Towards the preparation of a MMA-PEO block copolymer for the microencapsulation of mammalian cells

755

Towards the preparation of a MMA-PEO block copolymer for the microencapsulation of mammalian cells Thomas Eisa* and Michael V. Sefton Department of Chemical Engineering and Applied Toronto, Toronto, Ontario M5S lA4. Canada

Chemistry

and Centre for Biomaterials,

University

of

Polymethyl methacryiate-polyethylene glycol-polymethyl methacrylate triblock copolymers (PMMA-PEO-PMMA) were synthesized by reductive amination coupling of preformed aldehydeterminated PEO and amine-terminated PMMA. These were intended for use as high water content and therefore high permeability, biocompatible encapsulating materials for mammalian cells. Evidence for the formation of the block copolymer was obtained indirectly from precipitation experiments and IR analysis of the water-soluble extract of the polymer. Unfortunately the pure copolymer could not be separated from the homopolymers, because of the difficulty in finding appropriate non-solvents and the apparently limited yield of the product. Further work is necessary to confirm the underlying hypothesis of this work, i.e. that such a block copolymer would have a high permeability to the small proteins critical to microencapsulated cell survival or function. Keywords:

Microencapsulation,

polyethylene

glycol, synthesis,

block copolymer

Received 30 July 1992; revised and accepted 5 March 1993

cells may be microencapsulated in a waterinsoluble polymer using organic solvents without damaging the cells’-4. These capsules, containing for example pancreatic islets’, 3, may be useful as a means of transplanting cells into a host (e.g. a diabetic) to provide therapy, while the permselective capsule wall acts as a protective barrier from the immune system for the ‘foreign’ cells (‘immunoisolation’). The semipermeable membrane prevents the large (molecular weight >150,000) antibodies from coming into contact with the cells (‘immunoisolation’). The membrane is permeable, however, to the low molecular weight nutrients, metabolites and, more importantly, the active agents or intermediate molecular weight (molecular weight <~O,OOO) protein products (e.g. insulin, growth factors, etc.). These encapsulated cells can then be transplanted into the appropriate target site (peritoneal cavity, brain, etc.) to release drug as needed using the natural physiological stimuli. Hence the capsule wall must also be biocompatible and this is the advantage of using polyacrylates. Furthermore the polymer must be useable under conditions that are consistent with the microencapsulation process, i.e. it must be soluble (and insoluble) in non-toxic liquids for precipitation processes. It also must precipitate quickly to form a reasonably sturdy capsule wall, yet have a low enough viscosity that it can be pumped easily and will Mammalian

Correspondence to Dr M.V. Sefton. *Thomas Eisa presently at Stirling Health,

0 1993 Butterworth-Heinemann 0142-9612/93/100755-07

Ltd

Toronto.

flow around the cells when the capsule droplet is formed. We have focussed on a particular HEMA-MMA copolymer (75 molb hydroxyethyl methacrylate-25% methyl methacrylate) because of its biocompatibility and reasonably high water content (-30%) the latter leading to adequate permeability to molecules the size of insulin and other small proteins’. The resulting capsules prepared by interfacial precipitation (from a solution in PEG 200 into phosphate buffered saline) had a heterogeneous wall structure: thin outer skin, a macroporous sublayer and a thick, apparently dense (but not necessarily non-porous) inner layer; the latter was -75% of the total thickness which was -150 pm. The thickness of the inner layer and the consequent concern over diffusion limitations (time lags rather than steady-state behaviour) led us to explore methods of increasing capsule permeability. One approach was to change precipitation conditions and change the capsule wall structure (i.e. make it more macroporous)‘. The other approach which we report here is to prepare a more hydrophilic polymer with a higher water content. The relationship between water content and permeability is well known5. Simply increasing the water content by increasing the HEMA content of the HEMA-MMA polymer6 is limited by the -40% water content of pure pHEMA. Using more hydrophilic comonomers (e.g. methacrylic acid) results in polymers which are ultimately water soluble or at least may not be stable (‘emulsifiable’?) in a biological Biomaterials

