Polymers for Biomedical Applications - American Chemical Society

Chapter 11

Downloaded by PENNSYLVANIA STATE UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch011

Synthesis and Properties of Functional Poly(vinylpyrrolidinone) Hydrogels for Drug Delivery 1

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Louise E. Smith , Stephen Collins , Zuifang Liu , Sheila Mac Neil , Rachel Williams , and Stephen Rimmer * 3

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Departments of Engineering Materials, Kroto Research Institute, and Chemistry, University of Sheffield, Sheffield S3 7HQ, United Kingdom Department of Clinical Engineering, University of Liverpool, Liverpool L69 3GA, United Kingdom 3

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Poly (N-vinylpyrrolidinone) has many uses in biotechnology and medicine. Here we review our recent work on materials based on this polymer that have applications in drug release or as potential mitogens. The synthesis of crosslinked PNVP hydrogels and highly branched polymers is covered.

Introduction Poly(N-vinylpyrrolidinone) (PNVP) has been used in medical applications for many years. Its history can be traced back to the Second World War where in its un-crosslinked form it was used as a blood plasma expander (I). This uncrosslinked PNVP when added to iodine forms a complex, in solution this complex is better known as Povidone-iodine or by its trade name Betadine®, a surgical antiseptic. PNVP is also used as a binder in many pharmaceutical tablets as low molecular weight PNVP can be removed from the body by the kidneys. These polymers have also been investigated for use as wound dressings, drug 196

© 2008 American Chemical Society In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


197 delivery devices and vitreous substitutes; PNVP is also a major constituent in soft, gas permeable contact lenses. Research into PNVP as a potential vitreous substitute by the group of T.V. Chirila (2-5) showed that in static cultures PNVP hydrogels with water contents comparable to that of natural vitreous « 99% could increase the viability of 3T3 Swiss mouse fibroblasts after the injection of the hydrogel into the culture system (3). However this was dependent on the crosslinker used. In serum free conditions cell viability increased when a PNVP hydrogel containing lwt% 2-Hydroxyethyl methacrylate (HEMA) and 0.25wt% DEGDMA (Diethyleneglycol dimethcarylate) having an equilibrium water content (EWC) of 95% was injected into the culture system to levels approaching that of cells cultured with serum. This hydrogel also increased cell viability in cultures containing serum. Conversely a PNVP hydrogel containing 10% HEMA when added to the culture system decreased cell viability in both serum free and with serum culture systems. When this data is combined with the in vivo data obtained by Vijayasekaran et al (5) and Hong et al (4) polymers based on PNVP appeared ideal to incorporate into our research on the development of wound dressings. Hydrogels also have uses in slow release systems, which allow for controlled administration of therapeutics. Thus prior to using these polymers for drug release we first investigated the biocompatibility / potential mitogenic effect of the PNVP polymer in contact with cells and then looked at release of drugs from this polymer.

Biocompatibility In our recently published biocompatibility study (6) we showed that PNVP hydrogel networks crosslinked with lwt% ethyleneglycol dimethacrylate [1] with an EWC of 92.2% were less effective at increasing the viability of human dermal fibroblasts than PNVP crosslinked with lwt% diethylene glycol bisallylcarbonate [2] with an EWC of 96.2%. This study also showed that physical separation between the cells and the hydrogel was required to reliably obtain this increase in cell viability. The results from cultures carried out in the presence of P(NVP-co-l) or P(NVP-co-2) are summarised in figure 1. After 30 repeated experiments we observed a clear difference between these two series of polymers with P(NVP-co-2) being more effective at enhancing cell viability. The effect of the detailed structure of PNVP on cell viability appears to be associated with polymer microstructure. NVP copolymerises with 2 in an almost random, statistical manner whereas copolymerization of NVP with 1 is only effective at low concentrations of 1. The effect of this mismatch of the reactivity ratios (r p and r ) is to produce networks in which units of 1 are clustered together in blocky structures. Although, the mechanistic details behind these NV

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In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


Figure 1. MTT (cell viability) data-the pie-charts show the results of 3 repeated experiments. Positive results indicate an enhancement of viabili negative results indicate a decrease in viability. No effect indicates that v was not significantly differentfromthe control cell cultures. The + orindicates that the experiment was conducted in the presence (+) or abs offoetal calf serum in the culture medium.



