Beta Hairpin Peptide Hydrogels as an Injectable Solid Vehicle for Neurotrophic Growth Factor Delivery

Abstract

There is intense interest in developing novel methods for the sustained delivery of low levels of clinical therapeutics. MAX8 is a peptide-based beta-hairpin hydrogel that has unique shear thinning properties that allow for immediate rehealing after the removal of shear forces, making MAX8 an excellent candidate for injectable drug delivery at a localized injury site. The current studies examined the feasibility of using MAX8 as a delivery system for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two neurotrophic growth factors currently used in experimental treatments of spinal cord injuries. Experiments determined that encapsulation of NGF and BDNF within MAX8 did not negatively impact gel formation or rehealing and that shear thinning did not result in immediate growth factor release. ELISA, microscopy, rheology, and Western blotting experiments collectively demonstrate the functional capabilities of the therapeutic-loaded hydrogels to (i) maintain a protective environment against in vitro degradation of encapsulated therapeutics for at least 28 days; and (ii) allow for sustained release of NGF and BDGF capable of initiating neurite-like extensions of PC12 cells, most likely due to NGF/BDGF signaling pathways. Importantly, while the 21 day release profiles could be tuned by adjusting the MAX8 hydrogel concentration, the initial shear thinning of the hydrogel (e.g., during injection) does not induce significant premature loss of the encapsulated therapeutic, most likely due to effective trapping of growth factors within structurally robust domains that are maintained during the application of shear forces. Together, our data suggests that MAX8 allows for greater dosage control and sustained therapeutic growth factor delivery, potentially alleviating side effects and improving the efficacy of current therapies.

Figure 1. Encapsulated NGF and BDNF is continuously released from MAX8

Various concentrations of either NGF (A, B) or BDNF (C, D) were encapsulated in 1.0 wt% MAX8. Media was sampled at the given intervals and the amount of growth factor released was measured by ELISA. Shown is the total amount (A, C) and rate (B, D) of growth factor release (n=3). Error bars represent the SEM for all experiments in this figure.

Figure 2. Encapsulated NGF and BDNF release is dependent upon the concentration of MAX8

1 μg of either NGF (A, B) or BDNF (C, D) was encapsulated in 0.5 wt%, 1.0 wt%, or 1.5 wt% MAX8. Media was sampled at the given intervals and the amount of growth factor released was measured by ELISA. Shown is the total amount (A, C) and rate (B, D) of growth factor release (n=3–4). Error bars represent the SEM for all experiments in this figure.

Figure 3. Shear thinning does not result in release of encapsulated NGF

1 μg of either NGF (A) or BDNF (B) was added directly to media or encapsulated within 0.5 wt%MAX8. MAX8 or MAX8 containing NGF/BDNF was allowed to gel before media was added or MAX8 or MAX8 containing NGF/BDNF was delivered via syringe into a well containing media; media was harvested after 5 minutes and growth factor release was quantified by ELISA. Error bars represent the SEM for all experiments in this figure.

Figure 4. Physical properties of MAX8 do not change after NGF encapsulation

Time sweep using oscillatory rheology to monitor the effects of NGF encapsulation (1μg) on gel formation (G′ and G″) of 1 wt% MAX8 before (A) and after (B) shear was applied. A dashed line represents when shear was applied. G′ after 180 seconds was 293 ± 68 for MAX8 and 479 ± 63 for MAX containing NGF for initially assembled gels; G′ 180 seconds after shearing was 350 ± 66 for MAX8 and 572 ± 48 for MAX8 containing NGF. Frequency sweep of 1.0 wt% MAX8 encapsulated without (C) or with (D) 1μg NGF using oscillatory rheology. At 1 rad/s, G′ was 805±186 for MAX8 and 1334±143 for MAX8 containing NGF.

Figure 5. PC12 cells develop neurite-like extensions in response to NGF released from MAX8

(A) Transwells containing 0.5% MAX8 with and without 1 μg encapsulated NGF were added to PC12 cells. Media was not replaced during this experiment and cells were imaged after 7 days. Shown are representative images; bars represent 50 μm. (B) Images from three separate experiments were analyzed and the number of cells with neurite-like extensions were recorded. Error bars represent the SEM for all experiments in this figure.

Figure 6. PC12 cells extend neuron-like processes while in direct contact with MAX8

(A) NUNC chamber slides were coated with either 0.5 wt% MAX8 or 0.5 wt% MAX8 containing 1μg NGF. PC12 cells were allowed to adhere on top of the MAX8 gel and imaged at the given intervals. Shown are representative images; bar represents 25 μm. After 3 (B) or 7 (C) days images were analyzed for neurite-like extensions. Error bars represent the SEM for all experiments in this figure.

Figure 7. Encapsulated NGF released from MAX8 is biologically active for up to 28 days

(A) NGF encapsulated within MAX8 was released for 14 or 21 days before the gels were moved to wells containing PC12 cells. 1 week later cells were imaged. Shown are representative images; bars represent 25 μm. (B). Images from three separate experiments were analyzed for PC12 cells with neurite-like extensions. (C) 1 μg of NGF or BDNF was encapsulated within 0.5 wt% MAX8 or 1 μg of NGF or BDNF was added directly to serum free media. Media was collected at various intervals and the amount of growth factor was determined by ELISA analyses. The ratio of growth factor (compared to d0) is shown. Error bars represent the SEM for all experiments in this figure.

Figure 8. Encapsulation within MAX8 does not disrupt growth factor signaling

Serum-starved 3T3 cells were allowed to adhere in a 24-well dish for one hour before the addition of either deinoized water or 1μg NGF (A) or transwells containing 0.5 wt% MAX8 plus DI water or 1μg NGF (B). Cells were counted 24 hours later. (C) Media collected from transwells containing 0.5 wt% MAX8 plus DI water or 1 μg NGF were added to serum-starved PC12 cells. Cell lysates were harvested after 1 hour and used in Western analyses. Error bars represent the SEM for all experiments in this figure.