Biodegradable Ionomers for the Loading and Release of Proteins

Chapter 15

Biodegradable Ionomers for the Loading and Release of Proteins: Formation, Characterization, Mechanism, and Consequence of Water Uptake Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch015

1

1

1

2

Fredrik Nederberg , Björn Atthoff , Tim Bowden , Ken Welch , Maria Strömme , and Jöns Hilborn 2

1

1

Department of Materials Chemistry, Polymer Chemistry, The Ångström Laboratory, Uppsala University, Uppsala, Sweden Department of Engineering Sciences, The Ångström Laboratory, Uppsala University, Uppsala, Sweden 2

The increased understanding of proteins and the human genome point towards a future in which selective proteins may replace synthetic drugs in the general cure of disease. In this scenario the ability of controlled and gentle release of proteins provide the key for successful treatment. To address the ability of full protein delivery we have developed a series of telechelic biodegradable ionomers based on poly (trimethylene carbonate) carrying zwitterionic, anionic or cationic functional groups. The introduction of polar end-groups provides a material with unique properties that directs the introduced functionality within the material bulk but also to the material surface if water is introduced. Bulk aggregation provide a low elastic modulus material and the ability to surface enrich provide the on-set of water swelling. The latter finally results in a co-continuous water-ionomer structure that engulfs and stores proteins simply by soaking the material in an aqueous protein solution. Following protein loading the material can be dried and re-immersed in water so that release occurs. Our results, including both the careful synthesis and the ability to load and release proteins, provides new possibilities for full protein delivery.

250

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

251

Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch015

In this chapter the increased understanding of the water swelling properties and the subsequent formation of bulk water domains in biodegradable poly (trimethylene carbonate) (PTMC) ionomers is presented. The recent discovery that Afunctional PTMC may be functionalized with polar co-phosphoryl choline (PC) end groups and that the resulting telechelic zwitter ionomer forms an interesting low elastic modulus material has encouraged and directed the use of biodegradable ionomers in new areas of biomaterial research (I). Present findings now suggest that the scope of the synthesis may be broadened to provide telechelic ionomers with additional functionalities (2) and also that the water absorbing properties of such ionomers indicate their potential to serve as novel carriers for the loading and release of proteins (3).

Introduction In an effort to extend the scope from synthetic biodegradable polymers to materials that are able to dissolve or disperse both hydrophilic and hydrophobic substances, we have developed biomimetic zwitterionic, cationic and anionic ionomers. In order for those materials to perform as temporary hosts, to load and release active substances, pathways of their transport must be provided for. Possibly the most intriguing transport pathway is a linear swelling front of water which can provide an open porous material with a resulting co-continuous structure of both water and the swollen material. Such swelling mechanism is unique in that it may provide for zero order release kinetics or a constant rate of release from a biodegradable reservoir. This chapter describes the synthesis of such materials, their interaction with hydrophobic and hydrophilic substances, and the successful linear propagation of a distinct swelling front to produce an open porous structure. Finally we apply these materials by demonstrating how loading and release of proteins is achieved without the use of solvents or elevated temperatures. In our initial work on biodegradable ionomers it was postulated that the unique material properties of these ionomers originated from bulk aggregates of polar PC groups embedded in a hydrophobic PTMC matrix such that physical cross links were formed (I). A schematic representation is shown in Figure 1. This was further supported by the fact that the resulting material, as compared to non-functional PTMC, swelled significantly in water. Still the question has been: How does this swelling actually occur and what are the consequences for the material? (3a) To address this question we will currently introduce new results regarding the mechanism by which the material absorbs water and providing additional information on how the interior co-continuous (water and ionomer) structure is formed and what it looks like. Furthermore, supporting observations from dielectric spectroscopy measurements are provided as a direct means for

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


252

Figure 1. Schematic representation of polar PC bulk aggregates in the hydrophobic PTMC matrix.

studying of bulk PC aggregates and the dynamics of such aggregates with the introduction of water (4).