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MMA-PEG

environment; pure pHEMA was avoided for this reason, especially since a high molecular weight thermoplastic pHEMA is difficult to prepare. Unfortunately the polymer must be thermoplastic so that capsules can be prepared, which makes the twin properties of high water content yet water insolubility difficult to combine in a single polymer. Hence we chose to explore the possibility of preparing a high water content block or graft copolymer, whose microphase structure might allow us to meet our requirements: because of the requirement for biocompatibility we chose to prepare a polyethylene oxide (PEO) copolymer because of this polymer’s unique biological properties’, it’s availability in a variety of sizes and the potential for changing the terminal functional groups. The expectation that the final polymer might be soluble in our encapsulation solvent (PEG ZOO), which was one of the few ‘organic’ solvents that cells tolerate’, also influenced our choice; we noted that existing PEO polyurethanes would also have been satisfactory were they soluble in more ‘cell compatible’ solvents (i.e. not hexafluoroisopropanol). Methyl methacrylate (MMA) was used to form the water insoluble block, primarily because it served a similar role in the random HEMAMMA copolymer. Rather than prepare a graft copolymer with the commercially available polyethylene glycol methacrylate monomer, we chose to try and prepare a PMMA-PEOPMMA triblock copolymer. It was expected that a PEOrich triblock copolymer would have a continuous high water content phase stabilized by discrete PMMA domains. Hence this material was expected to have a higher permeability than the corresponding graft copolymer or at least, because of a higher water insolubility, could be prepared with a higher permeability than the graft copolymer. Difficulties in preparing and in particular, isolating the polymer, prevented us from testing this hypothesis.

MATERIALS AND METHODS Polymer

preparation

The polymerization strategy was divided into three parts: (1) preparation of amine-terminated PMMA with functionality at one end: (2) preparation of PEO with aldehyde functionality at both ends; and (3) coupling of these oligomers through their terminal functionalities (Figure 1). AIBN,

60°C

.

MMA

H2NCH2CH2-[MMAI,

HZNCHZCHZSH

Acetic

HO-PEO-OH

w

O=CH-PEO-CH=O

anhydride NaCNBH3 O=CH-PEO-CH=O

+

H2NCH2CH2-[MMAI,

t

Figure 1 Outline of preparation of PMMA-PEO-PMMA block copolymer. MMA, methyl methacrylate; (MMA], polymethyl methacrylate.

Biomaterials

1993, Vol. 14 No. 10

block copolymers:

T. Eisa and M.V. Sefton

Preparation of amine-terminated PMMA To prepare the amine-terminated MMA oligomer, predetermined amounts of previously distilled MMA monomer (2.5 M), azobisisobutyronitrile (AIBN, Polysciences, Warrington, USA) [purified by recrystallization) and aminoethanethiol hydrochloride (AESH, Aldrich, Milwaukee, WI, USA) chain transfer agent were reacted in a solvent for 3.5 h at 60% The method was based on that used by othersg’ lo. Two sets of experiments were conducted: (1) with ethanol (95%) as solvent, the AIBN concentration was constant at 5 X 10m3 M, and the AESH concentration varied from 0 to 1.25 M; and (2) with methanol as solvent, concentrations of both AIBN (1 X lop3 to 50 X 10e3 M) and AESH (0.025-0.625 M) varied with the ratio of AESH to AIBN remaining constant at 25. Most reactions were conducted under nitrogen with magnetic stirring with a 20 ml volume in a 25 ml flask equipped a rubber/Teflon seal cap (Pierce Chemical Co., Rockford, USA); three were conducted with -100 ml volume in a 250 ml sealed round-bottomed flask. In one case (M20) a 11 reaction volume was polymerized in a standard 2 1 Pyrex reaction kettle under nitrogen and with mechanical agitation. At the end of the reaction the mixture was precipitated in a lo-fold excess of either methanol or water. Mixtures were allowed to settle and the supernatant was decanted and discarded: some samples were centrifuged to expedite separation. Oligomers precipitated in methanol were further purified by a subsequent wash in water. One polymer (M20) was further purified by dialysing (SOOO8000 molecular weight cut-off, Spectrum Medical Ind., Los Angeles, USA] in water for 10 d. Volatiles were removed and the oligomers were freeze dried to a constant weight. Aldehyde- termina ted PEO Hydroxyl-terminated PEO (molecular weight of 18,500, Polysciences, or molecular weight of 35,000, Fluka, Ronkonkoma, USA) was dissolved in minimum DMSO (Caledon, Georgetown, purified by distillation and stored dry) with mild heat and then cooled to room temperature. The maximum concentration that readily stayed in solution at room temperature depended on the PEO molecular weight: 20% w/v for PEO 18,500 and 10% w/v for PEO 35,000. Excess acetic anhydride was added to this PEO/DMSO mixture to start the reaction. As per Llanos and Sefton” and Albright and Goldman”, an acetic anhydride to hydroxyl concentration of 20 was used, i.e. the molar ratio of acetic anhydride to PEO was 10. For each molecular weight of PEO, reaction times were varied to optimize the oxidation of hydroxyl units to aldehyde units. Two experiments were performed in which reactions were quenched immediately [zero time). Reactions were conducted at room temperature in stoppered round-bottomed flasks and stirred vigorously by magnetic agitation. At the end of the reaction, the solution was precipitated in a 2%fold excess of diethyl ether (BDH). The ether was decanted and volatiles in the precipitated polymer were allowed to evaporate. To purify this polymer further, it was dissolved in the minimum methylene chloride (Mallinckrodt, Paris, USA) and reprecipitated in a lofold excess of ether. The diethyl ether was again