199 phenomena are yet to be elucidated, these block structures appear to be less effective at increasing cell viability than the random copolymer structures produced by copolymerization of NVP and 2. These observations have prompted us to prepare soluble analogues of P(NVP-co-2). In order to prepare these polymers we used a modification of our route to highly branched polyvinyl acetate) (7), which involves copolymerization of NVP with allyl 2-isopropoxyethyl carbonate, 3. Polymerizations of NVP in the presence of 3 carried out at 60 °C, in butyl acetate, produced crosslinked networks. However, increasing the reaction temperature to 150 °C produced completely soluble branched polymers. The gelation process at 60 °C is a result of bimolecular termination by combination of propagating branches. At 150 °C this process is slower than termination by transfer to 3 so that gelation does not occur. Figure 2 shows an H-NMR spectrum of a typical polymer prepared in this manner. Resonances derived from methylene groups in residues of 3 (protons 1 and 3 in figure 2) were observed at 5*4.2 and 3.6.

PPM

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3.6

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Figure 2. H NMR spectrum of poly (NVP-co-3), synthesised at 150°C

Branching fractions of 0.036 and 0.064 branches per repeat unit were obtained by using 0.104 and 0.091 mole fractions of branching agent respectively. Cytotoxicity testing of these polymers is currently underway and will be reported in due course.


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Drug release from Hydrogels Two drugs were explored; 5-Fluouracil (5-FU) and 4-methyl umbelliferone (4-MU). Functionalized hydrogels incorporating the cytotoxic drug 5-Fluouracil (5-FU) were synthesised by copolymerizing derivatives of oxycarbonyloxymethyl-5-fluorouracil monomers with NVP (8). Methylolation of 5-FU gave mixtures of N-l-hydroxymethyl-5-fluouracil, N-3-hydroxymethyl-5fluouracil, 3-bis(hydroxymethyl-5-florouracil. The N-1 -hydroxymethyl-5fluouracil and N,NM,3-bis(hydroxymethyl-5-florouracil monomers were modified with allyl chloroformate or isopropenyl chloroformate to yield carbonate monomers 4, 5, 6 and 7. 4, 5, 6 and 7 could be readily copolymerised with NVP to give either linear polymers (copolymerization with 4 or 6) or hydrogel networks (copolymerization 5 or 7).

Polymerization with the monofiinctional monomers (4 and 6) was used to estimate the reactivity ratios for copolymerization with NVP (r = 0.32, r p = 0.97 and r = 0.61, r = 1.31). This study showed that both these allyl carbonate and isopropenyl carbonate monomers copolymerize in an almost statistical manner and that copolymerization with 5 and 7 could be predicted to produce random copolymer networks without significant block formation. We produced materials containing 5, 10, 15 and 20 wt% of 5-FU by copolymerization with 5 and 7. However, degradation of the allyl carbonate crosslinks in copolymers with 5 appears to be very slow and these materials were 5

7

NVP


NV

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not examined further. Drug release from copolymers of NVP with 7 occurred over 50 days (9). The degradation of the carbonate groups followed the increase in swelling as the crosslink density decreased, as shown in figure 3.

W

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time/hours Figure 3. Increase in swelling ratio (W ) over time due to degradation of carbonate crosslinking groups at 37°C. Wt% 7 in feed: • = 5; • = 10; • = 15; 0=20 mi0

