in the urine of rats implanted with (0) 0.24gm OC-90 (0) 0.24gm OC-50. Two rats housed in each cage implanted with 0. ffigm on either side. 1: counts tend to zero at this point

20

Biomaterials

19B2, Vol3

January

10

11

Hoffman, AS., Medical Applications of Polymeric Fibers, Appt. Polym. Symp., 31 (Fiber Sci.), 1977,313334 Kronenthal, R.L., Biodegradable Polymers in Medicine and Surgery, in Polymers in Medicine and Surgery, (Eds. R.L. Kronenthal, 2. Oser and E. Martin), Plenum Press, New York, 1975, pp. 119-137 Nevell, T.P., Oxidation, in Methods in Carbohydrate Chemistry, (Ed. R.L. Whistler), Academic Press, New York, 1963, Vol. 3, pp. 164-185 Richards, G.N., Alkaline Degradation, in ibid, pp. 154-164 Green, J.N., Determination of Carbonyl Groups, in ibid., pp. 49-54 Lewis, K.P. and Weinhouse, S., Determination of Glycolic, Glyoxylic and Oxalic Acids, in Methods in Enzymology, (Eds. S.P. Colowick and N.O. Kaplan), Academic Press, New York, 1966, Vol.3, pp. 269-276 Niederweiser, A., Matasovic, A. and Leuman, E.P., Glycolic acid in Urine: A calorimetric method with values in normal adult controls and in patients with primary Hyperoxaluria, Clin. Chim. Acta. 1978,89,13-23 Gavrilov, M.Z. and Ermolenko, I.N., Electronic Spectra of Dialdehyde Cellulose, Vyskomol. Soedin,. Ser. A. 1967,9, 1688-1692, (Russ.). Singh, Maninder, Ray, A.R., Vasudevan, P., Verma, K. and Guha, S.K., Potential biosoluble carriers: biocompatibility and biodegradability of oxidized cellulose, Biomat., Med. Dev., Art. Org. 1979,7,495-512 Singh, Maninder, Cellulosic Systems for Drug Delivery, Ph.D. Thesis, Indian Institute of Technology, New Delhi, India, 1980 Singh, Maninder, Vasudevan, P., Ray, A.R. and Guha, S.K., Biosoluble Polymers for Drug Delivery, Makromol. Chem. 1980,181,2433-2439