MMA-PEG

block copolymers:

757

T. Eisa and M.V. Sefton

decanted, the polymer dried in a vacuum desiccator, and stored in a sealed jar at -20°C. To check purity levels, one polymer was precipitated from diethyl ether four times rather than the usual two times. Furthermore, after the fourth precipitation, it was dialysed (2000 molecular weight cut-off, Sigma, St Louis, USA) for 8 d in water.

PEO and PMMA coupling The amine-terminated PMMA was reacted. to the aldehyde ends of PEO in the presence of sodium cyanoborohydride through the method of reductive amination. Predetermined quantities of functional PEO and PMMA were dissolved in stoppered erlenmeyer flasks with either methanol (BDH) or dimethyl formamide (DMF, Aldrich) as solvent. These solvents were among the few suitable for both prepolymers; because of the limited solubility of PEO in methanol it was used only with 5% PEO 18,500. With the solutions at room temperature and with continued agitation, a known quantity of predissolved sodium cyanoborohydride (NaCNBH,; 90-95% Sigma) was added to mark the start of the reaction, In some reactions, predissolved potassium hydroxide (BDH) was also added to alter the initial PH. Reactions were conducted with either aldehydeterminated PEO 18,500 or PEO 35,000 at a concentration of about 3.33% w/v for 0-15 d; zero time reactions were used to assess the efficacy of the separation procedure. Amine-terminated MMA oligomer (molecular weight: 7400 or 19,000) was used in excess at a PMMA to PEO mole ratio of eight [nominal amine to aldehyde ratio of four). Since the desire was to produce PEO-rich copolymers, only short MMA chains were investigated. Sodium cyanoborohydride was also used in excess (10 moles of NaCNBH,/mol PEO (nominal NaCNBH, to aldehyde ratio of five]). In initial experiments, various recovery and characterization techniques were investigated to find a suitable protocol. From these investigations, it was decided to add the reaction solution to a 3-fold excess of diethyl ether to end the reaction. This volatile ether mixture was then evaporated over several hours. Excess water (300 ml/g PEO) was added to the remaining mixture of dry polymer and salt (NaCNBH, and KOH) and allowed to mix thoroughly for a period of about 12 h. The mixture was then centrifuged at 3800 rev/min for 20 min at 8°C to separate the insolubles. Both the supernatant and the water-insoluble portion were freeze dried to a constant weight and stored at -20°C.

Molecular weight averages (A4,) were calculated the following Mark-Houwink constants: PMMA in DMF at 35”C13 a = 0.72 PMMA in acetone at 25”C14 a = 0.70 PEO in water at 30°C14 a = 0.78