The release of 5-FU from these system was monitored using the power law relationship developed by Peppas (10) as shown in figure 5, and exponents of 0.13 - 0.23. These values indicate that as expected the diffusion is not Fickian but is dominated by degradation of the carbonate crosslinking groups. The total amount of 5-FU realised from these hydrogels is also a function of initial composition of the monomer feed, i.e. the amount released is a function of the loading. 5-FU is a cytotoxic drug that has applications in cancer and other disease states and post-operative conditions involving over proliferation. In order to examine the effect of the release of 5-FU, cytotoxicity studies were performed on hydrogels prepared by copolymerization with 5, 10, 15 and 20wt% of 7 in the monomer feed. Cytotoxicity studies were performed using retinal pigment epithelial cells in a static model. Cell contact assays were performed by seeding cells 12 hours prior to the addition of the polymers in either a Labtek® chamber slide system (Nunc) for live/dead staining with calcein A M and Ethd-1 or in a 24


202 0.09H 0.08 0.07 0.06


0,05 0.04 0.03H 0.02 0

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1m

15cT^200

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Time/hours 10: Figure 4. Plot offractional release of 8 against time Wt% 7 in feed: • =/5; A =20

well plate for the trypan blue assay. After 12 hours the polymers were placed on top of the cells and cell viability was assessed at 12, 24 and 48 hours. The trypan blue assay suggested that the wells containing polymer with the 5FU functionality were not viable and this was confirmed by the calcein AM/Ethd-1 staining. It was also apparent that cell proliferation but not viability was reduced in the presence of the polymers lacking the 5-FU group, i.e. hydrogels that were similar in structure to those studied in reference 6. Preliminary data from current studies of the P(NVP-co-2) hydrogels show that these hydrogels can also be used for drug delivery. Delivery of another model compound, 4-methyl umbelliferone (4-MU), an inhibitor of hyaluronan synthesis which may be useful in the treatment of scaring, loaded into P(NVPco-2) hydrogels from solutions containing ImMol or 4mMol 4-MU, see figure 5, shows non-Fickian release with exponents of 0.655 and 0.614 respectively. In summary we find PNVP hydrogels to have a slight mitogenic effect when adjacent to cells and to give promising hydrogels for sustained release of drugs over several days.

Acknowledgements The authors would like to thank the EPSRC for doctoral training awards for L. Smith and S Collins and a post doctoral fellowship for Z Liu.


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A

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6

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0.8• A

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1mMol 4mMo!

E"


E" 0.4 0.2-

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Figure 5. Fractional release of the model compound 4-methyl umbelliferon from P(NVP-co-2) hydrogels over a period of 6 days assessed using fluorescence, excitation 360nm emission 440/460nm, n~6.

References 1.

Robinson, B.V.; Sullivan, F.M.; Borzelleca, J.F.; Schwartz, S.L.; PVP: a critical review of the kinetics and toxicology of polyvinylpyrrolidone (povidone). 1 ed. Lewis Publishers, Michigan, USA, 1990. 2. Chirila, T.V.; Hong, Y.; Dalton, P.D.; Constable, I.J.; Refojo, M.F. Prog. Polym. Sci. 1998, 23, 475-508. 3. Hong, Y.; Chirila, T.V.; Fitton, H.; Ziegelaar, B.W.; Constable, I.J. BioMed Mater Eng. 1997, 7, 35-47. 4. Hong, Y.; Chirila, T.V.; Vijayasekaran, S.; Shen, W.; Lou, X.; Dalton, P.D. J. Biomed. Mater. Res. 1998, 39(4), 650-659. 5. Vijayasekaran, S.; Chirila, T.V.; Hong, Y.; Tahija, S.; Dalton, P.D.; Constable, I.J.; McAllister, I.L. J. Biomater. Sci.-Polym. Ed. 1996, 7(8), 685-696. 6. Smith, L.E.; Rimmer, S.; MacNeil, S. Biomaterials, 2006, 27(14), 28062812. 7. Rimmer, S.; Collins, S.; Sarker, P. Chem Comm. 2005, 48, 6029-6031. 8. Liu, Z.; Fullwood, N.; Rimmer, S. J. Mater. Chem. 2000, 10, 1771-1775. 9. Liu, Z.; Rimmer, S. J. Control. Release. 2002, 81, 91-99 10. Peppas, N.A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur. J. Pharm. Biopharm 2000, 50, 27-46.


Polymers for Biomedical Applications - American Chemical Society

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