Thermosensitive Biodegradable Hydrogels for Local and Controlled Cerebral Delivery of Proteins: MRI-Based Monitoring of In Vitro and In Vivo Protein Release

Abstract

Hydrogels have been suggested as novel drug delivery systems for sustained release of therapeutic proteins in various neurological disorders. The main advantage these systems offer is the controlled, prolonged exposure to a therapeutically effective dose of the released drug after a single intracerebral injection. Characterization of controlled release of therapeutics from a hydrogel is generally performed in vitro, as current methods do not allow for in vivo measurements of spatiotemporal distribution and release kinetics of a loaded protein. Importantly, the in vivo environment introduces many additional variables and factors that cannot be effectively simulated under in vitro conditions. To address this, in the present contribution, we developed a noninvasive in vivo magnetic resonance imaging (MRI) method to monitor local protein release from two injected hydrogels of the same chemical composition but different initial water contents. We designed a biodegradable hydrogel formulation composed of low and high concentration thermosensitive polymer and thiolated hyaluronic acid, which is liquid at room temperature and forms a gel due to a combination of physical and chemical cross-linking upon injection at 37 °C. The in vivo protein release kinetics from these gels were assessed by MRI analysis utilizing a model protein labeled with an MR contrast agent, i.e. gadolinium-labeled albumin (74 kDa). As proof of principle, the release kinetics of the hydrogels were first measured with MRI in vitro. Subsequently, the protein loaded hydrogels were administered in male Wistar rat brains and the release in vivo was monitored for 21 days. In vitro, the thermosensitive hydrogels with an initial water content of 81 and 66% released 64 ± 3% and 43 ± 3% of the protein loading, respectively, during the first 6 days at 37 °C. These differences were even more profound in vivo, where the thermosensitive hydrogels released 83 ± 16% and 57 ± 15% of the protein load, respectively, 1 week postinjection. Measurement of volume changes of the gels over time showed that the thermosensitive gel with the higher polymer concentration increased more than 4-fold in size in vivo after 3 weeks, which was substantially different from the in vitro behavior where a volume change of 35% was observed. Our study demonstrates the potential of MRI to noninvasively monitor in vivo intracerebral protein release from a locally administered in situ forming hydrogel, which could aid in the development and optimization of such drug delivery systems for brain disorders.

Keywords: IVIVR; contrast MRI; drug delivery; in situ hydrogel; protein release; sustained release.

Scheme 1. Chemical Structure of Partially Thiolated Hyaluronic Acid (HA-SH)

The degree of thiolation was 60%.

Scheme 2. Chemical Structure of ABA-Triblock Copolymer Based on a Polyethylene Glycol (PEG₁₀₀₀₀) B-Block Flanked by Two Acrylated Poly(N-(2-hydroxypropyl) Methacrylamide Mono/Dilactate) (pHPMA-lac) A-Blocks

Characteristics of the A-blocks: M_n was 14 kDa as determined by ¹H-NMR (DMSO), molar ratio of mono/dilactate was 75:25 and the degree of acrylation was 15%. The average molecular weight (M_n) of the ABA-triblock polymer (pHPta), determined by ¹H-NMR (DMSO), was 38.8 kDa, and the cloud point was 15 °C.

Figure 1

Regions-of-interest on in vitro and in vivo MR images of hydrogels: (A) Illustration of in vitro MRI setup for measurement of the release of Galbumin from hydrogels (identified at the bottom of the tube by high signal intensity on GE3D image) into the supernatant. Regions-of-interest (ROIs) are depicted in the hydrogel (green) and the PBS supernatant (blue). (B) T₁ map of a coronal rat brain slice after stereotaxic injection of nonlabeled hydrogel in the right striatum. The corresponding ROIs for further image analysis are overlaid on the image. Green color represents implanted hydrogel; blue is surrounding peri-injection tissue. The red line indicates the 2-voxel wide rim area that was excluded from analyses.

Figure 2

Rheological properties of the hydrogels. Storage modulus (G′) and loss modulus (G″) of low concentration (LC Thermo) and high concentration (HC Thermo) thermosensitive hydrogels in time measured in situ for 5 h at 37 °C. At time = 0, the liquid pregel samples were inserted in the rheometer.

Figure 3

MRI volumetry of LC Thermo and HC Thermo gels: (A) in vitro, from day 0 to day 27, and (B) in vivo, from day 0 to day 21. In vitro measurements were started 75 min after the initiation of the gelation process. The first in vivo MRI data was acquired approximately 60 min after the stereotaxic gel injection. Volumetric analysis was performed on GE3D images and T₁ maps for the in vitro and in vivo MRI measurements, respectively. Data are shown as mean ± SD (n = 3).

Figure 4

MRI of protein release from in vitro hydrogel samples: GE3D images (top) and T₁ maps (bottom) of PCR tubes filled with 40 μL of Galbumin-loaded and nonloaded (blank) LC Thermo and HC Thermo gels, covered with 160 μL of PBS. The images were acquired 1 (left) and 24 (right) h after gelation. The signal intensity of the supernatant increased in GE3D images and T₁ values in supernatant decreased (T₁ maps) with time as a result of Galbumin release (white arrows).

Figure 5

In vitro release of Galbumin from the LC and HC hydrogels: in vitro cumulative release (%) of Galbumin from the LC Thermo and HC Thermo gels at 37 °C over 27 days after gelation. Each point represents the mean value ± SD (n = 3).

Figure 6

In vitroGalbumin release from the hydrogels at 37 °C: Galbumin concentration of LC Thermo and HC Thermo as a function of the square root of time up to 24 h after gelation. Each point represents the mean of three samples. The lines represent the linear fit of the data.

Figure 7

In vivo MRI of hydrogels after injection in rat brain. Representative GE3D images of axial (left), coronal (top right), and sagittal (bottom right) rat brain sections directly after stereotaxic Galbumin-loaded gel (LC Thermo) injection into the right striatum.

Figure 8

Serial T₁ maps of rat brain after injection of different blank or Galbumin-loaded hydrogels. T₁ maps of a coronal rat brain slice at different time points after stereotaxic injection of nonloaded (blank) or Galbumin-loaded hydrogel in the right striatum. Directly after injection (day 0), the Galbumin-loaded hydrogel appears as an area with relatively low T₁ values due to significant Galbumin-induced T₁ shortening. Hydrogel volume remained relatively stable (LC Thermo), or increased (HC Thermo), up to day 21 after injection. T₁ normalization in Galbumin-loaded thermosensitive gels during the first week after injection pointed toward release of Galbumin. A significant prolongation of T₁ was observed in tissue surrounding the injection area of Galbumin-loaded or blank LC Thermo gel on day 2 and 7 after implantation (white arrows). Additionally, a prominent rim with relatively low T₁ values enveloping the LC Thermo gels injection site formed around day 7 and remained present up to day 21 (black arrows).

Figure 9

In vivo Galbumin release from intracerebrally injected hydrogels. In vivo cumulative Galbumin release from LC Thermo and HC Thermo gels as a function of time after intracerebral injection in a rat brain. Data are shown as mean ± SD (LC Thermo, n = 3; HC Thermo, n = 3).

Figure 10

T₁ of surrounding ipsilateral and homologous contralateral brain tissue at different time points after unilateral implantation of the hydrogels. *P < 0.05 vs day 0.