using

K = 6.50 X 1O-3 ml/g K = 7.50 X 10e3 ml/g

K = 12.5 X 10e3 ml/g

IR spectroscopy Fourier transform IR spectroscopy (FTIR) (Nicolet 6OSX, Nicolet Instrument Corp., Madison, USA] was used to examine samples for both PMMA and/or PEO. PMMA was identified through its ester link at 1730 cm-‘. A strong peak at 1110 cm-’ was characteristic of the PEO ether link. Furthermore, the aldehyde functionality, at 1735 cm-’ , was used to identify oxidized PEO. Typically, 32 scans per sample were ratioed to background at a resolution of f4 cmm4. The extent of PEO oxidation was measured semiquantitatively by IR spectroscopy at a constant polymer concentration (0.2 or 0.15 g/ml) in chloroform (Mallinckrodt). The area under the aldehyde peak from wavenumber 1770 to 1715 was determined automatically with baseline correction. Copolymers in chloroform (0.010 g/ml) were analysed between salt windows at a fixed solution thickness. The area under the ester peak from wavenumber 1780 to 1690 was determined automatically with baseline correction. This area related only to the PMMA content since the terminal PEO aldehyde contribution was negligible in comparison, A calibration curve determined with pure PMMA was used to relate the area to MMA content.

Gel permeation chrome tograph y A liquid chromatograph (HP 1090, Hewlett Packard, Toronto, Canada] equipped with an HP 1037A differential refractive index detector was used to produce chromatograms. Results were plotted by an HP 3392A integrator and processed using an HP 85B input/output board. Three 30 cm Polymer Laboratories (PL) high performance gel permeation chromatography (GPC) columns (Rexdale, Ontario, Canada) were used in series for molecular size separation in solution: a pair of mixed gel bed columns with 15 pm particle size and a column with a 100 A pore size and 10 pm particle size. Samples in tetrahydrofuran (0.1% w/v, HPLC grade, Aldrich) were filtered and injected into the HPLC. RESULTS

Characterization

techniques

Preparation

of amine-terminated

PMMA

Intrinsic viscosity

Figure 2 shows the results in terms of yield and molecular

Polymer viscosities were measured at a constant temperature using a Cannon Ubbelohde Semi-Micro Dilution Viscometer (Cannon Instrument Co., State College, USA). PMMA samples were measured either at 35°C with DMF (Gold Label grade, Aldrich) as solvent or at 25°C with acetone (BDH) as solvent. PEO samples were run at 30°C with deionized distilled water as solvent. The data for each polymer sample were then interpreted using the Huggins and Kraemer equations.

weight for both sets of experiments and using either methanol or water to recover the amine-terminated polymer. In both sets of experiments, as the concentration of the chain transfer agent increased the molecular weight decreased (Figure Zb). With methanol as the precipitation medium, however, yields generally decreased as the molecular weight decreased (Figure Za). Although precipitate in excess methanol separated from the supernatant more readily than the same in water, low Biomaterials

1993. Vol. 14 No. 10

758

MMA-PEG

block copolymers:

T. Eisa and M.V. Sefton

60

2.5

1827

1781 1735 Wavenumber

1689

10

L 0

3040 0

0.03 0.1 0.01 0.05 0.2

a

[AESHI

0.2 0.5

0.01 0.25 0.005 0.5

I [MMAI (mole

0.25 0.15 0.5

2520

a

2000

1480

960

Wavenumber 3.0

ratio)

2.5 z

2.0

5 f

1.5

z 2

1.0 0.5

IV-1

0

3040

I 2520

b 0.01 0.05 0.1 0.03 0.05 0.1

b

0.

0.05 0.01

[AESH]/[MMA](mole

0.15

0.25 0.5 0.15 0.5

ratio)

4r

molecular weight oligomers dissolved in methanol. Therefore, water was preferred for later experiments although recovery was difficult and hence yields were inconsistent. The slope of a plot of the reciprocal of the degree of polymerization against the ratio of chain transfer agent to monomer concentration yielded an approximate chain transfer of 0.025.

PEO

Reactions proceeded smoothly and recovery yields were high (>QO%). IR spectroscopy was used to determine the time for which the aldehyde content was maximized. A typical spectrum is shown inFigure 3. The aldehyde peak area is plotted against time in Figure 4 for reactions with PEO 18,500 and PEO 35,000. The aldehyde content increased rapidly but started to reach a limiting concentration after about 50-70 h. Hence reaction times of 50 h (PEO 18,500) or 75 h (PEO 35,000) were chosen to Biomaterials

1993,

Vol. 14 No. 10

960

Figure 3 FTIR spectrum of a, oxidized and b, unreacted PEO; the inset shows amplified aldehyde peak.

