The major goal of this study was to develop polyelectrolyte negatively charged hydrogels with properties that could be varied in a controlled manner, and to provide a controlled delivery system for DOX. OPF is an injectable and biodegradable material previously used in our group for cartilage tissue engineering and nerve regeneration [20
]. We have reported that the physical and chemical properties of OPF hydrogels can be modified with incorporation of a positively charged monomer 2-(methacryloyloxy) ethyl]-trimethylammonium chloride (MAETAC), and positively charged OPF promoted neurite outgrowth from primary neuronal cells[20
]. In this study, the OPF macromer, copolymerized with varying amounts of a pH-dependent ionic monomer (SMA), becomes a hydrogel that contains fixed charge. Zeta-potential and conductivity measurements confirmed the electrically charged nature of SMA-modified hydrogels and that the amount of charge was dependent upon the SMA concentration in the polymerization formulation (data not shown). Incorporation of the unsaturated SMA monomer increased the frequency of crosslinks between OPF polymer chains, as demonstrated by a decreased sol fraction of charge-modified OPF hydrogels compared to unmodified hydrogels. This decrease in the sol fraction is indicative of a highly crosslinked network.
Attenuated total reflectance FTIR data demonstrated that SMA was successfully incorporated into the OPF hydrogel. After hydration in deionized water, the carbon-oxygen (C-O) peak intensity decreased on hydrogel surfaces as the concentration of SMA increased. The change in C-O peak intensity was inversely related to the swelling ratio of hydrogels. When the C-O peak intensity decreased, the swelling ratio of SMA-modified hydrogels increased. Such correlation suggests a conformational change in the SMA-modified hydrogel backbone, which is involved in ion and solvent transport, in different solvents. This results in SMA-modified hydrogels sensitivity to ionic strength and pH of the solvents. The swelling ratio of charged hydrogels in deionized water was shown to be a function of SMA concentration in hydrogel precursor solution. However, in PBS, swelling ratios of SMA-modified hydrogels were unchanged. We demonstrated that swelling ratios of SMA hydrogels changed with the change in ionic strength of NaCl solutions as well. With an increase in the ionic strength of the solution, the swelling ratio of charged hydrogels decreased. Hydrogels became more sensitive to the salt concentration with an increase in SMA concentration in hydrogel formulations. In contrast, there was little to no response to the ionic strength of the solution from hydrogels without charge (HG-0). When the polymeric network contains charged moieties such as carboxylic acids, the swelling becomes more complex. The swelling pressure may be greatly enhanced as a result of the localization of charged moieties, setting up an electrostatic repulsion between charged polymers. Since soluble polymers in a crosslinked network are interconnected covalently, the mobility of fixed charge moieties on the polymer backbone is hindered and they cannot diffuse freely into the outer solution. Hence, counter ions (original bounded cations) released from the polymer chains are confined inside the gel due to an electroneutrality effect. Consequently, the total mobile ion concentration inside the gel will exceed that in the external solution, leading to an osmotic pressure difference which tends to drive solvent into the gel from the less concentrated external solution.
Furthermore, we demonstrated that SMA-modified hydrogels are responsive to the pH of the solution. SMA-modified hydrogels had low swelling ratios at pH<3, however, it significantly increased at pH=3 and greater. Again, the hydrogels containing higher amounts of SMA appeared to exhibit more pH sensitivity. The swelling ratio of HG-SMA300 at pH=3 was about three times higher that that at pH=1. However, it did not change significantly with an increase in pH from 3 to 12. As expected for charged hydrogels, compressive modulus decreased with increasing swelling ratio of SMA-modified hydrogels. The compressive modulus of these hydrogels decreased with increasing SMA concentration in their formulations.
SMA-modified hydrogels could be effectively loaded with DOX due to the electrostatic interactions between negatively charged hydrogels and the protonated primary amine group on DOX. Uptake of DOX by SMA-modified hydrogels was shown to be an active process. These hydrogels had a high loading efficiency and absorbed about 99% of the drug from the loading solution within a few hours. Loading kinetics were shown to be a function of SMA concentration; for example 99% of the DOX in loading solution was taken up by HG-SMA300 hydrogel after 2h, while 95%, 80% and 60% was absorbed by HG-SMA200, HG-SMA100 and HG-SMA50, respectively. After 4 h, most of the drug was absorbed by SMA-modified hydrogels. This is favorable for clinical use of this material as the drug can be loaded in a short time period prior to administration. For comparisons, we measured the equilibrium loading efficiency of hydrogels after 24 hours. The loading efficiency of unmodified hydrogels decreased from 43% to about 25% as the concentration of DOX increased in loading solution. Unlike unmodified hydrogels, the loading efficiency of SMA-modified hydrogels did not change significantly with an increase in concentration of DOX in loading solution. SMA-modified hydrogels exhibited a high capacity for DOX uptake due to the electrostatic interactions between the drug and charged functional groups within the hydrogels. Our data revealed that SMA-modified hydrogels were capable of absorbing about 25mg of DOX per gram of hydrogel from loading solution at a concentration of 400 μg/ml. With an increase in concentration of DOX in loading solution (1mg/ml), charged hydrogels were still able to uptake about 99% of DOX and hydrogels absorbed about 60 mg/g from the solution (data not shown). Loading efficiency is an important factor in drug delivery approaches. The materials with high loading efficiency reduce drug waste and make it possible to deliver a very high local dose by using only a small amount of polymer.
The responsiveness of SMA-modified hydrogels to the ionic strength and pH of the surrounding environment is useful for drug delivery applications. As is typical of polyelectrolyte hydrogels, SMA-modified hydrogels swell significantly in deionized water and dilute solutions, while behaving as a neutral polymer with limited swelling in high ionic strength or low pH solutions. Given these data, an ion-exchange mechanism is suggested for release of DOX from SMA-modified hydrogels in which the rate of release can be controlled by the ionic strength of the eluting solution. The main counter ion present in PBS and NaCl is Na+, which is exchanged with DOX bound to the charged hydrogels. The rate and extent of DOX release was shown to correlate well with the amount of SMA in the hydrogel formulation. With incorporation of SMA the rate of release was significantly reduced and only 20% of the loaded amount was released from HG-SMA300 after 15 days. However, most of the DOX absorbed by HG-0 was released in the first few days of study. Our results revealed that the burst release significantly decreased with incorporation of SMA in OPF hydrogels. There was almost no burst release with increasing concentrations of SMA to 30% in HG-SMA300. Comparison of the total DOX release at two different loading doses (50 and 200 μg/ml) suggested that loading dose did not have a significant effect on the release kinetics of DOX. The ionic strength of the salt solution had a significant effect on the extent and rate of DOX release from SMA-modified hydrogels. However, the effect of salt concentration was less significant at high concentrations of the salt (1M) due to a decrease in solubility of DOX in this solution. These data suggest that the release kinetics of DOX are not only influenced by the salt concentration but also by the solubility of the drug in the medium.
The antitumor efficacy of DOX released from the hydrogels was investigated by measuring MG63 osteosarcoma cell death over three days. It appears the process of DOX loading to the hydrogels had no effect on biological activity of the released DOX and it preserved its antitumor activity. In addition, the percentage of living cells in the presence of DOX released from HG-0 and HG-SMA50 was slightly less than other SMA-modified hydrogels. This could be due to the burst release from these hydrogels in the first days of treatment that reduced cell proliferation, leading to more cell death.