Synthesis Our ionomers are designed based on a hydrophobic PTMC backbone. The preparation consists of stannous octanoate catalyzed ring opening polymerization (ROP) of the cyclic trimethylene carbonate monomers initiated from butanediol. PTMC diols of predictable molecular weights were formed in accordance to Figure 2a. The resulting alcohols where further quantitatively converted to result in three different biomimetic telechelic oligomers, shown in Figure 2b. The first anionomer has a sulphate end functionality similar to that of heparin. Heparin has been shown to be responsible for the binding of a magnitude of proteins in-vivo (5). To prepare the anionic moiety having sulphonic acid at both chain ends, the PTMC diol was employed as a nucleophile to the electrophilic sulfur trioxide trimethylamine complex, giving the PTMC disulphonate as a product. The second material is a end functionalized cation with trimethylammonium carrying the positive charge. This functionality is similar to that of in-vivo carnitine, which function as a complexing carrier for the transport of long chain activated fatty acids into the mitochondrial matrix (6). The second material is prepared in two steps: initially 4-chlorobutyryl chloride is reacted onto the cohydroxyl end-group of PTMC, finally trimethylamine displaces the chloride to introduce the cationic ammonium group. The third material is equipped with zwitterionic chain ends by the initial phosphorylation with ethylene chlorophosphate in the presence of a base


253 followed by the ring opening of the intermediate cyclic phosphate with trimethylamine at 60°C under pressure (7). This results in the formation of chain end functionalized phosphoryl choline which is analogous to the polar head group in the most common phospholipids in cellular membranes. It is noteworthy that all post transformations on the PTMC diol resulted in complete conversion as judged by H NMR and formation of only the pure desired products (2,8). !


Properties and Characterization The newly synthesized materials showed completely new properties as compared to PTMC. While the starting material was tacky and with poor film forming capabilities, the functionalized material was firm, showed more elastic properties, and could be cast into solid films. We believe that the anticipated ionomeric behavior explained by microscopic phase separation into zwitterionic aggregates result in the drastic change of the material characteristics. In order to obtain more data regarding the mechanical performance of the material and thus support the presence of ionomers, rheology measurements were performed. Specifically, an oscillating torque experiment, in which the PC ionomer was compared with PTMC at a frequency of 1Hz was used. Figure 3 shows the storage shear modulus (G') as function of temperature of the two materials. At ambient conditions PTMC behaves as an amorphous melt with no mechanical integrity whereas the ionomer, as anticipated, behaves like a rubber with a G ' value of ~2 MPa. In addition, Figure 3 reveals a rubbery plateau of the ionomer, stretching from 5°C to about 40°C and the viscous region is not reached until 70°C. These results demonstrates that one could benefit from the rubbery behavior at physiological conditions (~37°C). Moreover, one can observe an interesting shift in the glass transition temperature (T ) of the two materials. For PTMC the T is located at -16°C since the contribution of flexible end-groups is significant. For the PC ionomer however, a large difference was observed, as the T shifted to -5°C. Most likely the end-groups of the ionomer are captured in zwitterionic aggregates forming physical cross-links that restrains molecular mobility that raise T instead of lowering it. The higher temperature needed for the onset of translational mobility agrees well with the behavior of ionomers reviewed by Eisenberg as early as 1971 (9). The behavior of the anionic and cationic ionomers was similar to the PC ionomer (1,2). The observed shift of the glass transition was further analyzed by thermal analysis. Differential scanning calorimetry (DSC) was used and the results are shown in Figure 4. A similar shift to that found in the rheometer was observed, Without the frequency dependence however, the T was -27°C for the starting PTMC as compared to -16°C for the PC ionomer. g

g

g

g

g





Figure 2. (a) Ring opening polymerization of trimethylene carbonate initiatedfrom butane diol and catalyzedfrom stannous octanoate. (b) Three different telechelic ionomers, anionomer, cationomer and zwitterionomer.