1

of aldehyde-terminated

I

1480

Wavenumber

Figure 2 Effect of chain transfer agent (AESH) on a, yield and b, molecular weight of amine-terminated PMMA. [MMA] = 2.5 M in ethanol (series 1; [AIBN] = 5 X 10e3 M) or methanol (series 2; [AESH]/[AIBN] = 25); 3.5 h reaction at 60°C. Polymer precipitated in n , methanol or 8, water. 0 indicates no recovery. When an error bar is shown this is mean + range/2 for duplicate experiments; otherwisen = 1, except for columns marked with an asterisk, for which the range was too small to show.

Preparation

2000

l/f-

e 0

A

A

I

I

I

50

100

150

Reaction

Figure 4 oxidation 35,000.

A

A

time (h)

Areaof thealdehydepeak (1770-1715cm-‘) versus reaction time for qi, PEO 18,500 and A, PEO

minimize degradation when producing products for subsequent coupling reactions. The presence of side reactions (e.g. ester formation) was not ruled out, although others have found aldehyde yields to be >gOW”, 15.16. As a measure of purity levels, one polymer was purified by several reprecipitations followed by dialysis. The aldehyde area was nearly equal after each of the precipitations with an average value of 1.42 + 0.08. After dialysis, this value dropped by 17% to 1.18. The

MMA-PEG

block copolymers: T. E&a

and

decrease in the aldehyde content through dialysis may be attributed to the removal of contaminants or of low molecular weight PEO, which were richer, per unit mass, in aldehyde. Degradation during reaction was estimated using intrinsic viscosity measurements (F&ure 5). For reactions of PEO 18 500 at room temperature, no degradation was evident for a minimum of 50 h, although by 145 h (PlO) it appears that mild degradation had occurred. Degradation also occurred at 30 h at the higher temperature of 46°C and at an increased acetic anhydride concentration. On the other hand, marked degradation was evident with PEO 35,000; by 26 h the molecular weight had dropped by 17% and by 111 h it had dropped by 48%. Note, however, that with further reaction to 157 h the polymer product appeared to have unexpectedly increased in molecular weight. Loss of low molecular weight fragments was not considered important since the PEO was relatively narrow in molecular weight distribution and the same work-up was used in all cases. Extensive polymer degradation in PEO 35,000 was also evident from GPC studies (Figure 56). As reaction

28

_

PEO 18 500

10

a

30

30*

Reaction

50

50 dialysed

145

time (hl

45 PEO 35000 40 g

35

.z

30

2 .L?

25

S!

20

!!3

15

2 r”

10 5 n ”

0

b

0

10

26 Reaction

50

759

M.V. Sefton

85

111

157

time (h)

Figure5 Effect of oxidation time on PEO molecular weight (from viscosity measurements). a, PEO 18,500; b, PEO 35,000. Reaction at room temperature at a l&l mole ratio (acetic anhydride:PEO) except where noted otherwise (*, 4O”C, 2O:l mole ratio). In b the numbers at the tops of bars are GPC retention times for the same products.

times increased the chromatograms were broader and shifted to progressively longer retention times indicative of lower molecular weights. Since the aldehyde content started to level off after 70 h, a 75 h reaction time was considered to an appropriately conservative reaction time for further PEO 35,~O oxidations; the consequences of polymer degradation were ignored.

PEO/PMMA coupling Reactions proceeded smoothly but block copolymer recovery was hampered by the difficulty of separating it from the unreacted homopol~ers. In fact we were unable to separate the block copolymer from unreacted PMMA, with the result that evidence for block copolymer formation was indirect only. First the reaction solvent (methanol or DMF) was removed by precipitation in diethyl ether followed by evaporation of the supe~atant since the precipitate was difficult to collect directly. The dry polymer product was then added to water to dissolve the unreacted PEO, yielding water-soluble and water-insoluble fractions. While water could be removed easily by freeze drying, the PMMA, although insoluble in water, remained dispersed within the water phase as a fine colloid. Initially then, other selective solvents (ethanol, ethyl acetate, methyl isobutyl ketone) were used but PEO solubility was limited and subsequent recovery more difficult; hence water was preferred. Total recoveries (Tkbfe 1 ] were generally excellent (>90%) with much of that material (>80%) in the water-insoluble phase, except when there appeared to be poor separation of the two phases (e.g. PEO 35,00O/PMMA 7400). Howeyer, much of the insoluble phase could have been unreacted PMMA homopolymer. The water-soluble portion was expected to contain only PEO and salts (NaCNBH~ and KOH), whereas the water-insoluble portion should contain only PMMA. Consequently, as a result of oligomer coupling reactions, each fraction was expected to contain not only a homopolymer but also some block copolymer. Therefore, the extent of coupling was evaluated by determining the amount of PMMA in the water-soluble phase. Thus, the extent of coupling was estimated from the size of the PMMA ester peak at 1736 cm-’ in the wash fraction. Unfortunately, analogous detection of PEO in the waterinsoluble fraction was not possible with IR spectroscopy because the PEO ether peak at 1110 cm-’ was not sufficiently distinct. Because PMMA remained partly dispersed within the water phase (stabilized in part presumably by the block copolymer), it was necessary to check that the two phases were separated adequately or at least to take into account the extent of entrainment. GPC was used to analyse the wash fraction to determine qualitatively the adequacy of the separation, while coupling results were compared with equivalent zero time reactions to correct for entrainment. By careful examination of the results in Table I, evidence of coupling was found. In particular, when GPC indicated a low amount of PMMA physically blended within the water-soluble fraction, but subsequent IR analysis gave a high percentage of PMMA, then the difference was presumed to represent covalently coupled PMMA. Reactions with PEO 18,500 and PMMA 7400 Biomaterials

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MMA-PEG

Table 1

block copolymers: T. Eisa and M. V. Sefion

Yields from coupling reactions

PEO reaction time (h)

Coupling reaction Solvent

Time (d)

Recovery (%)

PH* Total’

PEO 16,50O/PMMA 7400 MeOH MeOH MeOH

30 P 30 7 PEO 16,5OO/PMMA 19 000 ::

Pi\ 350001PMMA 157400 75 0 75 :d PEO 35,OOOfPMMA 19 000 5: 14 0 5:

14

111

14

t: 95

11.2 11.3 11.2

MeOH MeOH DMF

97 97 46

37.3 37.6 37.6

DMF DMF MeOH MeOH DMF

90 95

21 4

95 94 93

1: 20

9 7.1

indicated an increase in PMMA by 13% relative to the zero reaction/separation only run in one case (21% versus 6%), and by 23% in another case (31% versus 6%); see the first series in Figure 6. Since GPC indicated good separation with a low amount of PMMA [Figure 7a), it was assumed that this increase was due to covalently bound PMMA in the PEO-rich wash fraction. On the other hand, reactions with PEO 18,500 and PMMA 19,000 suggested a large increase in PMMA content in the water-soluble phase (the second series of Figure 6); however, since GPC indicated poor separation with a

!z

r

Poor*

Poor Poor

PEO

II 18 5001PMMA 7400 1 PEO 35 DOO/PMMA7_4QO 1 PEO 18500/PMMA 19000 PEO 35000/PMMA

19000

Figure6 Estimated (by IR) MMA content of water soluble phase for the four series of coupled polymers listed in Table 1. a, PEO 16,500fPMMA 7400; b, PEO 16,500fPMMA 19,000; c. PEO 35,OOO/PMMA 7400; d, 35,OOO/PMMA 19,000. Where separation was poor as judged by GPC the bars are labelled as such. Bars with same shading represent replicate coupling experiments except the bar labelled with an asterisk which was reacted for 15 d. El, 0 d; 8,7 d; n , 14 d. Biomaterials

1993, Vol. 14 NO.

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tz 66

ii

Coupling reactions at room temperature with 0.0018 M PEO 18,500 or 0.0095 ‘KOH added to adjust pH to indicated value; if blank no KOH was added. tMass % of all initial reactants recovered in both phases. *Mass % of total recovered product which was water soluble. klass % of initial reactants that were water soluble (PEO. salts).

80

Water soluble*

26.6 26.6 26.4

MeOH MeOH DMF

P

Water-soluble reactants (6)’

19.6 19.0 19.0 19.0 19.0

M PEO.