256 10

G' (MPa)

PTMCdioi (o)

T ~-I6°C g

Ionomer (x) Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch015

-5°C

Rubbery plateau

I

Temperature (°C)

50

Figure 3. Results of oscillating rheology measurement of PTMC and PC ionomer. (Insets: Tg and rubbery plateau of PC ionomer)

To further analyze the bulk domains and the dynamics involved, dielectric spectroscopy measurements were performed. More specifically the real part of the dielectric loss was measured as function of frequency and temperature. The non functional PTMC was used as reference and any additional relaxation present in the spectra of the PC ionomer could be attributed to the dipolar vector of the PC group that interacts with the applied alternating electrical field. A common characteristic of essentially all amorphous polymers is that they exhibit both a principal relaxation related to the dynamic glass transition, termed the arelaxation, and a secondary relaxation called the p-relaxation. It is generally accepted that the a-relaxation is due to segmental motion (i.e. conformational changes) along the polymer chain. On the other hand, the dielectric p-relaxation stems from localized rotational fluctuations of the dipole vector. In addition to the presence of a- and P-relaxations (found in both PTMC and the PC


257

,

1


-35

1

-25

-15

Temperature (°C) Figure 4. DSC curves of PTMC (solid line) and PC ionomer (dotted line). T measured on 2 heating. nd

g

ionomer) two additional relaxations were found in the PC ionomer that were related to the presence of PC domains (4). These additional relaxations were called a-ionomer and X. The dielectric spectra of the PC ionomer and PTMC are shown below in Figure 5. The a-ionomer relaxation was found to be directly related to the conformational changes of individual PC dipoles, whereas the X relaxation most likely is related to movement of adjacent PC groups in the zwitterionic aggregates. This observation will be further discussed below.

Formation and Mechanism for Water Swelling As further evidence of the existence of polar bulk domains, a separate study has shown that the PC ionomer swells in water and that this behavior could be utilized to load and release proteins (3a). Initially it was believed that water was absorbed through gas phase diffusion such that the polar PC domains would swell and finally coalesce to form interior water channels in the material. Osmosis would trigger the swelling and the PC groups would minimize their energy by enriching the water interphase. The latter is similar to the surface enrichment of polar PC groups when introducing water to the material surface (8JO). In a study of a cationic ionomer however, a very interesting finding was observed that demonstrates how surface properties can affect water absorption (3b). When the cationic surface was pretreated with the negatively charged heparin (Mw 5000-30000 g/mol), the material did not swell at all even upon prolonged (5 months) storage in water. The lack of swelling is most likely due to


258


a

Figure 5. Dielectric loss £

der

spectra as a function offrequency and

temperature for a) PC ionomer and b) PTMC.



259 polyelectrolyte adsorption which creates a charged blocking layer on the surface. Thus, an immobilized layer of a few nm thickness inhibits the mechanism responsible for the entrance of water into the structure. If one takes into account the rapid surface rearrangement upon water immersion due to the migration of ionic chain ends to the material-water interface, it seems plausible that this mobility is associated with the swelling phenomena. Following, or accompanying, the surface rearrangement is the formation and opening of channels guiding water into the structure. Hence, water soluble species may be charged into these materials by simply placing a solution of this in direct contact. The water "channels" will open allowing passage into the material. Evaporation by drying removes only water and leaves the cargo inside the material. In fact, as the material is well above its Tg, the channels will close to encapsulate the content. Re-immersion into water will again open the diffusion pathways to allow for a release that might be possible to be controlled by the swelling front only. A schematic picture of how the material engulfs and store proteins is shown in Figure 6. These materials may offer attractive opportunities for delivery of macromolecules, where the delivery system protects the protein against denaturation by body fluids and provides for constant and sustained release. A microscopic investigation of the swelling revealed a sharp swelling front having a linear propagation rate of between 0.5|im/min (PC ionomer) to 20um/min (cationic ionomer). The formed interior structure left by the propagating water front shows a porous structure with pore sizes between 1 and 10 jim, as determined by cryo SEM (Figure 7). By dissolving the red emitting hydrophobic fluorophore Rhodamine DHPE (Lissamine™ rhodamine B l,2-dihexadecanoyl-Mglycero-3-phospho ethanolamine trimethylammonium salt) into the polymer and the green emitting BODIPY 492/515 disulfonate (4,4-difluoro-l,3,5,7,8-pentamethyl-4-bora-3a,4adiaza-5-indacene-2,6-disulfonic acid disodium salt) in the swelling water, shown in Figure 8, it becomes evident that the porosity is open allowing free passage of green water into the structure. The dynamics involved when water is absorbed was further supported by comparing the dielectric spectra of a dry sample with a sample which was left in the measurement cell for a period of one month and allowed to equilibrate with the surroundings at 37% relative humidity. As can be seen in Figure 9 the ctionomer relaxation has shifted to a higher frequency, indicating that the moiety responsible for this relaxation now has a higher mobility. Moreover, the X relaxation has almost completely disappeared. If this relaxation is associated with adjacent PC groups in the aggregate as was suggested earlier, the swelling of the aggregates could result in a decreased interaction between PC end-groups and a subsequent decrease in the relaxation strength.