Figure? Sample GPC chromatograms of the PEO-rich water-soluble wash fraction depicting a, good or b, poor separation of physi~lly blended (i.e. entrained) PMMA. a, PEO 16,5OO/PMMA 7400; 7 d reaction in MeOH at pH 9. b, PEO 16,5OO/PMMA 19,000; 15 d reaction in DMF.

high amount of PMMA homopolymer present in the water-soluble phase (Figure 7b), no conclusive interpretation can he made. Since the zero time reaction resulted in a good separation by GPC, the poorer separation with reaction may be attributed to the stabilization of emulsified PMMA in the aqueous phase by an amphipathic block copolymer. In reactions with PEO 35,000 and PMMA 7400 there was no evidence of coupling. This was consistent with the fact that the extent of oxidation of the PEO was limited. Several reactions were conducted with PEO 35,000 and PMMA 19,000. In those reactions in which GPC indicated a low amount of emulsified homopolymer, increases in PMMA ranged from negligible to 5% to 18% (Figure 6, last series), Since separation was apparently good, these increases in PMMA were taken to indicate the presence of at least a limited amount of covalently bound block copolymer. CONCLUSIONS The attempt to prepare a PMMA-PEO-PMMA triblock copolymer for use in encapsulation was inconclusive due

MMA-PEG

block copolymers:

T. Eisa and M.V.

761

Seiton

failure to isolate the block copolymer and in particular to separate the copolymer from unreacted homopolymer. This suggests a need for improved, perhaps structure sensitive, molecular weight insensitive separation techniques or, even better, a higher reaction yield. While the reductive amination chemistry warrants further investigation, it is more likely that alternative techniques may prove more fruitful: for example, isocyanate-terminated PEO may be preferred because of its reactivity.

selectivity

to the

of dialysis membranes,

Makromol.

Cbem.

1969, lZ6,177-186 6

7

Stevenson, W.T.K., Evangelista, R.A., Broughton, R.L. and Sefton, M.V., Preparation and characterization of thermoplastic polymers from hydroxyalkyl metharylates, 1. Appl. Polym. Sci. 1987, 34, 65-83 Merrill, E.W. and Salzman, E.W., Polyethylene oxide as a biomaterial, Am. Sot. Artif. Intern. Organs J. 1983, 6(2), 60-64

8

Stevenson, W.T.K., Evangelista, R.A., Sugamori, M.E. and Sefton, M.V., Microencapsulation of mammalian cells in a hydroxyethyl methacrylate-methyl methacrylate copolymer: preliminary development, Biomater. Artif. Cells Artif. Organs 1988, 16, 747

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council.

9

10

Ikada, Y., Iwata, H. and Nagaoka, S., Chain transfer in radical polymerizations and end group content of resultant polymers, Macromolecules 1977, 10, 1364-1371 De Boos, A.G., Preparation and characterization of some polymers terminated with primary amino groups, Polymer 1973,14,

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Uludag, H. and Sefton, M.V., Metabolic activity of CHO fibroblasts in HEMA-MMA microcapsules, Biotechnol. Bioeng. 1992, 39, 672-678

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J.D. and Goldman, L., Dimethyl sulfoxide-acid mixtures. New reagents for oxidation of Am. Chem. Sot. J. 1965, 87,4214-4216 L.C. and Katime, I., Comportemet viscosidu PMMA dans le dimethylformamie en de la temperature, Eur. Polym. J. 1984, 29,

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Yasuda, H., Peterlin, A., Colton, C.K., Smith, K.A. and Merrill, E.W., Permeability of solutes through hydrated polymer membranes III. Theoretical background for the

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Llanos, G.R. and Sefton, M.V., Immobilization of polyethylene glycol onto a polyvinyl alcohol hydrogel. 1. Synthesis and characterization, Macromolecules 1991,

Brandrup, J. and Immergut, E.H. (Eds), Polymer Handbook, 2nd Edn, John Wiley, NY, USA, 1975 Harris, J.M., Struck, E.C., Case, M.G., Paley, MS., Yalpani, M., Alstine, J.M.V. and Brooks, D.E., Synthesis and characterization of poly(ethylene glycol) derivatives, J. Polym. Sci.: Polym. Chem. Ed. 1984, 22, 341-352

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Harris, J.M., Laboratory synthesis of polyethylene glycol derivatives, Rev. Macromol. Chem. Phys. 1985, C25, 325-373

Biomaterials 1993, Vol. 14 No. 10