Figure 6. Schematic representation of the mechanism ofprotein loading and release.


s


261

Figure 7. Cryo SEM revealing the inner structure of swelled ionomers.

Figure 8. Confocal micrograph Hydrophobic PTMC stained with red Rhodamine DHPE and the water phase stained with green B0D1PY.



262

Frequency (Hz)

Figure 9. Two s"der spectra for the same PC ionomer sample collected at 30 °G, but with different moisture content. In the spectra corresponding to higher moisture content, the a-ionomer relaxation has shifter to higher frequencies while the X relaxation appears to have disappeared.

Protein Loading and Release Following the careful investigation of how our telechelic ionomers absorb water and how the swollen structure forms, and appears we wanted to investigate if this absorption ability could be utilized for protein loading and release. To test this question, discs of known dimensions were compression molded and allowed to equilibrate for a known time in different aqueous protein solutions. Following loading and sometimes drying (freeze drying or air drying) the discs were reimmersed in water and the release was measured. In these studies various proteins were used including insulin, albumin, carboxyhemoglobin (COHb), and cytochrome C. The latter two both carry the red iron containing heme group and upon denaturation change their colour from red to green, blue, or brown (11). COHb and cytochrome C therefore provide adventagous visual markers when studying if protein activity is retained following release from the substrate. Shown in Figure 10 is a selective release profile after freeze drying of discs incubated in an aqueous albumin solution. In this example, the release is about 60% after one week. Under milder drying conditions (air drying) or without any drying step, the protein release was near quantitative as was shown using COHb and cytochrome C (Figure 11). In addition, the released proteins maintained their activity since their respective UV spectra maintained constant before and following release (Figure 12) (3b).



263

o*50

100

150

200

250

300

Time (hrs)

Figure JO. Release profile of albumin viewed as the release at time t (Mj) normalized with the maximum release (M ) as function of time. max

Conclusion Throughout the course of this work we have demonstrated a new route to biomimetie and telechelic ionomers carrying either phosphoryl choline, sulphonate, or quaternary ammonium functional end-groups. The careful synthesis allows for complete conversion of the hydroxyl functionalised PTMC to the PTMC containing desired functionality, which results in a low elastic modulus material due to the bulk agglomeration of polar groups. The presence of physical cross-links has been confirmed using both spectroscopy and mechanical analysis. Water swelling is an additional feature of the resulting materials and a thorough evaluation has shown that water is absorbed from the surface, leaving an open co-continuous (water-ionomer) structure. The ability to absorb water and the open swollen structure was utilized for protein loading simply by letting the material swell in an aqueous protein solution. Near quantitative release was achieved either directly after loading or following an additional drying step. Protein activity is maintained following release, suggesting that the material may favourably interact with guest proteins such that denaturation is suppressed. The gentle and overall behaviour in which proteins are loaded and released has shown that full protein delivery is possible.



100

-i

Time (h)



—

,

50

, 150

, 100

,

—

, 250

Time (h)

200

, 300

, 350

, 400

, 450

Figure 11. Curves showing the release of COHb and Cytochrome C from dried discs. Top, cationomers of three different molecular weight 2000, 4000 and 12000 g/mol, below the release from 4000 and 12000g/mol anionomer.

0

o 4—

50 -j


266 A—Cytochrome C o o c

CD

o

n

Biodegradable Ionomers for the Loading and Release of Proteins

Recommend Documents